JP2009504157A - Albumin fusion protein - Google Patents

Abstract

  The present invention includes albumin fusion proteins. Nucleic acid molecules encoding albumin fusion proteins of the present invention are also included in the present invention, vectors containing these nucleic acids, host cells transduced with these nucleic acid vectors, and albumin fusion proteins of the present invention Also included are methods of making and methods of using these nucleic acids, vectors, and / or hosts. Furthermore, the present invention includes a pharmacological composition comprising an albumin fusion protein and a method for treating, preventing or reducing a disease, illness or condition using the albumin fusion protein of the present invention.

Description

  The present invention relates generally to therapeutic proteins fused to albumin or albumin fragments or variants, including but not limited to at least one polypeptide, antibody, peptide or fragment and variant thereof. The present invention contemplates polynucleotides encoding therapeutic albumin fusion proteins, therapeutic albumin fusion proteins, compositions, pharmacological compositions, formulations and kits. A host cell transduced with a polynucleotide encoding a therapeutic albumin fusion protein may also comprise the present invention, as well as methods of using these polynucleotides and / or host cells to form the albumin fusion protein of the invention. Is contemplated by.

Reference to Sequence List on Compact Disc This document refers to the “Sequence List” listed below, which includes three individual compact discs labeled “Copy 1”, “Copy 2” and “Copy 3” ( CD-R) as an electronic document. Each of these compact discs includes “PF617 PCT Sequence Listing.txt” (1,192,036 bytes, created July 28, 2006), which is incorporated herein by reference.

BACKGROUND ART Human serum albumin (HSA, HA), a 585 amino acid protein in its mature form (as shown in FIG. 1 (SEQ ID NO: 1)) is responsible for a significant proportion of serum osmotic pressure. And also functions as a carrier for endogenous and foreign ligands. Currently, HA for clinical use is produced by extraction from human blood. Production of recombinant HA (rHA) in microorganisms is described in EP 330451 and 361991.

  Their natural state, or recombinantly produced therapeutic proteins, such as interferons and growth hormones, are unstable molecules that typically exhibit a short half-life, especially when formulated in aqueous solution. It is. The instability of these molecules when formulated for administration indicates that many of the molecules should be lyophilized and frozen at all points during storage, thereby It makes it difficult to transport and / or store molecules. The storage problem is particularly acute when the pharmacological prescription must be stored and dispensed outside the hospital environment.

  Few practical solutions to the problem of preserving unstable protein molecules have been proposed. Thus, there is a need for stable, long-lasting formulations of proteinaceous therapeutic molecules that are preferably simple to formulate with minimal manipulation required after storage.

  Upon in vivo administration, their native state, such as interferon and growth hormone, or recombinantly produced therapeutic proteins exhibit short plasma stability due to rapid clearance from the bloodstream. Thus, the therapeutic effect provided by these proteins is also short-lived. Therefore, in order to maintain these desired therapeutic effects in vivo, the therapeutic molecules must be administered more frequently or at higher doses due to the rapid clearance of these proteins from the blood. Is instructed. However, increasing the dosing schedule for the administration of therapeutic proteins often results in increased injection site reactions, side effects, and toxicity in the patient. Similarly, administration of therapeutic proteins at higher doses also generally results in increased toxicity and side effects in the patient.

  Few practical solutions to increased plasma stability of therapeutic molecules, including chemical conjugates, have been proposed, limiting patient benefit. In general, in most cases, these chemically modified therapeutic molecules are still administered on a frequent dosing schedule, leaving significant injection site reactions, side effects and toxicity in patients. Thus, it maintains higher plasma stability in vivo than natural or recombinantly produced treatments alone and can be administered less frequently, thereby reducing potential side effects on the patient. There is a need for stabilized forms of therapeutic molecules.

SUMMARY OF THE INVENTION The present invention contemplates an albumin fusion protein comprising a therapeutic protein (eg, a polypeptide, antibody or peptide or fragment or variant thereof) fused to albumin or a fragment (part) or variant of albumin. ing. The invention also includes nucleic acids encoding therapeutic proteins (eg, polypeptides, antibodies or peptides or fragments or variants thereof) fused to albumin or a fragment (part) or variant of albumin or A polynucleotide consisting of is also contemplated. The present invention also provides solutions for extending the lifetime of a therapeutic protein, increasing plasma stability of a therapeutic protein, and / or in vitro and / or in vivo compared to its unfused state. A therapeutic protein (in a pharmacological composition) fused to albumin or a fragment (part) or variant of albumin sufficient to stabilize its activity (or in its pharmacological composition) Contemplated are polynucleotides that comprise or consist of nucleic acid molecules that encode proteins, including, eg, polypeptides, antibodies or peptides or fragments or variants thereof. An albumin fusion protein encoded by a polynucleotide of the present invention is also contemplated by the present invention, and also produces a host cell transduced with the polynucleotide of the present invention, and an albumin fusion protein of the present invention, and Methods using these polynucleotides and / or host cells are also contemplated.

  In a preferred aspect of the invention, albumin fusion proteins include, but are not limited to, those disclosed in Table 2 and polynucleotides encoding such proteins.

  The present invention also includes a pharmacological formulation comprising an albumin fusion protein of the present invention and a pharmacologically acceptable diluent and carrier. Such a formulation can be in a kit or container. Such kits or containers are packaged with instructions relating to the extended lifetime of the therapeutic protein. Such a formulation is in a method for treating, preventing, ameliorating or diagnosing a disease or disorder symptoms, comprising administering to a patient, preferably a mammal, most preferably a human, a pharmacological formulation. Can be used.

  In another embodiment, the present invention contemplates a method for preventing, treating or ameliorating a disease or condition. In a preferred embodiment, the present invention is an amount effective to treat, prevent or ameliorate a disease or condition (as listed in the “Preferred Indication: Y” column of Table 1). (In the same column as the disease or condition to be treated) (or the portion corresponding to the therapeutic protein or therapeutic protein disclosed in the “Therapeutic Protein: X” column of Table 1) Diseases listed in the “Preferred indications: Y” column of Table 1 comprising administering an albumin fusion protein of the invention comprising a fragment or variant) to a patient in need of treatment, prevention or amelioration thereof Or a method of treating a disease is contemplated.

  In one embodiment, the albumin fusion protein described in Table 1 or 2 has an extended lifetime.

  In a second embodiment, the albumin fusion protein described in Table 1 is more stable than the corresponding non-fusion therapeutic molecule described in Table 1 or 2.

  The present invention further further expresses an albumin fusion protein of the present invention that has been modified to include a nucleic acid molecule of the present invention (including but not limited to the polynucleotides described in Tables 1 and 2). Including transgenic organisms modified.

The amino acid sequence of the mature form of human albumin (SEQ ID NO: 1) and the polynucleotide encoding it (SEQ ID NO: 2) are shown. Nucleotides 1-1755 of SEQ ID NO: 2 encode the mature form of human albumin (SEQ ID NO: 1). The amino acid sequence of the mature form of human albumin (SEQ ID NO: 1) and the polynucleotide encoding it (SEQ ID NO: 2) are shown. Nucleotides 1-1755 of SEQ ID NO: 2 encode the mature form of human albumin (SEQ ID NO: 1). The amino acid sequence of the mature form of human albumin (SEQ ID NO: 1) and the polynucleotide encoding it (SEQ ID NO: 2) are shown. Nucleotides 1-1755 of SEQ ID NO: 2 encode the mature form of human albumin (SEQ ID NO: 1). The amino acid sequence of the mature form of human albumin (SEQ ID NO: 1) and the polynucleotide encoding it (SEQ ID NO: 2) are shown. Nucleotides 1-1755 of SEQ ID NO: 2 encode the mature form of human albumin (SEQ ID NO: 1). Figure 2 shows the restriction map of pPPC0005 cloning vector ATCC deposit PTA-3278. pSAC35 yeast A restriction map of the cerevisiae expression vector is shown (Sleep et al., BioTechnology 8:42 (1990)). 3 compares the antiproliferative activity of IFN albumin fusion proteins encoded by CID 3165 (CID 3165 protein) and recombinant IFNa (rIFNa) on Hs294T melanoma cells. Cells were cultured with various concentrations of either CID 3165 protein or rIFNa and proliferation was measured by BrdU incorporation after 3 days of culture. The CID 3165 protein that caused measurable inhibition of cell proliferation at a concentration of 10 ng / ml and higher at 50% inhibition was achieved at approximately 200 ng / ml. (■) = CID3165 protein, (♦) = rIFNa. FIG. 9 shows the effect of various dilutions of IFNa albumin fusion protein on SEAP activity in ISRE-SEAP / 293F reporter cells. One preparation upstream of IFNa fusion of albumin (♦) and two different preparations downstream of IFNa fusion of albumin (●) and (■) were tested. FIG. 6 shows the time and / or effect of IFNa albumin fusion protein encoded by DNA contained in construct 2249 (CID2249 protein) on the level of OAS (p41) mRNA in treated monkeys (see Example 76). thing). By time point, 1st bar = vehicle control, 2nd bar = 30 ug / kg CID2249 protein, 1st day iv, 3rd bar = 30 ug / kg CID2249 protein, 1st day sc, 4th bar = 300 ug / kg CID 2249 protein, 1st day sc, 5th bar = 40 ug / kg recombinant IFNa 1st, 3rd and 5th day, sc. FIG. 4 shows the / trip responsive relationship of BNP albumin fusion protein encoded by DNA contained in constructs CID3691 and 3681 (CID3691 and 3618 proteins) in activated cGMP formation in NPR-A / 293F reporter cells (implementation). See Examples 78 and 79). BNP peptide (■) and two different preparations (□) and (●) upstream of the BNP fusion of albumin were tested. FIG. 6 shows the effect of BNP albumin fusion protein on mean arterial pressure in spontaneously hypertensive rats (see Example 78). Excipient (□), BNP peptide (●), or BNP albumin fusion protein (◯) was delivered via tail vein injection. Systolic and diastolic blood pressure were recorded by the cuff-tail method. Shown are plasma cGMP levels in 11-12 week old male C57 / BL6 mice after intravenous injection of BNP peptide (●) or BNP albumin fusion protein (◯) (see Example 78). cGMP levels were measured from sera prepared from tail blood collected at various time points after intravenous injection. FIG. 8 shows the / trip responsive relationship of BNP peptide and BNP albumin fusion protein encoded by the DNA contained in constructs CID 3769 and 3959 in the formation of active cGMP in NPR-A / 293F reporter cells (Example 80). checking). The BNP peptide (■) and two different preparations (□) and (◇) downstream of the BNP fusion of albumin were tested. FIG. 6 shows the BNP / ANP peptide / times responsive relationship with or without treatment with neprilysin for 24 hours in active cGMP formation in NPR-A / 293F reporter cells (see Example 81). . FIG. 6 shows the dose response relationship of ANP peptide in activated cGMP formation in NPR-A / 293F reporter cells after treatment with neprilysin or control MES buffer for 20 minutes, 1 hour or 24 hours (Example 81). See ANP fusion of albumin encoded by DNA contained in construct CID3484 in activated cGMP formation in NPR-A / 293F reporter cells after treatment with neprilysin or control MES buffer for 20 minutes, 1 hour or 24 hours FIG. 5 shows the A / P albumin fusion protein / upstream response relationship including upstream (see Example 81). Shows the percentage of intact natriuretic peptide after treatment with neprilysin at specific time intervals. Two albumin fusion proteins were tested, including ANP and BNP peptides, and BNP fusion upstream of albumin via glycine (CID3809) and ANP fusion upstream to albumin (CID3484) (see Example 81). Infected with chronic hepatitis C genotype 1 and treated with HSA-IFNα2b in combination with ribavirin for 0-24 weeks, then at least one treatment regimen (PEG-RBV) of pegylated interferon α in combination with ribavirin 2 shows a decrease in HCV RNA titer as measured by median HCV RNA charge (log ₁₀ IU / ml) in patients who had no prior response to (non-responders). FIG. 4 shows the effect of HSA-BNP (construct: ID # 3959) on plasma and urine cGMP levels after administration of 5 mg / kg IV bolus in normal healthy pigs (n = 4-6 / group), respectively. FIG. 4 shows the effect of HSA-BNP (construct: ID # 3959) on plasma and urine cGMP levels after administration of 5 mg / kg IV bolus in normal healthy pigs (n = 4-6 / group), respectively. FIG. 6 shows the effect of administration of an intravenous bolus of 2 mg / kg or 6 mg / kg HSA-BNP (construct ID # 3959) on terminal diastolic diameter changes in a porcine experimental heart failure model (n = 10 / group). Heart failure is induced in the pig by ventricular pacing. Terminal diastolic diameter was measured by echocardiography. Significant changes from vehicle or baseline (p <0.05) are indicated (respectively & and #). FIG. 9 shows the effect of administering an intravenous bolus of 2 mg / kg or 6 mg / kg HSA-BNP (construct ID # 3959) on shortening rate in a porcine experimental heart failure model (n = 10 / group). Heart failure is induced in the pig by ventricular pacing. Significant changes from vehicle or baseline (p <0.05) are indicated (respectively & and #). FIG. 9 shows the hemodynamic effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Cardiac output (CO), mean arterial pressure (MAP), pulmonary capillary edge pressure (PCWP) and pulmonary artery pressure (PAP) were 0.5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group) or Measurements were made at baseline before intravenous bolus of 5 mg / kg HSA-BNP (construct ID # 3959) and at 30, 60, 90, 150, 210 and 270 time points after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 9 shows the hemodynamic effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Cardiac output (CO), mean arterial pressure (MAP), pulmonary capillary edge pressure (PCWP) and pulmonary artery pressure (PAP) were 0.5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group) or Measurements were made at baseline before intravenous bolus of 5 mg / kg HSA-BNP (construct ID # 3959) and at 30, 60, 90, 150, 210 and 270 time points after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 9 shows the hemodynamic effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Cardiac output (CO), mean arterial pressure (MAP), pulmonary capillary edge pressure (PCWP) and pulmonary artery pressure (PAP) were 0.5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group) or Measurements were made at baseline before intravenous bolus of 5 mg / kg HSA-BNP (construct ID # 3959) and at 30, 60, 90, 150, 210 and 270 time points after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 9 shows the hemodynamic effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Cardiac output (CO), mean arterial pressure (MAP), pulmonary capillary edge pressure (PCWP) and pulmonary artery pressure (PAP) were 0.5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group) or Measurements were made at baseline before intravenous bolus of 5 mg / kg HSA-BNP (construct ID # 3959) and at 30, 60, 90, 150, 210 and 270 time points after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 9 shows the hemodynamic effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Cardiac output (CO), mean arterial pressure (MAP), pulmonary capillary edge pressure (PCWP) and pulmonary artery pressure (PAP) were 0.5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group) or Measurements were made at baseline before intravenous bolus of 5 mg / kg HSA-BNP (construct ID # 3959) and at 30, 60, 90, 150, 210 and 270 time points after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 9 shows the hemodynamic effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Cardiac output (CO), mean arterial pressure (MAP), pulmonary capillary edge pressure (PCWP) and pulmonary artery pressure (PAP) were 0.5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group) or Measurements were made at baseline before intravenous bolus of 5 mg / kg HSA-BNP (construct ID # 3959) and at 30, 60, 90, 150, 210 and 270 time points after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 9 shows the hemodynamic effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Cardiac output (CO), mean arterial pressure (MAP), pulmonary capillary edge pressure (PCWP) and pulmonary artery pressure (PAP) were 0.5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group) or Measurements were taken at baseline before intravenous bolus of 5 mg / kg HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 9 shows the hemodynamic effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Cardiac output (CO), mean arterial pressure (MAP), pulmonary capillary edge pressure (PCWP) and pulmonary artery pressure (PAP) were 0.5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group) or Measurements were taken at baseline before intravenous bolus of 5 mg / kg HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 5 shows renal effects of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Urine flow rate (rate / recovery for 30 minutes), sodium excretion, renal blood flow, and glomerular filtration rate (GFR) were 0.5 mg / kg or 5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group). Measurements were taken at baseline before intravenous bolus of HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 5 shows renal effects of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Urine flow rate (rate / recovery for 30 minutes), sodium excretion, renal blood flow, and glomerular filtration rate (GFR) were 0.5 mg / kg or 5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group). Measurements were taken at baseline before intravenous bolus of HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 5 shows renal effects of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Urine flow rate (rate / recovery for 30 minutes), sodium excretion, renal blood flow, and glomerular filtration rate (GFR) were 0.5 mg / kg or 5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group). Measurements were taken at baseline before intravenous bolus of HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 5 shows renal effects of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Urine flow rate (rate / recovery for 30 minutes), sodium excretion, renal blood flow, and glomerular filtration rate (GFR) were 0.5 mg / kg or 5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group). Measurements were taken at baseline before intravenous bolus of HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 5 shows renal effects of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Urine flow rate (rate / recovery for 30 minutes), sodium excretion, renal blood flow, and glomerular filtration rate (GFR) were 0.5 mg / kg or 5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group). Measurements were taken at baseline before intravenous bolus of HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 5 shows renal effects of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Urine flow rate (rate / recovery for 30 minutes), sodium excretion, renal blood flow, and glomerular filtration rate (GFR) were 0.5 mg / kg or 5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group). Measurements were taken at baseline before intravenous bolus of HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 5 shows renal effects of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Urine flow rate (rate / recovery for 30 minutes), sodium excretion, renal blood flow, and glomerular filtration rate (GFR) were 0.5 mg / kg or 5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group). Measurements were taken at baseline before intravenous bolus of HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 5 shows renal effects of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Urine flow rate (rate / recovery for 30 minutes), sodium excretion, renal blood flow, and glomerular filtration rate (GFR) were 0.5 mg / kg or 5 mg / kg in anesthetized normal hybrid dogs (n = 8 / group). Measurements were taken at baseline before intravenous bolus of HSA-BNP (construct ID # 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 6 shows the hormonal effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Plasma aldosterol, renin and angiotensin II levels were measured in anesthetized normal hybrid dogs (n = 8 / group) before an intravenous bolus of 0.5 mg / kg or 5 mg / kg HSA-BNP (construct ID # 3959). Measurements were taken at baseline and at 30, 60, 90, 150, 210 and 270 after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 6 shows the hormonal effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Plasma aldosterol, renin and angiotensin II levels were measured in anesthetized normal hybrid dogs (n = 8 / group) before an intravenous bolus of 0.5 mg / kg or 5 mg / kg HSA-BNP (construct ID # 3959). Measurements were taken at baseline and at 30, 60, 90, 150, 210 and 270 after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 6 shows the hormonal effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Plasma aldosterol, renin and angiotensin II levels were measured in anesthetized normal hybrid dogs (n = 8 / group) before an intravenous bolus of 0.5 mg / kg or 5 mg / kg HSA-BNP (construct ID # 3959). Measurements were taken at baseline and at 30, 60, 90, 150, 210 and 270 after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 6 shows the hormonal effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Plasma aldosterol, renin and angiotensin II levels were measured in anesthetized normal hybrid dogs (n = 8 / group) before an intravenous bolus of 0.5 mg / kg or 5 mg / kg HSA-BNP (construct ID # 3959). Measurements were taken at baseline and at 30, 60, 90, 150, 210 and 270 after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 6 shows the hormonal effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Plasma aldosterol, renin and angiotensin II levels were measured in anesthetized normal hybrid dogs (n = 8 / group) before an intravenous bolus of 0.5 mg / kg or 5 mg / kg HSA-BNP (construct ID # 3959). Measurements were taken at baseline and at 30, 60, 90, 150, 210 and 270 after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). FIG. 6 shows the hormonal effect of HSA-BNP (construct ID # 3959) administered via a single intravenous bolus at 0.5 mg / kg or 5 mg / kg in a normal dog model. Plasma aldosterol, renin and angiotensin II levels were measured in anesthetized normal hybrid dogs (n = 8 / group) before an intravenous bolus of 0.5 mg / kg or 5 mg / kg HSA-BNP (construct ID # 3959). Measurements were taken at baseline and at 30, 60, 90, 150, 210 and 270 after injection. The asterisk indicates a statistically significant change from baseline (p <0.05). 5 mg / day in systolic and mean arterial blood pressure in normal, healthy, awakened Bagel dogs surgically implanted with a Data Science International telemetry transmitter with systemic arterial blood pressure, heart rate and ECG data collection capability Figure 8 shows the effect of a single intravenous bolus of kg HSA-BNP (construct ID # 3959). Changes from baseline in diastolic blood pressure (FIG. 18A), decrease in mean diastolic blood pressure (FIG. 18B), and change from baseline in mean arterial pressure (FIG. 18C) over 48 hours of continuous data recording following infusion. ). The asterisk shows a statistically significant difference in baseline-adjusted mean for 5 mg / kg HSA-BNP (construct ID # 3959) compared to vehicle (p <0.05). 5 mg / day in systolic and mean arterial blood pressure in normal, healthy, awakened Bagel dogs surgically implanted with a Data Science International telemetry transmitter with systemic arterial blood pressure, heart rate and ECG data collection capability Figure 8 shows the effect of a single intravenous bolus of kg HSA-BNP (construct ID # 3959). Changes from baseline in diastolic blood pressure (FIG. 18A), decrease in mean diastolic blood pressure (FIG. 18B), and change from baseline in mean arterial pressure (FIG. 18C) over 48 hours of continuous data recording following infusion. ). The asterisk shows a statistically significant difference in baseline-adjusted mean for 5 mg / kg HSA-BNP (construct ID # 3959) compared to vehicle (p <0.05). 5 mg / day in systolic and mean arterial blood pressure in normal, healthy, awakened Bagel dogs surgically implanted with a Data Science International telemetry transmitter with systemic arterial blood pressure, heart rate and ECG data collection capability Figure 8 shows the effect of a single intravenous bolus of kg HSA-BNP (construct ID # 3959). Changes from baseline in diastolic blood pressure (FIG. 18A), decrease in mean diastolic blood pressure (FIG. 18B), and change from baseline in mean arterial pressure (FIG. 18C) over 48 hours of continuous data recording following infusion. ). The asterisk shows a statistically significant difference in baseline-adjusted mean for 5 mg / kg HSA-BNP (construct ID # 3959) compared to vehicle (p <0.05). 0.02 mg / kg non-fusion in systemic blood pressure in a normal, healthy, awakened bagel dog surgically implanted with a Data Science International telemetry transmitter with systemic arterial blood pressure, heart rate and ECG data collection capability Figure 2 shows a comparison of the effects of an intravenous bolus of BNP peptide and subcutaneous injection of 10 mg / kg HSA-BNP (construct ID # 3959). Shows the change from baseline in diastolic blood pressure over the 48 hour data recording following administration of BNP (FIG. 19A) and HSA-BNP (construct ID # 3959). The asterisk shows a statistically significant difference in baseline-adjusted mean for 5 mg / kg HSA-BNP (construct ID # 3959) compared to vehicle (p <0.05). 0.02 mg / kg non-fusion in systemic blood pressure in a normal, healthy, awakened bagel dog surgically implanted with a Data Science International telemetry transmitter with systemic arterial blood pressure, heart rate and ECG data collection capability Figure 2 shows a comparison of the effects of an intravenous bolus of BNP peptide and subcutaneous injection of 10 mg / kg HSA-BNP (construct ID # 3959). Shows the change from baseline in diastolic blood pressure over the 48 hour data recording following administration of BNP (FIG. 19A) and HSA-BNP (construct ID # 3959). The asterisk shows a statistically significant difference in baseline-adjusted mean for 5 mg / kg HSA-BNP (construct ID # 3959) compared to vehicle (p <0.05).

Detailed Description Definitions The following definitions are provided to facilitate understanding of specific terms used throughout this specification.

  As used herein, “polynucleotide” refers to at least one albumin (or fragment thereof) bound in frame to at least one therapeutic protein X (or fragment or variant thereof). Or a nucleic acid molecule having a nucleotide sequence encoding or consisting of a fusion protein, an amino acid sequence of SEQ ID NO: Y (as described in column 6 of Table 2), or a fragment thereof or A nucleic acid molecule having a nucleotide sequence encoding a fusion protein comprising or consisting of a variant, a nucleic acid molecule having a nucleotide sequence comprising or consisting of the sequence shown in SEQ ID NO: X, SEQ ID NO: A fusion protein comprising or consisting of the amino acid sequence of Z A nucleic acid molecule having a nucleotide sequence encoding, a nucleic acid molecule having a nucleotide sequence encoding an albumin fusion protein of the invention produced as described in Table 2 or the Examples, therapeutic of the invention A nucleic acid molecule having a nucleotide sequence encoding an albumin fusion protein, a nucleic acid molecule having a nucleotide sequence contained in an albumin fusion structure described in Table 2, or deposited at ATCC (as described in Table 3) Means a nucleic acid molecule having a nucleotide sequence contained in an albumin fusion structure.

  As used herein, an “albumin fusion construct” is in frame to at least one polynucleotide encoding at least one therapeutic protein (or fragment or variant thereof). A nucleic acid molecule comprising or consisting of a polynucleotide encoding at least one albumin molecule (or fragment or variant thereof) linked in, a therapy produced as described in Table 2 or the Examples At least one molecule of albumin (or fragment or variant thereof) linked in frame to at least one polynucleotide encoding at least one molecule of a target protein (or fragment or variant thereof) Polynucleotide coding A nucleic acid molecule comprising, or consisting of, or, for example, one or more of the following elements (1) (including but not limited to shuttle vectors, expression vectors, integration vectors, and / or replication systems) A functional self-replicating vector, (2) a region for initiation of transcription (eg, a regulatable or inducible promoter, a promoter region, such as a structural promoter), (3) a region for termination of transcription, (4) At least linked in frame to at least one polynucleotide encoding at least one molecule of a therapeutic protein (or fragment or variant thereof) comprising a leader sequence and (5) a selectable marker, One poly encoding one therapeutic protein (or fragment or variant thereof) Including Kureochido, or refers to a nucleic acid molecule consisting. A polynucleotide encoding a therapeutic protein and an albumin protein means as part of an albumin fusion structure, respectively, as “portion”, “region” or “site” of an albumin fusion construct Can be done.

  The present invention generally treats or prevents a disease or condition using a polynucleotide encoding an albumin fusion protein, an albumin fusion protein, and an albumin fusion protein or a polynucleotide encoding an albumin fusion protein Or relates to a method of mitigating. As used herein, an “albumin fusion protein” is at least one albumin (or fragment or mutation thereof) to at least one molecule of a therapeutic protein (or fragment or variant thereof). Means the protein formed by the fusion of the body) to the molecule. Albumin fusion proteins of the invention include at least one therapeutic protein fragment or variant, at least one human serum albumin fragment or variant linked together by genetic fusion (ie, the albumin fusion protein comprises: A polynucleotide encoding all or a portion of a therapeutic protein is produced by translation of a nucleic acid that binds in-frame with a polynucleotide encoding all or a portion of albumin). Therapeutic protein and albumin protein can be meant as part of an albumin fusion structure, respectively, as “portion”, “region” or “site” of an albumin fusion construct (eg, “therapeutic” “Protein portion” or “albumin protein portion”). In a highly preferred embodiment, the albumin fusion protein of the invention comprises at least one molecule of therapeutic protein X, including but not limited to a mature form of therapeutic protein X, or a fragment or variant thereof, and at least one Including, but not limited to, albumin molecules, including mature forms of albumin, or fragments or variants thereof.

  In a further preferred embodiment, the albumin fusion protein of the invention is processed by the host cell and secreted into the surrounding culture medium. Processing of nascent albumin fusion proteins that occur in the host secretory pathway used for expression includes, but is not limited to, signal peptide cleavage, disulfide bond formation, correct folding, (eg, N- and O-linked glucosylation). Carbohydrate addition and processing, specific proteolytic cleavage, and assembly into multiple proteins. The albumin fusion protein of the present invention is preferably in a processed form. In the most preferred embodiment, “processed form of an albumin fusion protein” means an albumin fusion protein product that has undergone N-terminal signal peptide cleavage, It is also called “mature albumin fusion protein”.

  In various examples, a representative clone containing the albumin fusion construct of the present invention has been deposited with the American Type Culture Collection (hereinafter referred to as “ATCC®”). Furthermore, the albumin fusion construct can be removed from the deposit by techniques known in the art and described elsewhere in this specification. ATCC® is located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC® deposit was conducted in accordance with the Budapest Treaty on the International Approval of Deposits of Microorganisms in Patent Procedures.

  In one embodiment, the present invention provides a polynucleotide encoding an albumin fusion protein comprising or consisting of a therapeutic protein and serum albumin protein. In a further embodiment, the present invention provides an albumin fusion protein comprising or consisting of a therapeutic protein and serum albumin protein. In a preferred embodiment, the present invention provides an albumin fusion protein comprising or consisting of a therapeutic protein and a serum albumin protein encoded by the polynucleotide described in Table 2. In a further preferred embodiment, the present invention provides a polynucleotide encoding an albumin fusion protein, the sequence of which is shown in Table 2 as SEQ ID NO: Y. In other embodiments, the present invention provides albumin fusion proteins comprising or consisting of biologically active and / or therapeutically active fragments of therapeutic proteins and serum albumin proteins. In other embodiments, the present invention provides albumin fusion proteins comprising or consisting of biologically active and / or therapeutically active variants of therapeutic protein and serum albumin protein. In a preferred embodiment, the serum albumin protein component of the albumin fusion protein is a mature portion of serum albumin. The invention further includes polynucleotides encoding these albumin fusion proteins.

  In a further embodiment, the present invention provides an albumin fusion protein comprising or consisting of a therapeutic protein and a biologically active and / or therapeutically active fragment of serum albumin. In a further embodiment, the present invention provides an albumin fusion protein comprising or consisting of a therapeutic protein and a biologically active and / or therapeutically active variant of serum albumin. In a preferred embodiment, the therapeutic protein portion of the albumin fusion protein is the mature portion of the therapeutic protein. In a further preferred embodiment, the therapeutic protein portion of the albumin fusion protein is the extracellular soluble domain of the therapeutic protein portion. In other embodiments, the therapeutic protein portion of the albumin fusion protein is the active form of the therapeutic protein. The invention further includes polynucleotides encoding these albumin fusion proteins.

  In further embodiments, the present invention provides biologically active and / or therapeutically active fragments or variants of therapeutic proteins, and biologically active and / or therapeutically of serum albumin. Albumin fusion proteins comprising or consisting of active fragments or variants are provided. In a preferred embodiment, the present invention provides an albumin fusion protein comprising or consisting of a mature portion of a therapeutic protein and a mature portion of serum albumin. The invention further includes polynucleotides encoding these albumin fusion proteins.

Therapeutic proteins As described above, the polynucleotides of the invention preferably comprise at least one fragment or variant of a therapeutic protein and at least one fragment or human serum albumin that bind to each other, preferably by genetic fusion. It encodes a protein comprising or consisting of variants.

  Further embodiments include encoding a protein comprising or consisting of at least one fragment or variant of a therapeutic protein and at least one fragment or variant of human serum albumin linked together by chemical coupling A polynucleotide.

  As used herein, a “therapeutic protein” is a protein, polypeptide, antibody, peptide or fragment thereof having one or more therapeutic and / or biological activities. Means a variant. Therapeutic proteins included herein include, but are not limited to, proteins, polypeptides, peptides, antibodies and biological materials. (The phrases peptide, protein and polypeptide are used interchangeably herein.) The phrase “therapeutic protein” is specifically intended to include antibodies and fragments and variants thereof. Thus, the proteins of the invention can include at least one fragment or variant of a therapeutic protein and / or at least one fragment or variant of an antibody. Furthermore, the phrase “therapeutic protein” can mean an endogenous or naturally occurring related substance of the therapeutic protein.

  A polypeptide that exhibits "therapeutic activity" or a protein that is "therapeutically active" as described herein or known in the art, or By one or more known biological and / or therapeutic activity polypeptides linked to a therapeutic protein, such as a further therapeutic protein. By way of non-limiting example, a “therapeutic protein” is a protein that is useful for treating, preventing or alleviating a disease, condition or disease. By way of non-limiting example, a “therapeutic protein” is one that specifically binds to a particular cell type (normal (eg, lymphocyte) or abnormal (eg, cancer cell)) and is therefore a compound (drug or cytotoxic) Drug) can be used to specifically target the cell type.

  For example, a non-exhaustive list of “therapeutic proteins” portions that can be included by the albumin fusion proteins of the present invention include, but are not limited to, IFNa, ANP, BNP, LANP, VDP, KUP, CNP, DNP, HCC-1 , Beta-defensin-2, fractalkine, oxyntomodulin, killer toxin peptide, TIMP-4, PYY, adrenomedullin, ghrelin, CGRP, IGF-1, neuraminidase, hemagglutinin, butyrylchlorin esterase, endothelin and mechanoproliferation Factors are included.

  The interferon hybrid can also be fused to the amino or carboxy terminus of albumin to form an interferon hybrid albumin fusion protein. Interferon hybrid albumin fusion proteins can enhance or suppress interferon activity, such as antiviral response, control of cell proliferation, and regulation of immune response (Lebleu et al., PNAS USA, 73: 3107-3111 (1976). Gresser et al., Nature, 251: 543-545 (1974), and Johnson, Texas Reports Biol Med, 35: 357-369 (1977)). Each interferon hybrid albumin fusion protein can be used to treat, prevent or reduce viral infection (eg, hepatitis (eg, HCV), or HIV), multiple sclerosis or cancer.

In one embodiment, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon alpha-interferon alpha hybrid (referred to herein as an alpha-alpha hybrid). For example, the alpha-alpha hybrid portion of an interferon hybrid albumin fusion protein consists of or comprises interferon alpha A fused to interferon alpha D. In a further embodiment, the A / D hybrid is fused to interferon alpha D at a common BgIII restriction site, the N-terminal site of the A / D hybrid corresponds to amino acids 1 to 62 of interferon alpha A, C- The terminal site corresponds to amino acids 64-166 of interferon alpha D. For example, the A / D hybrid amino acid sequence CDLPQTHSLGSRRTLMLLAQMRX ₁ ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNX ₂ DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO: 99)
Wherein X ₁ is R or K and X ₂ is A or V. In a further embodiment, the A / D hybrid is fused at a common PvuIII restriction site, the N-terminal site of the A / D hybrid corresponds to amino acids 1-91 of interferon alpha A, and the C-terminal site is interferon alpha. It corresponds to amino acids 93 to 166 of D. For example, the A / D hybrids, the amino acid sequence CDLPQTHSLGSRRTLMLLAQMRX ₁ ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVMQEERVGETPLMNX ₂ DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO: 100)
Wherein X ₁ is R or K, and the second X ₂ is A or V. These hybrids are further described in US Pat. No. 4,414,510, all of which is hereby incorporated by reference.

In a further embodiment, the alpha-alpha hybrid portion of the interferon hybrid albumin fusion protein consists of or comprises interferon alpha A fused to interferon alpha F. In a further embodiment, the A / F hybrid is fused at a common PvuIII restriction site, the N-terminal site of the A / F hybrid corresponds to amino acids 1-91 of interferon alpha A, and the C-terminal site is interferon It corresponds to amino acids 93 to 166 of alpha F. For example, an A / F hybrid is an amino acid sequence,
CDLPQTHSLGSSRRTMLLAQMRXISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELKQFLDRIKLKYRSQ
Wherein X is either R or K. These hybrids are described in US Pat. No. 4,414,510, all of which is hereby incorporated by reference. In a further embodiment, the alpha-alpha hybrid site of the interferon hybrid albumin fusion protein consists of or comprises interferon alpha A fused to interferon alpha B. In a further embodiment, the A / B hybrid is fused at a common PvuIII restriction site, the N-terminal site of the A / B hybrid corresponds to amino acids 1-91 of interferon alpha A, and the C-terminal site is interferon It corresponds to amino acids 93 to 166 of alpha B. For example, the A / B hybrid is an amino acid sequence,
_{CDLPQTHSLGSRRTLMLLAQMRX1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEX 2 X 3 X 4 X 5} QEVGVIESPLMYEDSILAVRKYFQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSINLQKRLKSKE ( SEQ ID NO: 102),
In which X ₁ is R or K, and X _{2 to} X ₅ are SCVM or VLCD. These hybrids are further described in US Pat. No. 4,414,510, all of which is hereby incorporated by reference.

In other embodiments, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises a beta-interferon alpha hybrid (referred to herein as a beta-alpha hybrid). For example, the beta-alpha hybrid portion of an interferon hybrid albumin fusion protein consists of or includes interferon beta-1 (also referred to as interferon alpha-1) fused to interferon alpha D. In a further embodiment, the beta-1 / alpha D hybrid is such that the N-terminal portion corresponds to amino acids 1-73 of interferon beta-1 and the C-terminal portion corresponds to amino acids 74-167 of interferon alpha D. To merge. For example, the beta-1 / alpha D hybrid is an amino acid sequence,
MSYNLLGFLQRSSNNFQCQCKLKLWQLNGRLEYYCLKDRMNFDIPIEIKQLQQFQKEDAALTIYEMMLQNIFAIFRQDSSAAWEDDLLDKFCTILYQQLNDLEACVMQLEVGETLKLMRDSILAVKLY
Wherein X is A or V. These hybrids are further described in US Pat. No. 4,758,428, all of which is hereby incorporated by reference.

In other embodiments, the interferon hybrid portion of the interferon hybrid albumin fusion protein comprises an interferon alpha-interferon beta hybrid (referred to herein as an alpha-beta hybrid). For example, the alpha-beta hybrid site of an interferon hybrid albumin fusion protein consists of or includes interferon alpha D (also referred to as interferon alpha-1) fused to interferon beta-1. In a further embodiment, the alpha D / beta-1 hybrid is fused such that the N-terminal portion corresponds to amino acids 1-73 of interferon alpha D and the C-terminal portion corresponds to amino acids 74-166 of interferon alpha D. To do. For example, the alpha D / beta-1 hybrid is an amino acid sequence,
MCDLPEHSLDNRRTMLMLAQMSRISSPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTYKDSSSTGWNETIVENLLANVYHQINHLKTVLEFLEKLTGRTHLY
including. These hybrids are further described in US Pat. No. 4,758,428, all of which is hereby incorporated by reference.

  In a further embodiment, the interferon hybrid site of the interferon hybrid albumin fusion protein can include additional combinations of alpha-alpha interferon hybrids, alpha-beta interferon hybrids, and beta-alpha hybrids. In a further embodiment, the interferon hybrid site of the interferon hybrid albumin fusion protein can be modified to include mutations, substitutions, deletions or additions to the amino acid sequence of the interferon hybrid. Modifications to such interferon hybrid albumin fusion proteins can be performed, for example, to improve the level of production, increase stability, increase or decrease activity, or confer new biological properties.

  Such interferon hybrid albumin fusion proteins are encompassed by the present invention, as are host cells and vectors comprising a polynucleotide encoding a polypeptide. In one embodiment, the interferon hybrid albumin fusion protein encoded by a polynucleotide as described above has an extended lifetime. In a further embodiment, the interferon hybrid albumin fusion protein encoded by the polynucleotide has a longer serum half-life and / or in solution compared to the corresponding non-fused interferon hybrid molecule in vitro and / or in vivo. More stable activity (or in pharmacological composition).

  In other non-limiting examples, a “therapeutic protein” is a protein with biological activity, particularly biological activity that is useful for the treatment, prevention or alleviation of a disease. A non-inclusive list of biological activities that can be retained by therapeutic proteins includes inhibition of cellular HIV-1 infection, stimulation of intestinal epithelial cell proliferation, decreased intestinal epithelial cell permeability, stimulation of insulin secretion, bronchodilation And induction of vasodilation, inhibition of aldosterone and renin secretion, blood pressure control, promotion of nerve growth, enhancement of immune response, enhancement of inflammation, suppression of appetite, or any one or more of the following “biological activities” And / or the biological activities disclosed for the therapeutic protein in Table 1 (column 2).

In one embodiment, the IFN-alpha-HSA fusion is a Category A-Filo (Ebola), Arena (Puchende), Category B-Category B- Virus drugs classified under Toga (VEE), or Category C-Bunya (Punto Toro), Flavi (yellow fever, West Nile) Inhibits. For example, CPE inhibition, neutral red staining and virus production assays were performed to assess antiviral activity downstream of HSA INF-alpha fusion (CID 3165 protein). The pharmacokinetic and pharmacodynamic activity of CID 3165 protein in cynomolgus monkeys and human subjects was evaluated. The results show that antiviral activity was achieved for all RNA viruses evaluated with favorable safety indicators. IC50 values ranged from <0.1 ng / ml (Puntatro A) to 19 ng / ml (VEE) in the CPR assay. In cynomolgus monkeys, the half-life of CID 3165 protein was 90 hours and was detectable up to 14 days after administration. In human subjects, CID 3156 protein was safe and well tolerated. C _max after single injection administration was dose proportional. The average C _max in the 500 ug cohort is 22 ng / ml and the average t _1/2 is 150 hours. Dosing more than once every 2-4 weeks was supported by pharmacokinetics. An antiviral response to hepatitis C was observed in most subjects in a single injection cohort (120-500 μg).

  In a further embodiment, the IFN-alpha-HSA fusion is used to treat patients with hepatitis C infection (HCV). Interferon alpha (alfa), also known as leukocyte interferon, is a standard therapy for the treatment of patients infected with HCV. The phrase “interferon alpha” refers to a family of very homogeneously related polypeptides with antiviral activity. The interferon alpha portion of the TFN-alpha-HSA fusion consists of or includes any interferon alpha or fragment thereof known in the art. Non-limiting examples of the interferon alpha portion of the IFN-alpha-HSA fusion proteins of the present invention include, but are not limited to, the interferon alpha proteins disclosed in the therapeutic protein column of Table 1. In certain embodiments, the interferon alpha moiety is interferon alpha-2a, interferon alpha-2b, interferon alpha-2c, consensus interferon, interferon alphacon-1, interferon alpha-n1, interferon alpha-n3, such as INTRON®. ) A (Schering Corp., Kenilworth, NJ), ROFERON® A (Hoffman-La Roche, Nutley, NJ), Boerofor alpha interferon (Baye) Ringer Ingelheim Pharmaceuticals (Boehringer Ingelheim Pharmaceu) ticals., Inc.), Ridgefied, Conn.), OMNIFERON ™ (Viragen, Inc., Plantation, FL), MULTIFERON ™ (Viragen, Inc.), Plantation, FL , WELLFERON (registered trademark) (GlaxoSmithKline, London, Great Britian), INFERGEN (registered trademark) (Amgen, Inc.), Thousands Oaks, CA, SUMIFERON (registered trademark) ), Japan), BELEROFON (registered trademark) (Nautilus Biotech), ), MAXY-ALPHA (TM) (Maxygen, Redwood City, CA / Hoffman-La Roche, Nutley, NJ), any commercially available form of interferon, Or consists of or comprises any purified interferon alpha product or fragment thereof. In a further embodiment, the interferon alpha portion of the IFN-alpha-HSA fusion protein consists of or comprises interferon alpha modified or formulated for extended or controlled release. For example, interferon alpha includes, but is not limited to, interferon-alpha-XL (Flamel Technologies, France) and LOCTERON ™ (BioLex Therapeutics / OctoPlus, Pittsbor, NC) comprising or comprising commercially available extended release or controlled release interferon alpha. In a further embodiment, the interferon alpha site of the IFN-alpha-HSA fusion protein can be modified by the addition of a chemical site. For example, the interferon alpha site can be modified by pegylation. Thus, in a further embodiment, the interferon alpha site of the IFN-alpha-HSA fusion protein consists of or includes, but is not limited to, a PEGylated form of interferon alpha-2a, 2b, or consensus interferon. INTRON® (Schering Corp., Kenilworth, NJ), PEGASYS® (Hoffman-La Roche, Nutley, NJ), PEG-OMNIFERON ( (Trademark) (Viragen, Inc., Plantation, FL) or commercially available pegylated interferon alpha. However, as used herein, an “IFN-alpha-HSA” fusion means a HAS fused to any interferon alpha protein or fragment thereof known in the art.

  Patients infected with HCV are divided into two categories based on prior exposure to the interferon regimen for treatment of HCV infection. “Treatment-naive patients” or “native patients” are patients who have never been treated with an interferon regimen. “Treatment-experienced patients” or “experienced patients” are patients who have been or are currently being treated with an interferon regimen. “Non-responders” are experienced patients who were previously treated with an interferon regimen but did not meet the primary endpoints of treatment such as initial viral load reduction (EVR) or end-of-treatment response (ETR) It is. “Relappers” are experienced patients who have already been treated with an interferon regimen and have achieved the primary endpoint of treatment, such as EVR or ETR, but have subsequently become positive for HCV at a later time point. However, as used herein, “experience” or “HCV patient” is either a non-responder or a relapser.

  Furthermore, hepatitis C is classified into a number of genotypes, with four genotypes being genotypes 1, 2, 3 or 4 being the most common. In general, hepatitis C virus that infects HCV patients contains a single genotype. However, the hepatitis virus may contain a combination of two or more genotypes. Furthermore, the hepatitis C genotype can also be a variant of a known HCV genotype. In a further embodiment, the HCV patient's hepatitis C virus is genotype 1 or a variant thereof. However, as used herein, “HCV” means any genotype of hepatitis C virus, combinations or variants thereof.

  Standard treatment regimens for patients with HCV include treatment with interferon alpha in combination with an antiviral agent such as ribavirin. In general, interferon alpha is administered daily, twice a week, or weekly, and ribavirin is administered daily. However, recent studies use interferon alpha in combination with other antiviral agents known in the art for the treatment of HCV. Thus, in a further embodiment, the IFN-alpha-HSA fusion may be administered to HCV patients alone or in combination with an antiviral agent such as ribavirin. In a further preferred embodiment, the IFN-alpha-HSA fusion may be administered to an HCV patient in combination with one or more antiviral agents, such as ribavirin and additional antiviral agents.

  As described above, the pharmacokinetics of CID 3165 protein supports a dosing schedule of once or more every 2-4 weeks. Thus, in a further embodiment, HCV patients are treated with an IFN-alpha-HSA fusion by administration once every 2-4 weeks, alone or in combination with an effective amount of an antiviral agent. In a preferred embodiment, HCV patients are treated with an IFN-alpha-HSA fusion by administration once every 2-4 weeks in combination with an effective amount of one or more antiviral agents. . In a further preferred embodiment, the IFN-alpha-HSA fusion is administered to HCV patients at least once every 4 weeks. In a further preferred embodiment, the IFN-alpha-HSA fusion is administered to HCV patients more than once every 4 weeks. In a further embodiment, the IFN-alpha-HSA fusion is administered to HCV patients at least once every 4 weeks, where the treatment also includes an effective amount of one or more antiviral agents. Administration is also included.

  In other embodiments, the IFN-alpha-HSA fusion may be used as a low dose monotherapy for maintenance treatment of HCV. In further additional embodiments, the IFN-alpha-HSA fusion may be used in combination with ribavirin and one or more other antiviral agents for the treatment of HCV. Alternatively, in other embodiments, the IFN-alpha-HSA fusion may be used in combination with one or more antiviral agents other than ribavirin for the treatment of HVC.

  In further embodiments, IFN-alpha-HSA fusions can be used for the treatment of other viral infections. For example, in one embodiment, an IFN-alpha-HSA fusion can be used for the treatment of hepatitis B (HBV). In a further embodiment, the IFN-alpha-HSA fusion can be used for the treatment of human papillomavirus (HPV). In further embodiments, the IFN-alpha-HSA fusion includes but is not limited to hairy cell leukemia, malignant melanoma, follicular lymphoma, chronic myelogenous leukemia, AIDS-related Kaposi's sarcoma, multiple myeloma or renal cell carcinoma Can be used in the treatment of cancer.

  In other embodiments, HSA fusions with natriuretic peptides may be used for the treatment of cardiovascular disease, including but not limited to ANP-HSA fusions or BNP-HSA fusions. For example, in preferred embodiments, HSA fusions with natriuretic peptides, including but not limited to ANP-HSA fusions or BNP-HSA fusions, may be used for the treatment of congestive heart failure. In further preferred embodiments, HSA fusions with natriuretic peptides, including but not limited to ANP-HSA fusions or BNP-HSA fusions, may be used in the treatment after myocardial infarction. In further embodiments, HSA fusions with natriuretic peptides, including but not limited to ANP-HSA fusions or BNP-HSA fusions, include but are not limited to hypertension, salt-sensitive hypertension, angina, peripheral arteries. It may be used for additional cardiovascular diseases including disease, hypotension, heart volume overload, heart failure, heart failure, left ventricular dysfunction, dyspnea, myocardial reperfusion injury or left ventricular remodeling. In other embodiments, aldosterone levels that can lead to HSA fusions with natriuretic peptides, including but not limited to ANP-HSA fusions or BNP-HSA fusions, can lead to vasoconstriction, reduced cardiac output and / or hypertension. Can be used in treatment for elevation of In further embodiments, HSA fusions with natriuretic peptides, including but not limited to ANP-HSA fusions or BNP-HSA fusions, include but are not limited to diabetic neuropathy, glomerular hypertrophy, glomerular injury, renal thread It can be used in the treatment of kidney disease, including spherical disease, acute and / or chronic renal failure. In further embodiments, HSA fusions with natriuretic peptides, including but not limited to ANP-HSA fusions or BNP-HSA fusions, can be used to treat stroke or excess fluid in tissues.

  In a further embodiment, the HSA can be fused with a natriuretic peptide variant, including but not limited to a BNP-HSA fusion in which the BNP component of the fusion protein is BNP amino acid residues 1-29. In one embodiment, the BNP component of the HSA fusion protein consists of two BNP variants (eg, BNP amino acid residues 1-29) in tandem. In other embodiments, the BNP component of the HSA fusion protein consists of 3, 4, 5 or more BNP variants (eg, BNP amino acid residues 1-29) in tandem. In preferred embodiments, HSA fusions with BNP variants (eg, BNP amino acid residues 1-29) may be used for the treatment of congestive heart failure. In a further preferred embodiment, HSA fusions with BNP variants (eg BNP amino acid residues 1-29) may be used in the treatment after myocardial infarction. In a further embodiment, the HSA fusion with a BNP variant (eg, BNP amino acid residues 1-29) includes but is not limited to hypertension, salt-sensitive hypertension, angina, peripheral arterial disease, hypotension, heart It can be used to treat additional cardiovascular diseases including volume overload, heart failure, heart failure, non-hemodynamic CHF, left ventricular dysfunction, dyspnea, myocardial reperfusion injury or left ventricular remodeling. In other embodiments, HSA fusions with BNP variants (eg, BNP amino acid residues 1-29) may be used to treat aldosterone levels that may lead to vasoconstriction, decreased cardiac output, and / or hypertension. Can be used. In preferred embodiments, HSA fusions with BNP variants (eg, BNP amino acid residues 1-29) include, but are not limited to, diabetic neuropathy, glomerular hypertrophy, glomerular injury, renal glomerular disease, acute and / or It can be used in the treatment of kidney disease or illness, including chronic renal failure. In further embodiments, HSA fusions with BNP variants (eg, BNP amino acid residues 1-29) may be used to treat stroke or excess fluid in tissue.

  In a related but different embodiment, the present invention is directed to a natriuretic peptide variant comprising, but not limited to, BNP amino acid residues 1-29, where the peptide is not fused to HSA. In one embodiment, a BNP variant of the invention has the sequence of two BNP variants (eg, BNP amino acid residues 1-29) in tandem. In other embodiments, the BNP variants of the invention have a sequence of 3, 4, 5, or more BNP variants (eg, BNP amino acid residues 1-29) in tandem. In a preferred embodiment, the BNP variants of the invention (eg BNP amino acid residues 1-29) may be used for the treatment of congestive heart failure. In a further preferred embodiment, the BNP variants of the present invention (eg BNP amino acid residues 1-29) may be used in the treatment after myocardial infarction. In a further embodiment, a BNP variant of the invention (eg, BNP amino acid residues 1-29) is not limited to hypertension, salt-sensitive hypertension, angina, peripheral arterial disease, hypotension, heart volume It can be used to treat additional cardiovascular diseases including overload, heart failure, heart failure, non-hemodynamic CHF, left ventricular dysfunction, dyspnea, myocardial reperfusion injury or left ventricular remodeling. In other embodiments, a BNP variant of the invention (eg, BNP amino acid residues 1-29) is treated in a treatment for elevated aldosterone levels that can lead to vasoconstriction, reduced cardiac output and / or hypertension. Can be used. In a preferred embodiment, the BNP variants of the invention (eg BNP amino acid residues 1-29) are not limited to diabetic neuropathy, glomerular hypertrophy, glomerular injury, renal glomerular disease, acute and / or chronic. It can be used in the treatment of kidney disease or illness, including renal failure. In further embodiments, the BNP variants of the present invention (eg, BNP amino acid residues 1-29) may be used to treat seizures or excess fluid in tissue.

  In further embodiments, the present invention includes, but is not limited to, a BNP variant (eg, BNP amino acid residue 1) modified to increase half-life, increase biological activity, and / or facilitate purification of the variant. Directed to natriuretic peptide variants comprising ˜29). In accordance with this embodiment, natriuretic peptide variants (eg, BNP amino acid residues 1-29) can be PEGylated, methylated, or chemically modified or conjugated using techniques known in the art. Alternatively, methods known in the art may be used to extend the half-life by recombinantly fusing the natriuretic peptide variants of the invention to other peptide sequences known in the art to increase half-life. It can be used to improve biological activity and / or to facilitate purification. For example, a natriuretic peptide variant of the invention may be fused or conjugated to an antibody Fc region, or portion thereof. An antibody portion fused to a natriuretic variant of the invention (eg, BNP amino acid residues 1-29) can be a constant region, hinge region, CH1 domain, CH2 domain and CH3 domain, or any combination of all domains, or a portion thereof. It may be fused or conjugated. Natriuretic variants may also be fused or conjugated to the antibody sites to form multimers. For example, Fc sites fused to a polypeptide of the invention (eg, BNP amino acid residues 1-29) can form dimers through disulfide bonds between Fc sites. Higher order multiple forms can be made by fusing variants to portions of IgA and IgM. Methods for fusion or conjugation to the antibody portion of the invention are known in the art. For example, US Pat. No. 5,336,603, US Pat. No. 5,622,929, US Pat. No. 5,359,046, US Pat. No. 5,349,053, US Pat. No. 5,447,851 U.S. Pat. No. 5,112,946, European 307,434, European 367,166, PCT Publication No. WO 96/04388, WO 91/06570, Ashkenazi et al. , Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991), Zheng et al. , J .; Immunol. 154: 5590-5600 (1995), and Vil et al. , Proc. Natl. Acad. Sci. USA 89: 113337-11341 (1992), which is incorporated by reference in its entirety. In a further embodiment, the modified BNP variants of the present invention have the sequence of two BNP variants (eg, BNP amino acid residues 1-29) in tandem. In a further embodiment, the BNP variants of the present invention have a sequence of 3, 4, 5, or more BNP variants (eg, BNP amino acid residues 1-29) in tandem. In a preferred embodiment, the modified BNP variants of the present invention (eg, BNP amino acid residues 1-29) may be used for the treatment of congestive heart failure. In a preferred embodiment, the modified BNP variants of the invention (eg, BNP amino acid residues 1-29) may be used in the treatment after myocardial infarction. In further embodiments, the modified BNP variants of the invention (eg, BNP amino acid residues 1-29) include, but are not limited to, hypertension, salt-sensitive hypertension, angina, peripheral arterial disease, hypotension, heart It can be used to treat additional cardiovascular diseases including volume overload, heart failure, heart failure, non-hemodynamic CHF, left ventricular dysfunction, dyspnea, myocardial reperfusion injury or left ventricular remodeling. In other embodiments, a modified BNP variant of the invention (eg, BNP amino acid residues 1-29) is used for treatment for increased aldosterone levels, which can lead to vasoconstriction, decreased cardiac excretion and / or hypertension. Can be used. In preferred embodiments, the modified BNP variants of the invention (eg, BNP amino acid residues 1-29) include, but are not limited to, diabetic neuropathy, glomerular hypertrophy, glomerular injury, renal glomerular disease, acute and / or It can be used in the treatment of kidney disease or illness, including chronic renal failure. In further embodiments, modified BNP variants of the invention (eg, BNP amino acid residues 1-29) may be used to treat stroke or excess fluid in tissue.

  In other embodiments, CNP-HSA fusions can be used in the regulation of endochondral ossification. For example, in a preferred embodiment, CNP-HSA fusions can be used in the treatment of skeletal dysplasia, including achondrogenesis, hyperchondrogenesis, and lethal dysplasia.

  As used herein, “therapeutic activity” or “activity” is an activity whose effect is consistent with the desired therapeutic outcome in humans, or in non-human mammals, or otherwise. May mean the desired effect on the species or bacteria. The therapeutic activity can be measured in vivo or in vitro. For example, the desired effect can be assayed in cell culture. Such in vitro or cell culture assays are generally available for many therapeutic proteins described in the art. Examples of assays include, but are not limited to, those described herein in the Examples section or in the “Experimental Activity Assay” column of Table 1 (column 3).

  Therapeutic proteins corresponding to the therapeutic protein portion of the albumin fusion proteins of the invention, such as cell surface and secreted proteins, are often modified by the attachment of one or more oligosaccharide groups. The modifications cited as glycosylation dramatically affect the physical characteristics of the protein and can be important in protein stability, secretion and localization. Glycosylation occurs at specific locations along the polypeptide backbone. Usually there are two major types of glycosylation: glycosylation characterized by O-linked oligosaccharides that bind to serine or threonine, and asparagine residues in Asn-X-Ser or Asn-X-Thr sequences. There is a glycosylation characterized by N-linked oligosaccharides, where X can be any amino acid other than proline. N-acetylneuraminic acid (also known as sialic acid) is usually the terminal residue of both N-linked and O-linked oligosaccharides. Variables such as protein structure and cell type affect the number and nature of glycan units in the chain at different glycosylation sites. Glycosylated isomers are also common at the same site within the cell type.

  Therapeutic proteins corresponding to the therapeutic protein portion of the albumin fusion proteins of the present invention, and analogs and variants thereof, are expressed by the host cell in which glycosylation at one or more sites is expressed, or by them Depending on other conditions of expression of these, they may be modified to vary as a result of manipulation of their nucleic acid sequences. For example, glycosylated isomers may be produced to delete or introduce glycosylation sites, eg, by substitution or deletion of amino acid residues, such as substitution of glutamine for asparagine, or non-glycosylated recombinant proteins. It can be produced by expressing the protein in a host cell that is not glycosylated, such as in E. coli or a glycosylation-deficient yeast. These approaches are described in more detail below and are well known in the art.

  Therapeutic proteins, particularly those disclosed in Table 1, and their nucleic acid and amino acid sequences are well known in the art and are available in public databases such as Chemical Abstract Services Services Database (eg CAS Registry), GenBank. And available in subscription-providing databases such as GenSeq (eg, Derwent). An exemplary nucleic acid sequence of a therapeutic protein that can be used to drive a polynucleotide of the invention is shown in column 7, “SEQ ID NO: X” in Table 2. The sequence shown as SEQ ID NO: X can be a wild type polynucleotide sequence encoding the therapeutic protein (eg, either full length or mature), in some examples, the sequence is Nucleotide sequence variants (eg, a polynucleotide encoding a wild-type therapeutic protein, wherein the DNA sequence of the polynucleotide is optimized, eg, for expression in a particular species, or wild Polynucleotides encoding variants of type therapeutic proteins (ie, site-specific mutations, allelic variants)). It is well within the ability of one skilled in the art to utilize the sequence shown as SEQ ID NO: X to drive the construct described in the same column. For example, if SEQ ID NO: X represents a full-length protein, but only a portion of that protein is used to produce a specific CID, a specific fragment is amplified depending on molecular biological techniques such as PCR However, it is within the ability of one skilled in the art to clone it into an appropriate vector.

  Additional therapeutic proteins corresponding to the therapeutic protein portion of the albumin fusion protein of the present invention include, but are not limited to, one or more of the “Therapeutic Protein X” columns (Column 1) of Table 1 Therapeutic proteins or peptides, or fragments or variants thereof.

  Table 1 provides a non-inclusive list of therapeutic proteins corresponding to the albumin fusion proteins of the invention, or the therapeutic protein portion of the albumin fusion proteins encoded by the polynucleotides of the invention. The first column “Therapeutic Protein X” discloses in parentheses containing the therapeutic protein molecule followed by the chemical and brand name of the protein comprising or consisting of the therapeutic protein molecule or fragment or variant thereof. ing. As used herein, “therapeutic protein X” can mean an individual therapeutic protein or a whole group of therapeutic proteins bound to the therapeutic protein molecules disclosed in this column. The “biological activity” column (column 2) describes the biological activity associated with the therapeutic protein molecule. Column 3, “Exemplary Activity Assay” is used to test the therapeutic and / or biological activity of an albumin fusion protein comprising a therapeutic protein X or therapeutic protein X (or fragment thereof) moiety. A reference describing possible assays is provided. Each reference cited in the “Exemplary Activity Assay” column is described in the reference for assaying the corresponding biological activity listed in the “Biological Activity” column of Table 1, among others. All of which are incorporated herein by reference in connection with descriptions of other representative activity assays (see, eg, the method section therein). The fourth column “Preferred indication: Y” is a disease, disease and / or disease that can be treated, prevented, diagnosed and / or alleviated by an albumin fusion protein comprising therapeutic protein X, or therapeutic protein X (or a fragment thereof) Or state is described. The “construct ID” column (column 5) is an exemplary albumin disclosed in Table 2 that encodes an albumin fusion protein comprising or consisting of the referenced therapeutic protein X (or fragment thereof) site. Provide a connection to the fusion construct.

  Table 2 provides a non-comprehensive list of polynucleotides of the invention comprising or consisting of nucleic acids encoding albumin fusion proteins. The first column “Fusion Number” gives the fusion number of each polynucleotide. Column 2 “Construct ID” provides a unique numeric identifier for each polynucleotide of the invention. The construct ID includes or consists of a therapeutic protein portion corresponding to the therapeutic protein X listed in the corresponding column of Table 1, with the construct ID listed in column 5 It can be used to identify a polynucleotide encoding a protein. The “construct name” column (column 3) provides the name of the albumin fusion construct or polynucleotide.

  The fourth column “description” in Table 2 provides a general description of the albumin fusion construct, and the fifth column “expression vector” contains or consists of a nucleic acid molecule encoding the albumin fusion protein. The vectors in which the polynucleotides are cloned are listed. For example, as described in the Examples, one or more of (1) a polynucleotide encoding the albumin fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a transcription terminator An "expression cassette" comprising or consisting of is assembled into a convenient cloning vector, followed by other vectors such as, for example, yeast expression vectors or mammalian expression vectors, such as expression vectors You can move on. In one embodiment, S.I. For expression in S. cerevisiae, an expression cassette containing or consisting of a nucleic acid encoding an albumin fusion protein is cloned into pSAC35. In other embodiments, an expression cassette containing or consisting of a nucleic acid molecule encoding an albumin fusion protein is cloned into pC4 for expression in CHO cells. In a further embodiment, a polynucleotide comprising or consisting of a nucleic acid encoding a therapeutic protein portion of an albumin fusion protein is cloned into pC4: HSA. In yet a further embodiment, an expression cassette containing or consisting of a nucleic acid molecule encoding an albumin fusion protein is cloned into pEE12 for expression in NS0 cells. Other useful cloning and / or expression vectors are known to those skilled in the art and are within the scope of the present invention.

  The column “SEQ ID NO: Y” provides the full-length amino acid sequence of the albumin fusion protein of the present invention. In most instances, SEQ ID NO: Y represents the unprocessed form of the encoded albumin fusion protein, in other words, SEQ ID NO: Y is a signal sequence, HSA portion, and all encoded by a particular construct. Indicates the therapeutic part. Specifically encompassed by the present invention are all polynucleotides that encode SEQ ID NO: Y. When these polynucleotides are used to express a protein encoded from a cell, the natural secretion and processing steps of the cell are proteins that lack the signal sequence listed in columns 4 and / or 11 of Table 2. Produce. The specific amino acid sequences of the listed signal sequences are shown after the specification and are well known in the art. Thus, the most preferred embodiment of the present invention includes an albumin fusion protein produced by a cell (lacking the leader sequence shown in columns 4 and / or 11 of Table 2). A polypeptide comprising SEQ ID NO: Y is most preferred without the specific leader sequence listed in columns 4 and / or 11 of Table 2. Also preferred are compositions comprising these two preferred embodiments, including pharmacological compositions. In addition, the signal sequence listed in columns 4 and / or 11 of Table 2 is replaced with a different signal sequence, as described later in the specification, to facilitate secretion of the processed albumin fusion protein. Is well within the ability of those skilled in the art.

  The seventh column “SEQ ID NO: X” provides the parental nucleic acid sequence from which the polynucleotide encoding the therapeutic protein portion of the albumin fusion protein is derived. In one embodiment, the parent nucleic acid sequence from which the polynucleotide encoding the therapeutic protein portion of the albumin fusion protein is derived includes the wild-type gene sequence encoding the therapeutic protein shown in Table 1. It is. In other embodiments, the parent nucleic acid sequence from which the polynucleotide encoding the therapeutic protein portion of the albumin fusion protein is derived includes, for example, synthetic codon optimized mutations of the wild type gene sequence encoding the therapeutic protein Variants or derivatives of the wild-type gene sequence encoding the therapeutic proteins shown in Table 1 are included.

  The eighth column “SEQ ID NO: Z” provides the predicted translation of the parent nucleic acid sequence (SEQ ID NO: X). This parent sequence can be a full-length parent protein used to guide a particular construct, a mature portion of the parent protein, a variant or fragment of a wild-type protein, or an artificial sequence that can be used to create the described construct. . One skilled in the art can use this amino acid sequence shown in SEQ ID NO: Z to determine which amino acid residues of the albumin fusion protein encoded by the construct were provided by the therapeutic protein. It is. Furthermore, it is well within the ability of one skilled in the art to utilize the sequence shown as SEQ ID NO: Z to derive constructs described in the same column. For example, if SEQ ID NO: Z represents a full-length protein, but only a portion of that protein is used to produce a particular CID, a particular fragment is amplified and cloned into an appropriate vector It is within the skill of the artisan to rely on molecular biological techniques, such as PCR, to achieve

  The amplification primers provided in columns 9 and 10, respectively “SEQ ID NO: A” and “SEQ ID NO: B”, contain nucleic acid molecules encoding the therapeutic protein portion of the albumin fusion protein or they An exemplary primer used to produce a polynucleotide consisting of In one embodiment of the present invention, an oligonucleotide primer having the sequence shown in columns 9 and / or 10 (SEQ ID NO: A and / or B) is used as template DNA to provide nucleotides provided in column 7 of the corresponding row A nucleic acid molecule comprising or consisting of the sequence (SEQ ID NO: X) is used to PCR amplify a polynucleotide encoding the therapeutic protein portion of the albumin fusion protein. PCR methods are well established in the art. Additional useful primer sequences can be readily envisioned and utilized by those skilled in the art.

  In other embodiments, oligonucleotide primers can be used in overlapping PCR reactions to produce mutations within the template DNA sequence. PCR methods are known in the art.

  As shown in Table 3, certain albumin fusion constructs disclosed herein have been deposited with the ATCC®.

  The albumin fusion construct can be recovered from the deposit by techniques known in the art and described elsewhere herein (see Example 10). ATCC exists in 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. ATCC deposits were conducted in accordance with the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Procedures.

  In a further embodiment of the invention, it comprises one or more of (1) a polynucleotide encoding the albumin fusion protein, (2) a leader sequence, (3) a promoter region, and (4) a transcription terminator Or an “expression cassette” consisting of them can be transferred from one vector to another or “subcloned”. Fragments to be subcloned are well known in the art, such as PCR amplification (eg, using oligonucleotide primers having the sequence shown in SEQ ID NO: A or B) and / or restriction enzyme digestion. Can be produced by different methods.

In a preferred embodiment, the albumin fusion protein of the present invention corresponds to the therapeutic activity and / or biological activity of a therapeutic protein corresponding to the therapeutic protein portion of the albumin fusion protein listed in the corresponding column of Table 1. Is capable of therapeutic and / or biological activity. In a further preferred embodiment, the therapeutically active protein part of the albumin fusion protein of the invention is a fragment or variant of the protein encoded by the sequence shown in SEQ ID NO: X column of Table 2, correspondingly It is capable of therapeutic activity and / or biological activity of a therapeutic protein.

Polypeptide and polynucleotide fragments and variant fragments

  The present invention is further directed to the therapeutic proteins described in Table 1, albumin proteins of the present invention, and / or fragments of albumin fusion proteins.

  The present invention is also directed to polynucleotides encoding fragments of the therapeutic proteins described in Table 1, albumin proteins of the present invention, and / or albumin fusion proteins.

  Even when the deletion of one or more amino acids from the N-terminus of the protein results in a modification or absence of one or more therapeutic proteins, albumin proteins and / or albumin fusion proteins of the invention Other therapeutic and / or functional activities (eg, biological activity, ability to multiplex, ability to bind ligand) may also remain. For example, the ability of an N-terminal deficient polypeptide to induce and / or bind to an antibody that recognizes a complete or mature form of the polypeptide is such that less than most residues of the complete polypeptide are removed from the N-terminus. If removed, it can remain. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide remains immunologically active is described herein or can be readily determined by routine methods known in the art It is. Probably mutant proteins lacking a large number of N-terminal amino acid residues may leave biological or immunological activity. In fact, peptides consisting of only 6 amino acid residues often elicit an immune response.

  Accordingly, a fragment of the therapeutic protein corresponding to the therapeutic protein portion of the albumin fusion protein of the invention has one or more residues deleted from the amino terminus of the amino acid sequence of the full length protein as well as the reference polypeptide. Polypeptides (ie, therapeutic proteins cited in Table 1 or therapeutic protein portions of albumin fusion proteins encoded by polynucleotides or albumin fusion constructs described in Table 2) are included. In particular, the N-terminal deletion is described by the general formulas m to q, where q is a reference polypeptide (eg, a therapeutic protein cited in Table 1 or a therapeutic protein portion of an albumin fusion protein of the invention). Or a whole integer representing the total number of amino acid residues in the therapeutic protein portion of the albumin fusion protein encoded by the polynucleotide or albumin fusion construct described in Table 2, and m is 2 to q-6 Defined as any integer in the range Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Furthermore, the serum albumin polypeptide fragment corresponding to the albumin protein portion of the albumin fusion protein of the present invention lacks one or more residues from the amino terminus of the amino acid sequence of the full-length protein and the reference polypeptide. Polypeptides (eg, serum albumin or the serum albumin portion of an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). In a preferred embodiment, the N-terminal deletion may be described by the general formula m-585, where 585 is a whole integer representing the total number of amino acid residues in mature human serum albumin (SEQ ID NO: 1). And m is defined as any integer in the range of 2 to 579. Polynucleotides encoding these polypeptides are also encompassed by the present invention. In a further embodiment, the N-terminal deletion can be described by the general formula m-609, where 609 is a whole integer representing the total number of amino acid residues in full-length human serum albumin (SEQ ID NO: 3); m is defined as any integer in the range of 2 to 603. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Further, the fragments of albumin fusion proteins of the present invention include full-length albumin fusion proteins as well as polypeptides lacking one or more residues from the amino terminus of the albumin fusion protein (eg, as described in Table 2). Albumin fusion proteins encoded by polynucleotides or albumin fusion constructs, or albumin fusion proteins having the amino acid sequence disclosed in column 6 of Table 2). In particular, the N-terminal deletion is described by the general formulas m to q, where q is a whole integer representing the total number of amino acid residues in the albumin fusion protein, and m is in the range of 2 to q-6. Defined as an arbitrary integer. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Also as described above, the deletion of one or more amino acids from the N-terminus or C-terminus of a reference peptide (eg, a therapeutic protein of the invention, serum albumin protein, or albumin fusion protein) results in Other functional activities (eg, biological activity, ability to multiplex, ability to bind ligand) and / or therapeutic, even if it results in alteration or loss of one or more biological functions of the protein as Activity can remain. For example, the ability of a polypeptide with a C-terminal deletion to induce and / or bind an antibody that recognizes a complete or mature form of a polypeptide is generally less than the majority of residues of the complete or mature polypeptide. It can remain when removed from the C-terminus. Whether a particular polypeptide lacking the N-terminus and / or C-terminus of a reference polypeptide retains therapeutic activity is described in the present specification and / or is known in the art. It can be easily determined by the method.

  The invention further relates to therapeutic proteins corresponding to the therapeutic protein portion of the albumin fusion protein of the invention (eg, therapeutic proteins cited in Table 1 or polynucleotides or albumin fusion constructs described in Table 2). A polypeptide lacking one or more residues from the carboxy terminus of the amino acid sequence of the therapeutic protein portion of the albumin fusion protein encoded by In particular, the C-terminal deletion is described by general formulas 1-n, where n is any whole integer in the range 6-q-1 and q is a reference polypeptide (eg, cited in Table 1). All integers representing the total number of amino acid residues in the therapeutic protein or therapeutic protein portion of the albumin fusion protein encoded by the polynucleotide or albumin fusion construct described in Table 2. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Furthermore, the present invention provides an albumin protein corresponding to the albumin protein portion of the albumin fusion protein of the invention (eg, albumin protein of serum albumin or albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 2). A polypeptide lacking one or more residues from the carboxyl terminus of the amino acid sequence of (part). In particular, the C-terminal deletion is described by general formulas 1-n, where n is any whole integer in the range 6-584, where 584 is the amino acid residue in mature human serum albumin (SEQ ID NO: 1). All integers representing the total number of groups minus one. In particular, the C-terminal deletion is described by general formulas 1-n, where n is any whole integer in the range 6-608, and 608 is the amino acid residue in serum albumin (SEQ ID NO: 3). It is a whole integer representing the total number -1. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Furthermore, the present invention provides a polypeptide lacking one or more residues from the carboxy terminus of the albumin fusion protein of the present invention. In particular, the C-terminal deletion may be described by general formulas 1-n, where n is any whole integer in the range 6-q-1 and q is an amino acid residue in the albumin fusion protein of the invention Is a whole integer representing the total number of. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Furthermore, any of the above N- or C-terminal deletions can be mixed to produce an N- and C-terminal deletion reference polypeptide. The invention also provides a polypeptide in which one or more amino acids are deleted from both the amino and carboxyl termini, and a reference polypeptide (eg, a therapeutic protein cited in Table 1 or an albumin fusion of the invention). The therapeutic protein portion of the protein, or the therapeutic protein portion encoded by the polynucleotide or albumin fusion construct described in Table 2, or serum albumin (eg, SEQ ID NO: 1), or the albumin protein of the albumin fusion protein of the invention A portion, or an albumin protein portion encoded by a polynucleotide or albumin fusion construct described in Table 2, or an albumin fusion protein, or encoded by a polynucleotide or albumin fusion construct of the invention It was generally described and obtained as having residues m~n albumin fusion proteins), n and m in the formula is an integer as described above. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  The present specification also includes reference polypeptides listed herein (eg, therapeutic proteins cited in Table 1, or therapeutic protein portions of albumin fusion proteins of the invention, or polynucleotides described in Table 2). Or a therapeutic protein portion encoded by an albumin fusion construct, or serum albumin (eg, SEQ ID NO: 1), or an albumin protein portion of an albumin fusion protein of the invention, or a polynucleotide or albumin fusion construct described in Table 2 Albumin protein portion, or albumin fusion protein, or albumin fusion protein encoded by a polynucleotide or albumin fusion construct of the present invention) and at least 80%, 85%, 90%, 95%, 9 %, 97%, directed to protein or fragment thereof, 98% or 99% identical to a polypeptide. In a preferred embodiment, the present specification provides a reference polypeptide having an N- and C-terminal deletion amino acid sequence, as described above, and at least 80%, 85%, 90%, 95%, 96%, 97% , Directed to proteins containing 98% or 99% identical polypeptides. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  Preferred polypeptide fragments of the invention are amino acid sequences that exhibit therapeutic and / or functional activity (eg, biological activity) of therapeutic protein or serum albumin protein polypeptide sequences, wherein the amino acid sequence is a fragment. Is a fragment comprising or consisting of

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those that exhibit similar, but not necessarily identical, activity to the polypeptides of the present invention. The biological activity of the fragment includes an improvement in the desired activity or a reduction in unwanted activity.
Mutant

  “Variant” means a polynucleotide or nucleic acid that differs from a reference nucleic acid or polypeptide but retains its essential characteristics. In general, variants are very similar overall to a reference nucleic acid or polypeptide and are identical in many regions.

  As used herein, a “variant” refers to a therapeutic protein portion of an albumin fusion protein of the invention, an albumin portion of an albumin fusion protein of the invention, or a therapeutic protein (eg, “therapeutic” in Table 1). Column "), albumin protein, and / or albumin fusion protein that differ in sequence but at least one such as those described elsewhere herein or known in the art. It means an albumin fusion protein of the invention that retains its functional and / or therapeutic properties. In general, a variant is defined as a therapeutic protein corresponding to a therapeutic protein portion of an albumin fusion protein, an albumin protein corresponding to an albumin protein portion of an albumin fusion protein, and / or an amino acid sequence of an albumin fusion protein. It is very similar and is the same in many areas. Nucleic acids encoding these variants are also encompassed by the present invention.

  The present invention also includes, for example, the amino acid sequence of a therapeutic protein corresponding to the therapeutic protein portion of an albumin fusion protein of the present invention (eg, the therapeutic protein disclosed in Table 1: X amino acid sequence, or Tables 1 and 2). The amino acid sequence of the therapeutic protein portion of the albumin fusion protein encoded by the polynucleotide or albumin fusion construct described in 1) or a fragment or variant thereof), the albumin protein corresponding to the albumin protein portion of the albumin fusion protein of the invention Of the albumin protein portion of the albumin fusion protein encoded by the polynucleotide or albumin fusion construct described in Tables 1 and 2, for example the amino acid shown in SEQ ID NO: 1. Sequence, or fragments or variants thereof) and / or the amino acid sequence of the albumin fusion protein at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical Directed to proteins comprising or consisting of amino acid sequences. Fragments of these polypeptides are also provided (eg, fragments described herein). Additional polypeptides included in the present invention include hybridization to filter-bound DNA at about 45 ° C. in tight hybridization conditions (eg, 6 × sodium chloride / sodium citrate (SSC), followed by 0.2 × SSC, One or more washes at about 50-65 ° C. in 0.1% SDS) under very tight conditions (eg, 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C. Hybridization to filter-bound DNA, followed by 0.1 × SSC, 0.2% SDS, one or more washes at about 68 ° C.) or other tight hybridization conditions known to those skilled in the art (For example, Ausubel, FM et al., Eds., 1989 Current protocol in Molecular. (See Iolology, Green publishing associates, Inc., and John Wiley & Sons Inc., New York, at pages 6.3.1-6.3.6 and 2.10.3). A polypeptide encoded by a polynucleotide that hybridizes to the complement of a nucleic acid encoding a protein. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

  A polypeptide having an amino acid sequence that is at least 95% “identical” to the query amino acid sequence, for example, so that the subject polypeptide sequence can contain up to 5 amino acid changes for every 100 amino acids in the query amino acid sequence Except, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence. In other words, up to 5% amino acid residues in the subject sequence can be inserted, deleted or substituted with other amino acids to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence. These reference sequence changes may be made at the amino- or carboxy-terminal position of the reference amino acid sequence or at any position between these terminal sites and individually within the residues in the reference sequence or reference It can occur interspersed in one or more consecutive groups in the sequence.

  As a practical matter, a particular polypeptide is, for example, at least 80% of the amino acid sequence of an albumin fusion protein of the invention, or a fragment thereof (such as a therapeutic protein portion of an albumin fusion protein or an albumin portion of an albumin fusion protein) , 85%, 90%, 95%, 96%, 97%, 98% or 99% can be easily determined using a known computer program. A preferred method for determining an optimal total match between a query sequence (the sequence of the invention) and a subject sequence, also referred to as global sequence alignment, is described in Brulag et al. (Comp. App. Biosci. 6: 237-245 (1990)) and can be determined using the FASTDB computer program based on the algorithm. In sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of the global sequence alignment are shown as percent identity. Preferred parameters used in the FASTDB amino acid alignment are: Matrix = PAM 0, k-tuple = 2, Mismatch Penalty = 1, Joining Penalty = 20, Randomization Group Length = 0, Cutoff Score = 1, Window Size = Sequence Length, Gap Penalty = 5, Gap Size Penalty, Randomization Group Length = 0, Cutoff Score = 1, Window Size = Sequence Length, Gap Penalty = 5, Gap Size Penalty = 0.05, window size (Window Size) = 500, or shorter length of the target amino acid sequence.

  If the subject sequence is shorter than the query sequence because of an N- or C-terminal deletion rather than due to an internal deletion, a manual correction must be performed on the result. This is because the FASTDB program does not take into account N- and C-terminal truncations of the subject sequence when calculating global percentage identity. For the query sequence, with respect to the subject sequence shortened at the N- and C-termini, the percent identity is expressed as a percentage of the total base of the query sequence and does not match / sequence the corresponding subject residue N- and Correct by calculating the number of residues in the query sequence that are C-terminal. Whether a residue matches / sequences is determined by the results of the FASTDB sequence alignment. The percentage is then subtracted from the percentage identity calculated by FASTDB using specific parameters to arrive at the final percentage identity score. This final percent identity score is what is used for the purposes of the present invention. Only the N- and C-terminal residues of the subject sequence that do not match / sequence the query sequence are considered for the purpose of manually fitting the percent identity score. This is only the query residue site outside the farthest N- and C-terminal residues of the subject sequence.

  For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. A deletion occurs at the N-terminus of the subject sequence, and therefore the FASTDB alignment does not show a match / alignment of the first 10 residues of the N-terminus. Ten unpaired residues represent 10% of the sequence (number of non-matching N- and C-terminal residues / total number of residues in the query sequence), thus 10% was calculated by the FASTDB program. Subtract from percentage identity score. If the remaining 90 residues are a perfect match, the final percent identity can be 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. At this time, the deletion is an internal deletion, and therefore there is no residue at the N- or C-terminus of the subject sequence that does not match / sequence the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, as presented in the FASTDB alignment, only the residue positions outside the N- and C-terminal ends of the subject sequence that do not match / sequence the query sequence are manually corrected. No other manual correction is performed for the purposes of the present invention.

  A variant usually has at least 75% (preferably at least about 80%, 90%, 95% or 99%) sequence identity with the length of a normal HA or therapeutic protein of the same length as the variant. Homology or identity at the nucleotide or amino acid residue level is determined by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, J. Mol. Evol. 36: 290-300 (1993), fully incorporated by reference), BLAST (Basic Local). Alignment Search Tool) analysis.

The approach used by the BLAST program first considers similar segments between the query and database sequences, then evaluates the statistical significance of all matches that are identical, and finally determines the significance of the previously selected significance. Summarize only those matches that meet the threshold. Altschul et al., Fully incorporated by reference, for discussion of basic events in sequence database similarity searches. , (Nature Genetics 6: 119-129 (1994)). Histograms, descriptions, alignments, predictions (ie, statistical significance thresholds for reporting matches against database sequences), cutoff, matrix and filter search parameters are default settings. The default scoring matrix used by blastp, blastx, tblastn and tblastx is the BLOSUM62 matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992), fully incorporated by reference. Is). For blastn, the scoring matrix is set by the ratio of M (ie reward score for matching residue pairs) to N (ie penalty score for mismatched residues), where the default values for M and N are 5 and −, respectively. 4. The four blastn parameters may be adjusted as follows. Q = 10 (gap creation penalty), R = 10 (gap extension penalty), wink = 1 (generate word hits at each wink ^th position along the query), and gapw = 16 (generate gap alignment) Set window width). The equivalent Blastp parameter settings are Q = 9, R = 2, wink = 1 and gapw = 32. Bestfit comparison between sequences, available in GCG package version 10.0, uses DNA parameters GAP = 50 (gap creation penalty) and LEN = 3 (gap extension penalty), and the equivalent setting for protein comparison is GAP = 8 and LEN = 2.

  The polypeptide variants of the invention may contain changes in the coding region, non-coding region, or both. In particular, polynucleotide variants that include changes that produce silent substitutions, additions or deletions, but do not alter the properties or activities of the encoded polypeptide are preferred. Nucleotide variants produced by silent substitution due to the degeneracy of the genetic code are preferred. Furthermore, in any combination, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 to 50, 5 to 25, 5 to 10, 1 to 5, or 1 to 2 amino acids are substituted, deleted or added Also preferred are polypeptide variants. Polynucleotide variants can be produced for a variety of reasons, such as to optimize codon expression for a particular host (codons in human mRNA can be transformed into bacteria such as yeast or E. coli). Change to the one preferred by the host).

  In a preferred embodiment, the polynucleotide of the invention encoding the albumin portion of an albumin fusion protein is optimized for expression in yeast or mammalian cells. In a further preferred embodiment, the polynucleotide of the invention encoding the therapeutic protein portion of the albumin fusion protein is optimized for expression in yeast or mammalian cells. In yet a further preferred embodiment, the polynucleotide encoding the albumin fusion protein of the invention is optimized for expression in yeast or mammalian cells.

  In other embodiments, the codon-optimized polynucleotide encoding the therapeutic protein portion of the albumin fusion protein is a wild-type encoding therapeutic protein under tight hybridization conditions as described herein. Does not hybridize to the polynucleotide. In a further embodiment, the codon-optimized polynucleotide encoding the albumin portion of the albumin fusion protein is transformed into a wild-type polynucleotide encoding albumin protein under constrained hybridization conditions as described herein. Does not hybridize. In other embodiments, the codon-optimized polynucleotide encoding the albumin fusion protein is wild-type encoding a therapeutic protein portion or an albumin protein portion under constrained hybridization conditions as described herein. Does not hybridize to type polynucleotides.

  In a further embodiment, the polynucleotide encoding the therapeutic protein portion of the albumin fusion protein does not comprise or consist of the naturally occurring sequence of the therapeutic protein. In further embodiments, the polynucleotide encoding the albumin protein portion of the albumin fusion protein does not comprise or consist of the naturally occurring sequence of the albumin protein. In other embodiments, the polynucleotide encoding the albumin fusion protein does not comprise or consist of the therapeutic protein portion or the naturally occurring sequence of the albumin protein portion.

  Naturally occurring variants are referred to as “allelic variants” and refer to one of various alternating forms of a gene that occupies the locus on the chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)) These allelic variants may vary at either the polynucleotide and / or polypeptide level and are included in the present invention. included. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

  Using known methods of protein engineering and recombinant DNA technology, variants may be produced to improve or alter the properties of the polypeptides of the invention. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a polypeptide of the invention without an essential loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported a mutant KGF protein with heparin binding activity even after deletion of the 3, 8 or 27 amino-terminal amino acid residues. Similarly, interferon gamma showed up to 10-fold higher activity after deletion of 8-10 amino acid residues from the carboxy terminus of the protein (Dobeli et al., J. Biotechnology 7: 199-216 (1988). )).

  Furthermore, sufficient evidence indicates that variants often maintain biological activity similar to naturally occurring proteins. For example, Gayle and co-workers ((J. Biol. Chem. 268: 22105-22111 (1993)) performed extensive mutational analysis of the human cytokine IL-1a. Random mutagenesis was used to produce more than 3,500 individual IL-1a variants with an average of 2.5 amino acid changes per body, multiple mutagenesis was tested at each potential amino acid site. The investigator has found that “most of the molecules can change minor effects on either binding or biological activity.” In fact, 23 of the over 3,500 nucleotide sequences tested Only the unique amino acid sequence produced a protein that was significantly different from the activity from the wild type.

  In addition, deletion of one or more amino acids from the N-terminus or C-terminus of a polypeptide may result in other organisms even when one or more biological functions are altered or deleted. The pharmacological activity can remain maintained. For example, the ability of a deletion mutant to induce and / or bind an antibody that recognizes a secreted form can be maintained when less than a majority of the secreted form is removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking the N- and C-termini of a protein retains such immunological activity is described in a simple manner by conventional methods described herein and known in the art. Can be determined.

  Accordingly, the present invention further includes polypeptide variants having functional activity (eg, biological activity and / or therapeutic activity). In one embodiment, the present invention provides a functional activity (eg, corresponding to one or more biological and / or therapeutic activities of a therapeutic protein corresponding to a therapeutic protein portion of an albumin fusion protein) Variants of albumin fusion proteins with biological activity and / or therapeutic activity) are provided. In other embodiments, the invention provides a functional activity (eg, a biological activity) that corresponds to one or more biological and / or therapeutic activities of a therapeutic protein corresponding to a therapeutic protein of an albumin fusion protein. Variants of albumin fusion proteins with functional and / or therapeutic activity) are provided. Such variants include deletions, insertions, inversions, repeats and substitutions selected according to general rules known in the art to have a slight effect on activity. Polynucleotides encoding such variants are also included in the present invention.

  In a preferred embodiment, the variants of the invention have conservative substitutions. “Conservative substitutions” include substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile, substitution of hydroxyl residues Ser and Thr, substitution of acidic residues Asp and Glu, amide residues Asn and Gln. Intragroup intentions such as substitution of the basic residues Lys, Arg and His, substitution of the aromatic residues Phe, Tyr and Trp, and substitution of the small amino acids Ala, Ser, Thr, Met and Gly Exchange.

  Guidance on how to create phenotypically silent amino acid substitutions can be found in, for example, Bowie et al. , "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247: 1306-1310 (1990), changes to amino acid substitutions. It suggests that there are two main strategies to study sequence resistance.

  The first strategy takes advantage of resistance to amino acid substitutions by natural selection during the course of evolution. By comparing amino acid sequences of different species, conserved amino acids can be identified. These conserved amino acids are expected to be important with respect to protein function. On the other hand, amino acid positions where substitutions were tolerated by natural selection suggest that the positions are not essential for protein function. Thus, the position of tolerance to amino acid substitutions can be altered while maintaining the biological activity of the protein.

  The second strategy uses genetic engineering to introduce amino acid changes at specific positions in the cloned gene to identify regions essential for protein function. For example, site-directed mutagenesis or alanine-scanning mutagenesis (introduction of a single alanine mutation at each residue in the molecule) can be used. See Cunningham and Wells, Science 244: 1081-1085 (1989). The resulting mutant molecules can also be tested for biological activity.

  As the authors mentioned, these two strategies revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further suggest that amino acid changes may be tolerated at specific amino acid sites within the protein. For example, most of the buried amino acid residues (within the tertiary structure of the protein) require nonpolar side chains, while the few features of surface side chains are generally conserved. In addition, tolerant conservative amino acid substitutions include substitution of aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile, substitution of hydroxyl residues Ser and Thr, substitution of acidic residues Asp and Glu, substitution of amide residues Asn and Gln. Substitution, substitution of basic residues Lys, Arg and His, substitution of aromatic residues Phe, Tyr and Trp, and substitution of small size amino acids Ala, Ser, Thr, Met and Gly. In addition to conservative amino acid substitutions, variants of the invention include (i) a polypeptide comprising one or more substitutions of non-conservative amino acid residues, wherein the substituted amino acid residues are encoded by the genetic code. Or (ii) a polypeptide comprising a substitution of one or more amino acid residues with substituents, or (iii) stability and / or solubility of the polypeptide. A polypeptide that is fused or chemically conjugated to another compound, such as a compound (eg, polyethylene glycol) to increase the amount of (iv) a polypeptide comprising additional amino acids, such as an IgG Fc fusion region peptide included. Such variant polypeptides are considered within the scope of those skilled in the art from the teachings herein.

  For example, a polypeptide comprising an amino acid substitution of a charged amino acid with another charged or neutral amino acid can produce a protein with improved characteristics, such as less aggregation. Aggregation of pharmacological formulations reduces activity and increases clearance due to the immunological activity of the aggregate. Pinkard et al. , Clin. Exp. Immunol. 2: 331-340 (1967), Robbins et al. Diabetes 36: 838-845 (1987), Cleland et al. , Crit. Rev. See Therapeutic Drug Carrier Systems 10: 307-377 (1993).

  In certain embodiments, a polypeptide of the invention comprises or consists of an amino acid sequence fragment or variant of an albumin fusion protein, therapeutic protein and / or human serum albumin, wherein the fragment or variant The body has 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150 amino acid residue additions, substitutions and / or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Nucleic acids encoding these polypeptides are also encompassed by the present invention.

The polypeptides of the present invention may comprise amino acids joined to each other by peptide bonds or modified peptide bonds, ie peptide equivalent formulas, and may comprise amino acids other than the 20 gene-encoded amino acids. Polypeptides can be modified either by natural processes, such as post-transcriptional processing, or by chemical modification techniques well known in the art. Such modifications are well described in basic documents and more detailed monographs as well as extensive search literature. Modifications can occur throughout the polypeptide, such as the peptide backbone, amino acid side chains, and amino or carboxyl termini. It will be appreciated that the same type of modification may be present at various positions in the polypeptide, to the same or different degrees. The polypeptide may also include many types of modifications. Polypeptides may be branched and cyclic with or without branching, for example as a result of ubiquitination. Polypeptides that are cyclic, branched, and branched and cyclic are produced as a result of post-translation natural processes or are produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme site covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphotidylinositol Covalent bond, crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cysteine formation, pyroglutamic acid formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination Transfer-RNA-mediated amino acid addition to proteins such as methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitination included. (For example, PROTEINS-STRUCTURE AND MOLECULAR PROPERITES, 2nd Ed., T.E. Creaton, W. H. Freeman and Company, New York (C). , Academic Press, New York, pgs.1-12 (1983), Seifter et al., Meth.Enzymol.182: 626-646 (1990), Rattan et al., Ann.N.Y. : 48-62 (1992).
Functional activity

  “A polypeptide having functional activity” can present one or more known functional activities related to the full length, pro-protein and / or mature form of a therapeutic protein Means a polypeptide that is Such functional activities include, but are not limited to, biological activity, antigenic [ability to bind (or compete with polypeptides for binding) anti-polypeptide antibodies], immunogenicity (of the invention Ability to produce antibodies that bind to a particular polypeptide), the ability to form multimers with the polypeptides of the invention, and the ability to bind to a receptor or ligand for the polypeptide.

  A “polypeptide having biological activity” includes a mature form as determined in a particular biological assay, either in a time-dependent or independent manner. Means a polypeptide presenting activity that is similar to, but not necessarily identical to, the activity of the therapeutic protein. If there is / time-dependency exists, it need not be identical to that of the polypeptide, but is essentially similar to the dose-dependence in the activity as compared to the polypeptide of the present invention (ie, the candidate peptide Presents greater activity, but is less than or equal to about 25 times, preferably less than or equal to about 1/10, most preferably less than or equal to about 1/3 of the activity of the polypeptides of the invention .

  In a preferred embodiment, the albumin fusion protein of the invention has at least one biological and / or therapeutic activity associated with the therapeutic protein moiety (or fragment or variant thereof) when not fused to albumin.

  In a further preferred embodiment, the albumin fusion protein of the invention has increased plasma stability compared to the therapeutic protein portion (or fragment or variant thereof) in the unfused state. The plasma stability of an albumin fusion protein of the invention or of a non-fusion therapeutic protein portion (or fragment or variant thereof) can be assayed using assays known in the art or modified as usual. is there.

  The albumin fusion proteins of the invention can be assayed for functional activity (eg, biological activity) using assays known in the art, as well as assays described herein, or modified as usual. . In addition, those skilled in the art will identify fragments of therapeutic proteins corresponding to the therapeutic protein portion of the albumin fusion protein for activity using the assays referenced in the corresponding row of Table 1 (eg, column 3 of Table 1). You may assay as usual. In addition, one of ordinary skill in the art can determine fragments of albumin protein corresponding to the albumin protein portion of the albumin fusion protein with respect to activity using assays known in the art and / or as described in the Examples section below. You can assay as usual.

  For example, one embodiment of assaying for the ability of an albumin fusion protein to bind to or compete with a therapeutic protein for binding to an anti-therapeutic polypeptide antibody and / or anti-albumin antibody is known in the art. A variety of immunoassays can be used, including but not limited to radioimmunoassays, ELISAs (enzyme immunoassays), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays (eg, In situ immunoassay (using colloidal gold, enzyme or radionuclide labeling), Western blot, precipitation reaction, agglutination assay (eg gel aggregation assay, hemagglutination assay), complement fixation assay, immunofluorescence assay, protein A assay, and Immunity Using techniques such as gas migration assays include competitive and non-competitive assay systems. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In other embodiments, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

  In a preferred embodiment, when a therapeutic protein binding partner (eg, a receptor or ligand) is identified, binding to the binding partner by an albumin fusion protein comprising the therapeutic protein as the therapeutic protein portion of the fusion is, for example, reduction and It can be assayed by methods known in the art, such as non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally Physicky et al. , Microbiol. Rev. 59: 94-123 (1995). In other embodiments, the ability of the physiological correlates of the albumin fusion protein to bind to a substrate of a therapeutic polypeptide corresponding to the therapeutic protein portion of the fusion is routinely assayed using techniques known in the art. Is possible.

  In other embodiments, when assessing the ability of the albumin fusion protein to multiplex, the association of the multimer with other components can be determined by, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. It can be assayed by methods known in the art. See generally Physicky et al. See above.

  In a preferred embodiment, an albumin fusion protein comprising all or part of an antibody that binds to a therapeutic protein is associated with an antibody that binds to the therapeutic protein (or fragment or variant thereof) when not fused to albumin ( Has at least one biological and / or therapeutic activity (eg, specifically binds to a polypeptide or epitope). In other preferred embodiments, the biological and / or therapeutic activity of an albumin fusion protein, including all or a portion of an antibody that binds to a therapeutic protein, is a poly-specifically bound by an antibody that binds to the therapeutic protein. Inhibition (ie antagonism) or activation (ie agonism) of one or more biological and / or therapeutic activities associated with a peptide.

  Albumin fusion proteins comprising at least one fragment or variant of an antibody that binds to a therapeutic protein can be characterized in a variety of ways. In particular, an albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein can be obtained using the techniques described herein or by routinely modifying techniques known in the art. The antibody that specifically binds to the therapeutic protein corresponding to the therapeutic protein portion of the albumin fusion protein may be assayed for the ability to specifically bind to the same antigen that is specifically bound.

  Assays for the ability of albumin fusion proteins to bind (specifically) to a specific protein or epitope (eg, including at least one fragment or variant of an antibody that binds to a therapeutic protein) in solution (eg, Houghten, Bio / Techniques 13: 412-421 (1992)), on beads (eg, Lam, Nature 354: 82-84 (1991)), on chips (eg, Fodor, Nature 364: 555-556 (1993)), on bacteria (eg, U.S. Pat. No. 5,223,409), on spores (e.g., patents 5,571,698, 5,403,484, and 5,223,409), on plasmids (e.g., Cull et al. Proc. Natl. Aci. Sci. USA 89: 1865-1869 (1992)) or on phage (eg Scott and Smith, Science 249: 386-390 (1990), Devlin, Science 249: 404-406 (1990), Cwirl et al. Natl. Acad. Sci. USA 87: 6378-6382 (1990); and Felici, J. Mol. Biol. 222: 301-310 (1991)) (each of these references is All incorporated herein by reference). An albumin fusion protein comprising at least one fragment or variant of a therapeutic antibody is used to modify a particular protein or epitope using the techniques described herein or known in the art or modified as usual. May be assayed for their specificity and affinity for.

  An albumin fusion protein comprising at least one fragment or variant of a therapeutic antibody that binds to a therapeutic protein can be converted to other antigens (eg, a therapeutic protein of the albumin fusion protein of the invention by any method known in the invention). It can be assayed for cross-reactivity between a molecule specifically bound by an antibody that binds to a therapeutic protein corresponding to the moiety (or fragment or variant thereof) and a molecule with sequence / structure retention.

  Immunoassays that can be used to analyze (immunospecific) binding and cross-reactions include, but are not limited to, Western blots, radioiminoassays, ELISAs (enzyme immunoassays), to name just a few. Competitive and using techniques such as “sandwich” immunoassay, immunoprecipitation reaction, precipitation reaction, gel diffusion precipitation reaction, aggregation assay, complement immobilization assay, immunoradiometric assay, immunofluorescence assay, protein A assay Non-competitive assay systems are included. Such assays are routine and well known in the art (eg, Ausubel et al, eds, 1994, Current Protocols in Molecular, all of which are incorporated herein by reference). Biology, Vol. 1, see John Wiley & Sons, Inc., New York). An exemplary immunoassay is briefly described below (but is not intended to be limiting).

  Immunoprecipitation protocols generally involve RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxy) containing protein phosphatases and / or protease inhibitors (eg, EDTA, PMSA, aprotinin, sodium vanadate). A population of cells was lysed in a lysis buffer such as cholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate pH 7.2, 1% trasilol) Add an albumin fusion protein of the invention (eg comprising at least one fragment or variant of an antibody that binds to a therapeutic protein) at 0 ° C. and incubate at 40 ° C. for a while (eg 1 to 4 hours), eg Sepharose beads bound to anti-albumin antibody In addition to cyst lysate, incubate at 40 ° C. for about 1 hour or longer, wash beads in lysis buffer, and resuspend beads in SDS / sample buffer. The ability of an albumin fusion protein to immunoprecipitate a particular antigen can be assessed, for example, by Western blot analysis. One skilled in the art knows that the parameters can be modified to increase the binding of the albumin fusion protein to the antigen and reduce the background (eg pre-cleaning of cell lysates with Sepharose beads). For further discussion on immunoprecipitation protocols, see, eg, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc. , New York at 10.16.1.

Western blot analysis generally involves preparing protein samples, electrophoresis in polyacrylamide gels (eg, 8% -20% SDS-PAGE, depending on the molecular weight of the antigen), from polyacrylamide gels. A sample of protein to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in a blocking solution (eg PBS containing 3% BSA or non-fat milk), wash buffer (eg PBS-Tween 20) Washing the membrane in, applying the albumin fusion protein of the invention (diluted in blocking buffer) to the membrane, washing the membrane in wash buffer, diluting in blocking buffer, enzymatic substrate ( Eg horseradish peroxidase or alkaline phosphatase) or radioactive (Such as ^{32 P} or ^{125 I)} conjugated to (recognizes the albumin fusion protein, for example, anti-human serum albumin antibody) applying a secondary antibody, washing the membrane in wash buffer, and the presence of the antigen Detecting. Those skilled in the art know that parameters can be modified to increase the detected signal and reduce background noise. For further discussion on Western blot protocols, see, eg, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc. , New York at 10.8.1.

  ELISA consists of preparing the antigen, coating the wells of a 96-well microtiter plate with the antigen, washing away any antigen that did not bind to the well, enzymatic substrate (eg horseradish peroxidase or alkaline phosphatase) Adding an albumin fusion protein of the invention (eg comprising at least one fragment or variant of an antibody that binds to a therapeutic protein) conjugated to a detectable compound such as Or washing away non-specifically bound albumin fusion proteins and detecting the presence of albumin fusion proteins that specifically bind to the antigen coating the wells. In ELISA, the albumin fusion protein need not be conjugated to a detectable compound; instead, a secondary antibody may be added to the well that is conjugated to the detectable compound (recognizes the albumin fusion protein). Further, instead of coating the well with the antigen, the well may be coated with an albumin fusion protein. In this case, the detectable molecule can be an antigen conjugated to a detectable compound such as an enzymatic substrate (eg, horseradish peroxidase or alkaline phosphatase). Those skilled in the art are aware of parameters that can be modified to increase the detected signal and other variations of ELISA known in the art. For further discussion on ELISA, see, eg, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc. , New York at 11.2.1.

The binding affinity of an albumin fusion protein for a protein, antigen or eptop, and the off-rate of the albumin fusion protein-protein / antigen / epitope interaction can be determined by competitive binding assays. One example of a competitive binding assay is the incubation of a labeled antigen (eg ³ H or ¹²⁵ I) with an albumin fusion protein of the invention in the presence of increasing amounts of unlabeled antigen and an antibody that is bound to a labeled light source. A radioimmunoassay involving the detection of The affinity of the albumin fusion protein for a particular protein, antigen, or epitope, and the binding off-rate can be determined from the data by Scatchard plot analysis. As an albumin fusion protein, competition with a second protein that binds to the same protein, antigen or epitope can also be determined using radioimmunoassay. In this case, the protein, antigen or epitope is labeled as an albumin fusion protein of the invention in the presence of an increased amount of unlabeled second protein bound to the same protein, antigen or epitope (eg ³ H or ¹²⁵ Incubate with albumin fusion protein conjugated to I).

  In a preferred embodiment, BIAcore kinetic analysis is used to determine the on and off rates of binding of the albumin fusion protein of the invention to a protein, antigen or epitope. For BIAcore kinetic analysis, analysis of binding and separation of albumin fusion protein, or specific polypeptide, antigen or epitope from a chip having a specific polypeptide, antigen or epitope or albumin fusion protein immobilized on its surface, respectively. For example.

Antibodies that bind to a therapeutic protein corresponding to the therapeutic protein of the albumin fusion protein can also be described or specified for their binding affinity for the protein or antigen, preferably the antigen to which they bind. Preferred binding affinities include those with a dissociation constant of 5 × 10 ⁻² M, 10 ⁻² M, 5 × 10 ⁻³ M, 10 ⁻³ M, 5 × 10 ⁻⁴ M, 10 ⁻⁴ M or less or Kd. A more preferred binding affinity is a dissociation constant of ⁵ × 10 ⁻⁵ M, 10 ⁻⁵ M, 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M, 10 ⁷ M, 5 × 10 ⁻⁸ M or 10 ⁻⁸ M or less Kd's are included. More preferred is the binding affinity ^{5X10 -9 M, 10 -9 M,} 5X10 -10 M, 10 -10 M, 5X10 -11 M, 10 -11 M, 5X10 -12 M, 10- 12 M, 5X10 -13 M, 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5 × 10 ⁻¹⁵ M or 10 ⁻¹⁵ M or less of dissociation constant or Kd are included. In a preferred embodiment, taking into account the valency of the antibody that binds to the therapeutic protein of the albumin fusion protein (including at least one fragment or variant of the antibody that binds to the therapeutic protein) and the valency of the corresponding antibody An albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein, and corresponding antibody (not fused to albumin) that binds to the therapeutic protein against the protein or epitope Has the same affinity as In addition, assays described herein (see Examples and Table 1) or known in the art are routinely applied to albumin fusion proteins, and fragments, variants and derivatives thereof, The ability to induce biological activity and / or therapeutic activity (either in vitro or in vivo) associated with either the therapeutic protein portion and / or the albumin portion of the albumin fusion protein may be measured. Other methods are known to those skilled in the art and are within the scope of the present invention.

albumin

  As described above, albumin fusion proteins of the present invention include at least one fragment or variant of a therapeutic protein and at least one fragment or variant of human serum albumin, which are preferably genetically They are joined together by fusion.

  Further embodiments include at least one fragment or variant of a therapeutic protein and at least one fragment or variant of human serum albumin linked together by chemical conjugation.

  The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The phrases “albumin” and “serum albumin” more broadly refer to human serum albumin (and fragments and variants thereof), as well as albumin (and fragments and variants thereof) from other species.

  As used herein, “albumin” collectively refers to albumin protein or amino acid sequence, or an albumin fragment or variant having one or more functional activities (eg, biological activity) of albumin. Means. In particular, “albumin” refers to human albumin or fragments thereof (see, eg, European Patent No. 201 239, European Patent No. 322 094, International Patent No. WO 97/24445, International Patent No. WO 95/23857), In particular, it means the mature form of human albumin as shown in FIG. 1 and SEQ ID NO: 1, or albumin from other vertebrates or fragments thereof, analogs or variants of these molecules, or fragments thereof.

  In a preferred embodiment, the human serum albumin protein used in the albumin fusion protein of the invention comprises one or both of the following sets of point mutations with respect to SEQ ID NO: 1. Leu-407 to Ala, Leu-408 to Val, Val-409 to Ala, and Arg-410 to Ala; or Arg-410 to A, Lys-413 to Gln, and Lys-414 to Gln (eg, as used herein) (See International Patent Specification No. WO 95/23857, which is incorporated by reference in its entirety). In a more preferred embodiment, an albumin fusion protein of the invention comprising one or both of the above point mutation sets has improved stability / resistance to yeast Yap3p proteolytic cleavage and is a recombinant expressed in a yeast host cell. Production of albumin fusion protein is increased.

  As used herein, a portion of albumin sufficient to extend the therapeutic activity of a therapeutic protein, or plasma stability or life span, is the life span or plasma stability of the therapeutic protein portion of an albumin fusion protein. Is sufficient to stabilize or prolong the therapeutic activity or plasma stability of a protein so that it is prolonged or extended compared to lifetime or plasma stability in an unfused state Means the portion of albumin of length or structure. The albumin portion of the albumin fusion protein may include the full length of the HA sequence as described above, or may include one or more fragments thereof that are capable of stabilizing or extending therapeutic activity. Such a fragment may be 10 or more amino acids in length, or may comprise about 15, 20, 25, 30, 50 or more contiguous amino acids from the HA sequence, or a HA specific May include some or all of the domains. For example, one or more fragments of HA that span the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HA fragment is the mature form of HA.

  The albumin portion of the albumin fusion protein of the present invention may be a mutant of normal HA. The therapeutic protein portion of an albumin fusion protein of the invention may also be a variant of a therapeutic protein as described herein. The phrase “variant” includes such changes that exert one or more of albumin's swelling, useful ligand-binding and non-immunological properties, or active site, or therapeutic activity of a therapeutic protein. Including conservative or non-conservative insertions, deletions and substitutions that do not substantially alter the active domain of

  In particular, the albumin fusion proteins of the present invention include naturally occurring polymorphic variants of human albumin, and fragments of human albumin, such as those disclosed in European Patent No. 322094 (ie HA (Pn), where n 369-419). Albumin may be derived from vertebrates, especially any mammal, such as humans, cows, sheep or pigs. Non-mammalian albumin includes, but is not limited to, hens and salmon. The albumin portion of the albumin fusion protein can be from a different animal than the therapeutic protein portion.

  Generally speaking, an HA fragment or variant is at least 100 amino acids long, preferably at least 150 amino acids long. HA variants may comprise at least one whole domain of HA, such as domain 1 (amino acids 1-194 of SEQ ID NO: 1), domain 2 (amino acids 195-387 of SEQ ID NO: 1), domain 3 (SEQ ID NO: 1). Amino acids 388-585), domains 1 and 2 (1 to 387 of SEQ ID NO: 1), domains 2 and 3 (195 to 585 of SEQ ID NO: 1) or domains 1 and 3 (amino acids 1 to 194 of SEQ ID NO: 1) And amino acids 388-585) of SEQ ID NO: 1. Each domain is itself a flexible intradomain linker region containing residues Lys106-Glu119, Glu292-Va1315 and Glu492-Ala511, two homologous subdomains, 1-105, 120-194, 195-291. 316 to 387, 388 to 491, and 512 to 585 are configured.

Preferably, the albumin portion of the albumin fusion protein of the invention includes at least one HA subdomain or domain, or conservative modifications thereof. Where the fusion is based on subdomains, some or all of the adjacent linkers are preferably used to link to a therapeutic protein moiety.

An antibody that specifically binds to a therapeutic protein is also a therapeutic protein.

The invention also includes albumin fusion proteins comprising at least one fragment or variant of an antibody that specifically binds to a therapeutic protein disclosed in Table 1. In particular, the phrase “therapeutic protein” is intended to include antibodies that bind to the therapeutic protein (eg, as described in column 1 of Table 1), or fragments and variants thereof. Thus, an albumin fusion protein of the invention may include at least one fragment or variant of a therapeutic protein and / or at least one fragment or variant of an antibody bound to the therapeutic protein.

Antibody structure and background

  The basic antibody structural unit is known to contain a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair consisting of one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino terminal portion of each chain contains a variable region of about 100-110 or more amino acids and is primarily involved in antigen recognition. The carboxy-terminal portion of each chain defines a constant region and is primarily responsible for effector functions. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Generally, Fundamental Immunology Chapters 3-5 (Paul, W., ed., 4th ed. Raven Press, NY (1998)), all of which are incorporated by reference for all purposes. checking ... The variable region of each light / heavy chain pair forms the antibody binding site.

  Thus, an intact IgG antibody has two binding sites. Except for bivalent or bispecific antibodies, the two binding sites are identical.

  All strands exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. In general, a CDR region is a portion of an antibody that contacts an antigen and determines its specificity. The CDRs from the heavy and light chains from each pair are aligned by a framework region and can bind to a specific epitope. From the N-terminus to the C-terminus, both the light and heavy chain variable regions contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable region is linked to the heavy or light chain constant region. The amino acid assignments for each domain were as follows: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & L. Biol. 196: 901-917 (1987), Chothia et al. Nature 342: 878-883 (1989). According to the definition.

As used herein, an “antibody” is an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, ie, a molecule that contains an antigen binding site that specifically binds an antigen (eg, one Or a molecule containing the CDRs of more antibodies). Antibodies that can represent the therapeutic protein portion of an albumin fusion protein include, but are not limited to, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies (eg, single chain Fvs), Fab fragments, F (ab ′) fragments, fragments produced by a Fab expression library, anti-idiotype (anti-Id) antibodies (eg, including anti-Id antibodies specific for the antibodies of the invention), and any of the above Epeptop binding fragments (eg, VH domain, VL domain, or one or more CDR regions) are included.

Antibodies that bind to therapeutic proteins

  The invention includes albumin fusion proteins comprising at least one fragment or variant of an antibody that binds to a therapeutic protein or fragment or variant thereof (eg as disclosed in Table 1).

  The antibody that binds to the therapeutic protein (or fragment or variant thereof) can be from any animal, including birds and mammals. Preferably, the antibody is a human, murine (eg, mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken antibody. Most preferably, the antibody is a human antibody. As used herein, “human” antibodies include antibodies having the amino acid sequence of human immunoglobulins, and are genetically engineered to produce human immunoglobulin libraries and human antibodies. Antibodies isolated from the isolated xenomouse or other organisms.

  Antibody molecules that bind to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein of the invention can be any type of immunoglobulin (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In a preferred embodiment, the antibody molecule that binds to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein is IgG1. In another preferred embodiment, the immunoglobulin molecule that binds to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein is IgG2. In another preferred embodiment, the immunoglobulin molecule that binds to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein is IgG4.

  Most preferably, the antibody molecule that binds to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein is a human antigen-binding antibody fragment of the invention, including but not limited to Fab, Fab ′ and F ( ab ′) 2, Fd, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv) and fragments containing either VL or VH domains. Antigen-binding antibody fragments, including single chain antibodies, may contain only the variable region or a combination with all or part of the following hinge region, CH1, CH2 and CH3 domains.

  An antibody that binds to a therapeutic protein and may represent the therapeutic protein portion of an albumin fusion protein may be monospecific, bispecific, trispecific, or more multispecific. Multispecific antibodies can be specific for different epitopes of a therapeutic protein or specific for both a therapeutic protein and a heterologous epitope, such as a heterologous polypeptide or solid support material. Good. For example, PCT specifications WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt, et al. , J. et al. Immunol. 147: 60-69 (1991), US Pat. No. 4,474,893, US Pat. No. 4,714,681, US Pat. No. 4,925,648, US Pat. No. 5,573,920, US Patent No. 5,601,819, Kostelny et al. , J. et al. Immunol. 148: 1547-1553 (1992).

  An antibody that binds to a therapeutic protein (or fragment or variant thereof) is bispecific, meaning that the antibody is an artificial hybrid antibody with two different heavy / light chain pairs and two different binding sites May be sex or bifunctional. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or binding of Fab 'fragments. For example, Songsivirai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J Immunol. 148: 1547 1553 (1992). In addition, bispecific antibodies are described in “diabodies” (Holliger et al. “'Diabodies”: small divalent and bispecific antibody fragments ”PNAS AS US : 6444-6448 (1993)) or “Janusins” (Traunecker et al. “Bispecific single-stranded molecules (Janusins) target cytotoxic lymphocytes on HIV-infected cells (Bisspecific singles). chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cel ls) "EMBO J 10: 3655-3659 (1991) and Traunecker et al." Janusin: new molecular design for bispecific reagent (Ins. J51) Int. 1992).

  The present invention also provides albumin fusion proteins comprising fragments (including derivatives) or variants of antibodies described herein or known in the art and others. For example, standard techniques known to those skilled in the art, including site-directed mutagenesis and PCR-mediated mutagenesis, resulting in amino acid substitutions, are used to introduce mutations into the nucleotide sequence encoding the molecule of the invention. Is possible. Preferably, variants (including derivatives) have 50 or fewer amino acid substitutions, 40 or fewer amino acid substitutions, 30 or fewer, relative to a reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3 Amino acid substitution, 25 or less amino acid substitution, 20 or less amino acid substitution, 15 or less amino acid substitution, 10 or less amino acid substitution, 5 or less amino acid substitution, 4 or less amino acid substitution, 3 or less amino acid substitution, or 2 or less amino acid substitution Encodes an amino acid substitution. In preferred embodiments, the variant has conservative amino acid substitutions at one or more predicted non-essential amino acid residues.

  Antibodies that bind to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein are described or characterized with respect to the epitope (s) or portion (s) of the therapeutic protein that recognizes or specifically binds. It's okay. Antibodies that specifically bind to a therapeutic protein or specific epitope of a therapeutic protein can also be excluded. Thus, the present invention includes antibodies that specifically bind to therapeutic proteins, and the like can be excluded. In a preferred embodiment, an albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein binds to the same epitope as a non-fused fragment or variant of the antibody itself.

  An antibody that binds to a therapeutic protein and may represent the therapeutic protein portion of an albumin fusion protein may be described or characterized with respect to its cross-reactivity. Antibodies that do not bind to other analogs, orthologs, or homologs of therapeutic proteins are included. At least 95%, at least 90%, at least 85%, at least 80%, at least 75 (as calculated using methods known in the art and described herein) for therapeutic proteins Antibodies that bind to polypeptides having%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% sequence identity are also included in the invention. In a specific embodiment, an antibody that binds to a therapeutic protein and can correspond to the therapeutic protein portion of an albumin fusion protein is a murine, rat and / or rabbit homologue of human protein, and its corresponding epitope Cross-react with. 95% or less, 90% or less, 85% or less, 80% or less, 75% (as calculated using methods known in the art and described herein) for therapeutic proteins Hereinafter, antibodies that do not bind to polypeptides having sequence identity of 70% or less, 65% or less, 60% or less, 55% or less, and 50% or less are also included in the present invention. In certain embodiments, the cross-reactivity described above is any single specific antigenic and / or immunogenic polypeptide, or 2, 3, 4, 5 or more disclosed herein. Regarding the combination of specific antigenic and / or immunogenic polypeptides. In a preferred embodiment, an albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein has a similar or essentially identical crossover compared to a particular antibody itself fragment or variant. Has reactive properties.

Further included in the present invention is binding to a polypeptide encoded by a polynucleotide that hybridizes to a polynucleotide encoding a therapeutic protein under constrained hybridization conditions (as described herein). Antibody. Antibodies that bind to therapeutic proteins and can represent the therapeutic protein portion of an albumin fusion protein of the invention can also be described or characterized with respect to their binding affinity for the polypeptides of the invention. Preferred binding affinities include those having a dissociation constant or Kd of 5 × 10 ⁻² M, 10 ⁻² M, 5 × 10 ⁻³ M, 10 ⁻³ M, 5 × 10 ⁻⁴ M, 10 ⁻⁴ M or less. More preferred binding affinities are 5 × 10 ⁻⁵ M, 10 ⁻⁵ M, 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M, 10 ⁷ M, 5 × 10 ⁻⁸ M, or 10 ⁻⁸ M or less dissociation constant. Or the thing with Kd is included. More preferred binding affinities include 5 × 10 ⁻⁹ M, 10 ⁻⁹ M, 5 × 10 ⁻¹⁰ M, 10 ⁻¹⁰ M, 5 × 10 ⁻¹¹ M, 10 ⁻¹¹ M, 5 × 10 ⁻¹² M, 10 ⁻¹² M, 5 × 10 ⁻ Those having a dissociation constant or Kd of ¹³ M, 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5 × 10 ⁻¹⁵ M, or 10 ⁻¹⁵ M or less are included. In a preferred embodiment, the albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein comprises an albumin fusion protein (including at least one fragment or variant of an antibody that binds to the therapeutic protein). Taking into account the valency and the valency of the corresponding antibody, it has an affinity for the protein or epitope similar to that of the corresponding antibody (not fused to albumin) that binds to the therapeutic protein.

  The invention also competes for binding of an antibody to an epitope of a therapeutic protein, as determined by any method known in the art for determining competitive binding, such as the immunoassay described herein. An antibody that inhibits the activity is provided. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%. To do. In a preferred embodiment, an albumin fusion protein comprising at least one fragment or variant that binds to a therapeutic protein competitively inhibits binding of the second antibody to an epitope of the therapeutic protein. In other preferred embodiments, the albumin fusion protein comprising at least one fragment or variant that binds to a therapeutic protein is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% Competitively inhibits binding of the second antibody to an epitope of the therapeutic protein by at least 60%, or at least 50%.

  An antibody that binds to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein of the invention can act as an agonist or antagonist of the therapeutic protein. For example, the invention includes antibodies that disrupt receptor / ligand interactions with the polypeptides of the invention, either partially or completely. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies that do not prevent ligand binding but prevent receptor activation. Receptor activation (ie, signal transduction) may be determined by techniques described herein or known in the art. For example, receptor activation can be determined by detecting phosphorylation (eg, tyrosine or serine / threonine) of the receptor or its substrate by immunoprecipitation (eg, as described above) followed by Western blot analysis. . In certain embodiments, up to at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% at least 60%, or at least 50% in the absence of antibodies, ligand activity or receptor activity An antibody that inhibits is provided. In a preferred embodiment, an albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein prevents ligand binding compared to an unfused fragment or variant of an antibody that binds to the therapeutic protein. And / or have similar or essentially similar features with respect to preventing receptor activation.

  The invention also features receptor-specific antibodies that prevent both ligand binding and receptor activation, and antibodies that recognize receptor-ligand complexes and preferably do not specifically recognize unbound receptors or unbound ligands. To do. Similarly, neutralizing antibodies that bind to the ligand and prevent binding of the ligand to the receptor, as well as antibodies that bind to the ligand and thereby prevent receptor activation but do not prevent the ligand from binding to the receptor, are also disclosed herein. include. Antibodies that activate the receptor are further included in the present invention. These antibodies can act as receptor agonists, i.e., can promote or activate all or a subset of the biological activity of ligand-mediated receptor activation, e.g., by inducing receptor dimerization. An antibody may be characterized as an agonist, antagonist or inverse agonist for a biological activity, including a specific biological activity of a therapeutic protein (eg, as disclosed in Table 1). The above antibody agonist can be prepared using a method known in the art. See, for example, PCT specification No. WO 96/40281, US Pat. No. 5,811,097, Deng et al., All of which are hereby incorporated by reference. , Blood 92 (6): 1981-1988 (1998), Chen et al. , Cancer Res. 58 (16): 3668-3678 (1998), Harrop et al. , J. et al. Immunol. 161 (4): 1786-1794 (1998), Zhu et al. , Cancer Res. 58 (15): 3209-3214 (1998), Yoon et al. , J. et al. Immunol. 160 (7): 3170-3179 (1998), Prat et al. , J. et al. Cell. Sci. 111 (Pt2): 237-247 (1998), Pittard et al. , J. et al. Immunol. Methods 205 (2): 177-190 (1997), Liautard et al. Cytokine 9 (4): 233-241 (1997), Carlson et al. , J. et al. Biol. Chem. 272 (17): 11295-11301 (1997), Taryman et al. , Neuron 14 (4): 755-762 (1995), Muller et al. , Structure 6 (9): 1153-1167 (1998), Bartunek et al. , Cytokine 8 (1): 14-20 (1996). In a preferred embodiment, an albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein is similar or essentially identical to an unfused fragment or variant of the antibody that binds to the therapeutic protein. Have agonist or antagonist properties.

  Antibodies that bind to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein of the invention, eg, purify, detect and target therapeutic proteins, including both in vitro and in vivo diagnostic and therapeutic methods May be used to make For example, the antibodies are useful in immunoassays for quantitatively and qualitatively measuring the level of therapeutic proteins in a biological sample. For example, Harlow et al., Which is incorporated by reference herein in its entirety. , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Similarly, albumin fusion proteins comprising at least one fragment or variant of an antibody that binds to a therapeutic protein, eg, purifying, detecting and targeting therapeutic proteins, including both in vitro and in vivo diagnostic and therapeutic methods May be used to make

Antibodies that bind to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein include derivatives that are modified by covalently attaching any type of molecule to the antibody. For example, without limitation, antibody derivatives include, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, cellular ligands or other proteins Antibodies modified by binding or the like are included. Any number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative can include one or more non-classical amino acids. The albumin fusion protein of the invention may also be modified as described above.

Methods of producing antibodies that bind to therapeutic proteins

  Antibodies that bind to a therapeutic protein and may represent the therapeutic protein portion of an albumin fusion protein of the invention may be produced by any suitable method known in the art. Polyclonal antibodies against the antigen of interest can be produced by various procedures well known in the art. For example, therapeutic proteins may be administered to a variety of host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants are used to increase the immunological response, depending on the host species, including but not limited to floyd (complete or incomplete), mineral gels such as aluminum hydroxide, lysolecithin, etc. Potentially useful human adjuvants such as surfactant substrates, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and BCG (Bacilli Calmette Guerin) and Corynebacterium parvum are included. Such adjuvants are also well known in the art.

  Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant and phage display techniques, or combinations thereof. For example, monoclonal antibodies are known in the art, see, eg, Harlow et al. , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Hammerling, et al. , In: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981). Are incorporated by reference). The phrase “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The phrase “monoclonal antibody” means an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not a method of production.

  Methods for production and selection for specific antibodies using hybridomas are routine and well known in the art. In a non-limiting example, a mouse is immunized with a therapeutic protein or fragment or variant thereof, an albumin fusion protein, or a cell expressing such therapeutic protein or fragment or variant thereof, or an albumin fusion protein. sell. Once an immune response is detected, i.e., an antibody specific for the antigen is detected in the mouse serum, the mouse spleen is harvested and the spleen cells are isolated. The spleen cells are then fused by well known techniques with any known myeloma cells, such as cells from cell line SP20 available from ATCC. Hybridomas are selected and cloned by limiting dilution. Hybridoma clones are then assayed by methods known in the art for cells secreting antibodies capable of binding to the polypeptides of the invention. Ascites fluid, which generally contains high levels of antibodies, can be produced by immunizing mice with positive hybridoma clones.

  Accordingly, the present invention provides an antibody produced by a method of producing a monoclonal antibody, as well as a method comprising culturing hybridoma cells secreting the antibody, wherein preferably the hybridoma is an antigen of the present invention. Produced by fusing spleen cells isolated from immunized mice with myeloma cells and then selecting the hybridomas obtained from the fusion for hybridoma clones that secrete antibodies capable of binding to the polypeptides of the invention. .

  Another well-known method for producing both polyclonal and monoclonal human B cell lines is transduction with Epstein-Barr virus (EBV). Protocols for generating EBV-transduced B cell lines are described in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., For example, which is incorporated herein by reference in its entirety. Eds. , 1994, John Wiley & Sons, NY, as is generally known in the art. The source of B cells for transduction is generally human peripheral blood, but B cells for transduction are also not limited to lymph nodes, tonsils, spleen, tumor cells and infected tissues. May come from other sources, including Tissues are generally made into a single cell suspension prior to EBV transduction. Furthermore, since T cells from individual seropositive individuals against anti-EBV antibodies can suppress B cell immortalization by EBV, T cells (eg, by treatment with cyclosporin A) in a sample containing B cells Either a physical removal or inactivation step may be involved.

  Generally, a sample containing human B cells is inoculated into EBV and cultured for 3-4 weeks. A typical source of EBV is the cell supernatant of the B95-8 cell line (ATCC # VR-1492). Physical signs of EBV transduction are generally seen towards the end of the 3-4 week culture period. By phase contrast microscopy, transduced cells tend to be large and clearly covered with hair and aggregate within tight clusters of cells. Initially, EBV strains are generally polyclonal. However, during long-term cell culture, EBV strains can become monoclonal or polyclonal as a result of selective growth of specific B cell clones. Alternatively, polyclonal EBV transduced strains are subcloned (eg, by limiting dilution medium) or fused with a suitable fusion partner and plated at limiting dilution to obtain a monoclonal B cell line. Suitable fusion partners for EBV transduced cell lines include mouse myeloma cell lines (eg SP2 / 0, X63-Ag8.653), heteromyeloma cell lines (human x mouse, eg SPAM-8, SBC-H20). And CB-F7) and human cell lines (eg GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the present invention also provides a method for producing polyclonal or monoclonal human antibodies against a polypeptide of the present invention or a fragment thereof comprising EBV-transduction of human B cells.

  Antibody fragments that recognize specific epitopes may be produced by known techniques. For example, the Fab and F (ab ′) 2 fragments of the present invention can be immunized using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ′) 2 fragments). It may be produced by proteolytic cleavage of a globulin molecule. The F (ab ') 2 fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain.

  For example, antibodies that bind to therapeutic proteins can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that contain the polynucleotide sequences encoding them. In certain embodiments, such phage can be used to display an antigen binding domain expressed from a repertoire or combinatorial antibody library (eg, a human or murine). Phage expressing an antigen binding domain that binds to the antigen of interest can be selected or identified by antigen, for example, using labeled antigen or antigen bound or captured on a solid surface or bead. Phage used in these methods are typically fd and M13 binding expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either phage gene III or gene VIII protein. A filamentous phage containing a domain. Examples of phage display methods that can be used to generate antibodies that bind to therapeutic proteins are described by Brinkman et al., Each of which is incorporated herein by reference. , J. et al. Immunol. Methods 182: 41-50 (1995), Ames et al. , J. et al. Immunol. Methods 184: 177-186 (1995), Kettleborough et al. , Eur. J. et al. Immunol. 24: 952-958 (1994), Persic et al. Gene 187 9-18 (1997), Burton et al. , Advances in Immunology 57: 191-280 (1994), PCT Specification No. PCT / GB91 / 01134, PCT Specification No. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / No. 18619, WO 93/11236, WO 95/15982, WO 95/20401, and US Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5 , 580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516 , 637, 5,780,225, 5,658,727, 5,733,743 and 5, They include those disclosed in JP 69,108.

  As described in the above references, after phage selection, the antibody coding region from the phage is isolated and used to produce whole antibodies, including human antibodies, or other desired antigen-binding fragments, eg It can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, as described in detail. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments are also described in PCT Specification No. WO 92/22324, Mulinax et al. BioTechniques 12 (6): 864-869 (1992), and Sawai et al. , AJRI 34: 26-34 (1995), and Better et al. , Science 240: 1041-1043 (1988), which are available using methods known in the art (all of which are incorporated by reference). .

  Examples of techniques that can be used to produce single chain Fvs and antibodies include US Pat. Nos. 4,946,778 and 5,258,498, Huston et al. , Methods in Enzymology 203: 46-88 (1991), Shu et al. , PNAS 90: 7995-7999 (1993), and Skerra et al. Science 240: 1038-1040 (1988). For some uses, including in vivo use of antibodies in humans, and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. For example, Morrison, Science 229: 1202 (1985), Oi et al., All of which are hereby incorporated by reference. , BioTechniques 4: 214 (1986), Gillies et al. (1989) J. Am. Immunol. See Methods 125: 191-202, US Pat. Nos. 5,807,715, 4,816,567, and 4,816,397. A humanized antibody is an antibody from a non-human species antibody that binds to the desired antigen with one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. Is a molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example by modeling CDR and framework interactions, to identify unusual framework residues at a particular site In order to identify important framework residues for antigen binding and sequence comparison (eg, all incorporated herein by reference), Queen et al. U.S. Pat. No. 5,585,089, Riechmann et al. , Nature 332: 323 (1988)). Antibodies can be produced, for example, by CDR-conjugation (European Patent No. 239,400 PCT Specification No. WO 91/09967, US Pat. Nos. 5,225,539, 5,530,101, and 5,585). 089), beneling or reserfacing (European Patent No. 592,106, European Patent No. 519,596, Padlan, Molecular Immunology 28 (4/5): 489-498 (1991), Studnikka et al., Protein. Engineering 7 (6): 805-814 (1994), Roguska. Et al., PNAS 91: 969-973 (1994)), and chain shuffling (US Pat. No. 5,565,332). Various techniques known in And can be humanized.

  Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be produced by various methods known in the art, including the phage display method described above, using antibody libraries derived from human immunoglobulin sequences. US Pat. Nos. 4,444,887 and 4,716,111, and PCT specifications WO 98/46645, WO 98, each of which is incorporated herein by reference. No./50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

  Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes may be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, human variable regions, constant regions, and diverse regions may be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous defects in the JH region prevent endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized with a selected antigen, eg, all or a portion of a polypeptide of the invention, in the usual manner. Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes are grown by transgenic mouse rearrangement during B cell differentiation, followed by class switching and somatic mutation. Thus, using such techniques, it is possible to produce theoretically useful IgG, IgA, IgM and IgE antibodies. For a review of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995). For a detailed discussion of the technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, for example, PCT, all of which is incorporated herein by reference. Descriptions WO 98/24893, WO 92/01047, WO 96/34096, WO 96/33735, EP 0 598 877, US Pat. No. 5,413,923, 5 , 625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885 793, No. 5,916,771, No. 5,939,598, No. 6,075,181, and No. 6,114,598. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genfarm (Genpharm, San Jose, CA) were selected using techniques similar to those described above. It is possible to engage in providing human antibodies directed against antigens.

Fully human antibodies that recognize selected epitopes can be generated using techniques cited as “guide items”. In this approach, selected non-human monoclonal antibodies such as mouse antibodies are used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al., Bio / technology 12: 899-903). (1988)).

Polynucleotide encoding antibody

  The present invention further provides a polynucleotide comprising a nucleotide sequence encoding the antibody or fragment thereof. The present invention also provides antibodies that specifically bind to a therapeutic protein, preferably more preferably in Table 2, under tight or less tight hybridization conditions (such as those defined above). A polynucleotide that hybridizes to a polynucleotide that encodes an antibody that binds to a polypeptide having the amino acid sequence of “therapeutic protein X” disclosed in the column “SEQ ID NO: Z”.

  The polynucleotide is obtained by any method known in the art and the nucleotide sequence of the polynucleotide is determined. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody was chemically synthesized (eg, as described in Kutmeier et al., BioTechniques 17: 242 (1994)). Can be assembled from oligonucleotides, simply synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR. included.

  Alternatively, a polynucleotide encoding the antibody may be produced from the nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin is chemically synthesized or the sequences 3 'and 5' 'Use oligonucleotide probes specific for specific gene sequences by PCR amplification with synthetic primers that can hybridize to the ends or to identify cDNA clones from, for example, cDNA libraries encoding antibodies By cloning, a suitable source (e.g., an antibody cDNA library, or a cDNA library produced from any tissue or cell that expresses the antibody, such as a hybridoma cell selected to express the antibody, Or nucleic acids isolated from those tissues or cells, preferably Ku may be obtained from the poly A + RNA). Amplified nucleic acids produced by PCR may then be cloned into replicable cloning vectors using any method well known in the art (see Example 65).

  Once the nucleotide sequence of the antibody and the corresponding amino acid sequence have been determined, the antibody nucleotide sequence can be used, for example, to produce antibodies with different amino acid sequences, eg, to create amino acid substitutions, deletions and / or insertions For manipulation of nucleotide sequences, such as recombinant DNA technology, site-directed mutagenesis, PCR, etc., one may operate using methods well known in the art (eg, both Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, which is incorporated herein by reference. NY and Ausubel et al., Eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

  In certain embodiments, the amino acid sequences of the heavy and / or light chain variable domains are compared with known amino acid sequences of other heavy and light chain variable regions, for example, to determine regions of sequence hypervariability. Thus, it may be examined to identify the sequence of the complementarity determining region (CDR) by methods well known in the art. Using routine recombinant DNA technology, one or more CDRs may be inserted within the framework region, eg, as described above, into the human framework region to humanize non-human antibodies. Good. The framework region may be a naturally occurring or consensus framework region, preferably a human framework region (eg, for the listing of human framework regions, Chothia et al., J. Mol. Biol 278: 457-479 (1998)). Preferably, the polynucleotide produced by the combination of framework regions and CDRs encodes an antibody that specifically binds to a polypeptide of the invention. Preferably, as described above, one or more amino acid substitutions are made in the frame region, preferably the amino acid substitution improves the binding of the antibody to its antigen. In addition, such methods can be used to generate amino acid substitutions of one or more variable region cysteines that are involved in intrachain disulfide bonds to produce antibody molecules that lack one or more intrachain disulfide bonds. Or it may be used to create defects. Other modifications to the polynucleotide are encompassed by the present invention and within the skill of the art.

  In addition, a technique developed specifically for the production of “chimeric antibodies” by splicing genes from mouse antibody molecules of appropriate antigen specificity, together with genes from human antibody molecules of appropriate biological activity (Morrison et al., Proc. Natl. Acad. Sci. 81: 851-855 (1984), Neuberger et al., Nature 312: 604-608 (1984), Takeda et al., Nature 314: 452-4. 1985)) can be used. As described above, chimeric antibodies have different portions derived from molecules of different animal species, ie, variable regions derived from murine mAbs and human immunoglobulin constant regions, eg, humanized antibodies. It is.

Alternatively, techniques described for the production of single chain antibodies (US Pat. No. 4,946,778, Bird, Science 242: 423-42 (1988), Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988), and Ward et al., Nature 334: 544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for assembling functional Fv fragments in E. coli may also be used (Skerra et al., Science 242: 1038-1041 (1988)).

Recombinant expression of antibodies

  Recombinant expression of an antibody, or a fragment, derivative or analog thereof (eg, the heavy chain or light chain of an antibody or single chain antibody) requires the structure of an expression vector containing the polypeptide encoding the antibody. Once the polypeptide encoding the heavy chain or light chain of an antibody molecule of the invention, or an antibody or fragment thereof (preferably comprising a heavy chain or light chain variable domain) is obtained, for the production of antibody molecules Vectors may be produced by recombinant DNA techniques using techniques well known in the art. Accordingly, methods for preparing proteins by expressing a polypeptide comprising an antibody encoding nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibodies encoding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. To do. Such vectors include nucleotide sequences encoding the constant regions of antibody molecules (eg, PCT specification WO 86/05807, PCT specification WO 89/01036, and US Pat. No. 5,122,464). Antibody variable domains may be cloned into such vectors for expression of full heavy or light chains.

  The expression vector is delivered to the host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibody. Accordingly, the present invention includes host cells comprising a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody, operably linked to a heterologous promoter. In a preferred embodiment for the expression of double chain antibodies, vectors encoding both heavy and light chains are co-expressed in host cells for expression of whole immunoglobulin molecules, as detailed below. May be.

  A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such a host-expression system represents an excipient that can express the coding sequence of interest and is subsequently purified, but also when transduced or transfected with the appropriate nucleotide encoding sequence , Represents cells capable of expressing the antibody molecule of the present invention in situ. These include, but are not limited to, bacteria (eg, E. coli, B. subtilis) transduced with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences, antibody coding sequences Yeast transduced with a recombinant yeast expression vector containing (eg, Saccharomyces, Pichia), an insect cell line infected with a recombinant viral expression vector containing an antibody coding sequence (eg, baculovirus), recombinant virus expression Plant cells infected with a vector (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transduced with a recombinant plasmid expression vector (eg, Ti plasmid) containing an antibody coding sequence Or a mammalian cell line with a recombinant expression construct comprising a promoter derived from the genome of a mammalian cell (eg, a metallothionein promoter) or from a mammalian virus (eg, adenovirus late promoter, vaccine virus 7.5K promoter) Microorganisms such as COS, CHO, BHK, 293, 3T3 cells). Preferably, bacterial cells such as E. coli, more preferably eukaryotic cells, are used for the expression of recombinant antibody molecules, especially for the expression of whole recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO), together with vectors such as the major middle and early gene promoter elements from human cytomegalovirus, are an effective expression system for antibodies (Foecking et al. , Gene 45: 101 (1986), Cockett et al., Bio / Technology 8: 2 (1990)).

  In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, if large quantities of such proteins are to be produced, vectors that are directed to the expression of high level fusion protein products that are easily purified are desirable for the production of pharmacological compositions of antibody molecules. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al.), In which an antibody coding sequence can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein. , EMBO J. 2: 1791 (1983)), pIN vector (Inouye & Inouye, Nucleic Acids Res. 13: 3101-3109 (1985), Van Heke & Schuster, J. Biol. Chem. 24: 5503-50. )) Etc. are included. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites, and the cloned target gene product can be released from the GST site.

  In insect systems, Autographa calfornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences may be individually cloned into non-essential regions of the virus (eg, polyhedrosis genes) and under the control of an AcNPV promoter (eg, polyhedrosis promoter).

  A number of virus-based expression systems may be used in mammalian host cells. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription / translation control complex, eg, the late promoter and tripartite leader sequence. This chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region (eg, region E1 or E3) of the viral genome can result in a recombinant virus that is viable and capable of expressing antibody molecules in an infected host (eg, Logan & Shenk, Proc Natl.Acad.Sci.USA 81: 355-359 (1984)). A specific initiation signal may also be required for full translation of the inserted antibody coding sequence. These signals include the ATG start codon and adjacent sequences. Furthermore, the start codon must match the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins and can be both natural and synthetic. The efficiency of expression may be enhanced by insertion of suitable transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153: 51-544 (1987)).

  In addition, a host cell line may be selected which modulates the expression of the inserted expression or modifies and processes the gene product in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important with respect to protein function. Different host cells have features and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To achieve this goal, eukaryotic host cells with cellular machinery for the proper processing of the initial transcript, glycosylation and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, such as BT483, Hs578T, HTB2, BT20, and T47D, among others. Breast cancer cell lines, and normal breast cells such as CRL7030 and Hs578Bst.

  For long-term, high yield production of recombinant protein, stable expression is preferred. For example, cell lines that stably express antibody molecules may be designed. Rather than using an expression vector containing a viral origin of replication, transfect host cells with appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and DNA controlled by selectable markers Is possible. Following the introduction of foreign DNA, the engineered cells are grown in rich media for 1-2 hours and then changed to selective media. A selectable marker in the recombinant plasmid confers resistance to selection and allows the cell to integrate the plasmid into its chromosome, proliferate to form a nest, and then clone into the cell line. It is possible to increase. This method may be advantageously used to design cell lines that express antibody molecules. Such engineered cell lines may be particularly useful for screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

  Without limitation, herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine morphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. (1992)), and multiple selection systems may be used, including the adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817 (1980)) gene, using tk-, hgprt- or aprt-cells, respectively. To do. In addition, antimetabolite resistance can be used as a basis for selection for the following genes. Dhfr (Wigler et al., Natl. Acad. Sci. USA 77: 357 (1980), O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1981), which confers resistance to methotrexate. Gpt conferring resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (1981)), neo conferring resistance to aminoglycoside G-418 (Clinical Pharmacy 12: 488-505, Wu and Wu, Biotherapy 3: 87-95 (1991), Tolstoshev, Ann. Rev. Pharmacol. 32: 573-596 (1993), Mulligan, Science 260: 926-932 (1993), and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993), May, 1993, TIB TECH11. (5): 155-215 (1993)), hygro (Santare et al., Gene 30: 147 (1984)), which confer resistance to hygromycin. Methods generally known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clones, such as all of which are hereby incorporated by reference. As described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), Kriegler, Gene Transfer and Expression, A Laboratory, A Laboratory. (Eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Colberre-Garapin et al. , J. et al. Mol. Biol. 150: 1 (1981), chapters 12 and 13.

  The level of expression of antibody molecules can be increased by vector amplification (for review, Bebington and Hentschell, “Gene amplification based vectors for expression of cloned genes in mammalian cells in DNA cloning (See The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning), Vol. 3. (Academic. If the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the culture of the host cell will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production is also increased (Crouse et al., Mol. Cell. Biol. 3: 257 (1983)).

  Vectors using glutamine synthase (GS) or DHFR as selectable markers can be amplified in the presence of the drugs methionine sulfoxime or methotrexate, respectively. The advantage of a glutamine synthase-based vector is the availability of a cell line that is negative for glutamine synthase (eg, a murine myeloma cell line, NS0). The glutamine synthase expression system can also function in glutamine synthase expressing cells (eg, Chinese hamster oocytes (CHO) cells) by providing additional inhibitors that prevent foreign gene function. The glutamine synthase expression system and its compounds are described in PCT specifications WO 87/04462, WO 86/05807, WO 89/01036, WO 89/10404, and WO 91/06657, all of which are hereby incorporated by reference. It has been detailed. In addition, glutamine synthase expression vectors that can be used in accordance with the present invention are commercially available from suppliers such as, for example, Lonza Biologics, Inc. (Portsmouth, NH). The expression and production of monoclonal antibodies using the GS expression system in murine myeloma cells is described in Bebbington et al., Which is incorporated herein by reference in its entirety. , Bio / technology 10: 169 (1992) and Biblia and Robinson Biotechnology. Prog. 11: 1 (1995).

  A host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers that allow equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used that encodes and is capable of expressing heavy and light chain polypeptides. In such situations, the light chain is placed in front of the heavy chain to avoid excess toxic free heavy chain (Proudfoot, Nature 322: 52 (1986), Kohler, Proc. Natl. Acad. Sci. USA 77: 2197 (1980)). The heavy and light chain coding sequences may comprise cDNA or genomic DNA.

Once the antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, any method known in the art can be used to purify immunoglobulin molecules, for example, By chromatography (eg by ion exchange, affinity, especially affinity for a specific antigen after protein A, and by size column chromatography), by centrifugation, separation solubility or by any other standard technique for protein purification, It may be purified. In addition, antibodies that bind to therapeutic proteins and that may correspond to therapeutic protein portions of the albumin fusion proteins of the invention, and fragments thereof, are described herein to facilitate purification or the present technology. It can be fused to a heterologous polypeptide sequence known in the art.

Antibody modification

  An antibody that binds to a therapeutic protein or fragment or variant thereof can be fused to a marker sequence, such as a peptide, to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. It is. For example, Gentz et al. , Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), hexa-histidine provides convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, hemagglutinin tags (also referred to as “HA tag”) corresponding to an eptope derived from influenza hemagglutinin protein (Wilson et al., Cell 37: 767 (1984)). And "flag" tags.

  The invention further includes an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically, for example, as part of a clinical trial procedure, to monitor tumor development or progression, eg, to determine the effects of the treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substrate. Examples of detectable substrates include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions Is included. The detectable substrate can be directly attached to the antibody (or fragment thereof) or indirectly via an intermediate (eg, a linker known in the art) using techniques known in the art. Either may be bound or conjugated. See, eg, US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics in accordance with the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin, suitable Examples of fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, examples of luminescent materials include luminol, bioluminescent materials Examples of these include luciferase, luciferin and aequorin, and examples of suitable radioactive materials include 125I, 131I, 111In or 99Tc. Other examples of detectable substrates are described elsewhere herein.

  Furthermore, the antibodies of the present invention may be conjugated to therapeutic sites such as cytotoxins such as cytostatic or cytocidal agents, therapeutic agents or radioactive metal ions such as alpha-radiants such as 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, methine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, corcitine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxatron, misromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin and its derivatives or homologues are included. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechloretamine, thioepachlorambucil, melphalan, carmustine) (BSNU) and lomustine (CCNU), cyclosofamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and Doxorubicin), antibiotics (eg dactinomycin (formerly actinomycin), bleomycin, misramycin and ansolamycin (AMC) And anti-mitotic agents (eg, vincristine and vinblastine) is included.

  The conjugates of the invention can be used to modify the biological response and the therapeutic agent or drug moiety is not to be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, Pseudomonas etotoxin, or diphtheria toxin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminol Gen activators, apoptotic drugs such as TNF-alpha, TNF-beta, AIMI (see International Patent Specification No. WO 97/33899), AIM II (see International Patent Specification No. WO 97/34911) ), Fas ligand (Takahashi et al., Int. Immunol., 6: 1567-1574 (1994)), VEGI (see International Patent Specification No. WO 99/23105), for example angiostatin or endos Thrombotic agents such as tachin, or anti-angiogenic agents or, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL- 6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony stimulating factor (" G-CSF ") or other growth factors or other growth factors.

  The antibody may also be bound to a solid support and is particularly useful for immunoassays or target antigen purification. Such solid supports include but are not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

  Techniques for coupling such therapeutic sites to antibodies are well known. For example, Arnon et al. , “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapeutics”, in Monoclonal Antibodies And Cancer Recipients. (Eds.), Pp. 243-56 (Alan R. Liss, Inc. 1985), Hellstrom et al. "Antibodies for Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (Eds.), Pp. 623-53 (Marcel Dekker, Inc. 1987), Thorpe, “Antibody Carriers of Cytotoxic Agents in Cytotoxic Agents in Biotechnology: A Review”, 84 Clinical Applications, Pinchera et al. (Eds.), Pp. 475-506 (1985), “Analysis, Results, The Future of the Therapeutic Use of Radioactive Antibodies in Cancer.” And Therapy, Baldwin et al. (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al. “Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates”, Immunol. Rev. 62: 119-58 (1982).

Alternatively, an antibody is conjugated to a second antibody to form an antibody heteroconjugate as described by Segai in US Pat. No. 4,676,980, all of which is incorporated herein by reference. Is possible.

An antibody administered alone or in combination with cytotoxic factor (s) and / or cytokine (s) can be used therapeutically, with or without a therapeutic moiety conjugated thereto.
Antibody-albumin fusion

  Antibodies that bind to a therapeutic protein and can represent the therapeutic protein portion of an albumin fusion protein of the invention include, but are not limited to, the therapeutic protein disclosed in the “Therapeutic Protein X” column of Table 1, or Antibodies that bind to the fragment or variant are included.

  In certain embodiments, antibody fragments or variants that immunospecifically bind to a therapeutic protein and correspond to a therapeutic protein portion of an albumin fusion protein comprise or consist of a VH domain. In other embodiments, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein comprises one, two, or three VH CDRs Or consist of them. In other embodiments, the antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to the therapeutic protein portion of an albumin fusion protein comprises or consists of VH CDR1. In other embodiments, the antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to the therapeutic protein portion of an albumin fusion protein comprises or consists of VH CDR2. In other embodiments, the antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to the therapeutic protein portion of an albumin fusion protein comprises or consists of VH CDR3.

  In certain embodiments, antibody fragments or variants that immunospecifically bind to a therapeutic protein and correspond to a therapeutic protein portion of an albumin fusion protein comprise or consist of VL. In other embodiments, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein comprises one, two, or three VL CDRs Or consist of them. In another embodiment, the antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to the therapeutic protein portion of an albumin fusion protein comprises or consists of VL CDR1. In other embodiments, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein comprises or consists of VL CDR2. In other embodiments, the antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to the therapeutic protein portion of an albumin fusion protein comprises or consists of VL CDR3.

  In other embodiments, the antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein is one, two, three, four, five, Contains or consists of one or six VH and / or VL CDRs.

In a preferred embodiment, an antibody fragment or variant that immunospecifically binds to a therapeutic protein and corresponds to a therapeutic protein portion of an albumin fusion protein is: (Gly ₄ Ser) ₃ (SEQ ID NO: 4) A scFv containing or consisting of the VH domain of the therapeutic protein linked to the VL domain of the therapeutic antibody by a simple peptide linker.

Immunological marker diagnosis

  An albumin fusion protein of the present invention comprising at least one fragment or variant of an antibody that binds to an antibody of the invention, or a therapeutic protein (or fragment or variant thereof), an immunology of cell lines and biological samples. May be used for diagnostic marker diagnosis. The therapeutic proteins of the present invention may be useful as cell-specific markers, more particularly as cell markers that are differentially expressed at various stages of differentiation and / or maturation of a particular cell type. Screening for a population of cells expressing a marker by a monoclonal antibody directed against a specific eptop, or combination of epitopes (or an albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein) Is possible. Using a monoclonal antibody (or an albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein) to screen various techniques for cellular means expressing the marker (s) Available and includes magnetic separation using antibody-coated magnetic beads, “panning” with antibodies bound to a solid matrix (ie, plate), and flow cytometry (see, eg, US Pat. No. 5,985,660 and Morrison et al., Cell, 96: 737-49 (1999)).

These techniques allow for hematological malignancies (ie, minimal residual disease (MRD) in patients with acute leukemia) and transplantation versus host disease (GVHD) as seen in “non-self” cells in transplantation. Screening of a specific population of cells is possible. Alternatively, these techniques allow the screening of hematopoietic stem and progenitor cells that can proliferate and / or differentiate as can be seen in human umbilical cord blood.

Characterization of antibodies that bind to therapeutic proteins and albumin fusion proteins, including fragments or variants of antibodies that bind to therapeutic proteins

  An albumin fusion protein comprising at least one fragment or variant of an antibody that binds to an antibody of the invention, or a therapeutic protein (or fragment or variant thereof) may be characterized in a variety of ways. In particular, albumin fusion proteins comprising at least one fragment or variant of an antibody that binds to a therapeutic protein can be fused with albumin fusion using the techniques described herein or routinely known in the art. An antibody that binds to a therapeutic protein corresponding to an antibody that binds to a therapeutic protein portion of the protein may be assayed for its ability to specifically bind to the same antigen specifically bound.

  The ability to (specifically) bind to a specific protein or epitope of an albumin fusion protein comprising at least one fragment or variant of an antibody that binds to an antibody of the invention, or a therapeutic protein (or fragment or variant thereof). Assays are performed in solution (eg, Houghten, Bio / Techniques 13: 412-421 (1992)), on beads (eg, Lam, Nature 354: 82-84 (1991)), on a chip (eg, Fodor, Nature 364: 555-556 (1993)), on bacteria (eg, US Pat. No. 5,223,409), on spores (eg, patents 5,571,698, 5,403,484, and 5,223). 409) on plasmids (eg Cul) USA et al., Proc. Natl. Acad. Sci. 406 (1990), Cwirla et al., Proc. Natl. Acad. Sci. (Each of these references is hereby incorporated by reference in its entirety). Albumin fusion proteins comprising at least one fragment or variant of an antibody that binds to an antibody of the invention, or a therapeutic protein (or fragment or variant thereof) are also known in the art or in the art described herein. Routinely modifying techniques may be used to assay for their specificity and affinity for specific proteins or eptops.

  An albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a therapeutic protein can be transferred to other antigens (eg, to the therapeutic protein portion of an albumin fusion protein of the invention) by any method known in the art. Corresponding molecules that are specifically bound by an antibody that binds to a therapeutic protein (or fragment or variant thereof) may be assayed for cross-reactivity with molecules with sequence / structure conservation.

  Immunoassays that can be used to analyze (immunospecific) binding and cross-reactivity include, but are not limited to, Western blot, radioimmunoassay, ELISA (enzyme immunoassay), to name just a few examples. Includes "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel nucleic acid precipitin reactions, immunonucleic acid assays, agglutination assays, complement immobilization assays, immunoradiometric assays, propensity immunoassays and protein A immunoassays. Such assays are routine and well known in the art (eg, Ausubel et al, eds, 1994, Current Protocols in, all of which are incorporated herein by reference). See Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). An exemplary immunoassay is briefly described below (but is not intended to be limiting).

  Immunoprecipitation protocols generally include RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate) containing protein phosphatases and / or protease inhibitors (eg EDTA, PMSF, aprotinin, sodium vanadate). Lysing a population of cells in a lysis buffer such as 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate mpH 7.2, 1% Trasylol), antibody of the invention Or an albumin fusion protein of the invention comprising at least one fragment or variant of an antibody that binds to a therapeutic protein (or fragment or variant thereof) is added to the cell lysate for a period of time (eg 1-4 Time) Incubate at 4 ° C, tongue Cell lysate with protein A and / or protein G sepharose beads (or beads coated with an appropriate anti-idiotype antibody or anti-albumin antibody if the albumin fusion protein contains at least one fragment or variant of a therapeutic protein) Incubating at 40 ° C. for about 1 hour, washing the beads in lysis buffer, and resuspending the beads in SDS / sample buffer. The ability of the antibodies or albumin fusion proteins of the present invention to immunoprecipitate a particular antigen can be assessed, for example, by Western blot. Those skilled in the art know that parameters can be modified to increase the binding of antibody or albumin fusion protein to antigen and reduce background (eg, pre-cleaning of cell lysate with Sepharose Bose). For further discussion on immunoprecipitation protocols, see, eg, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc. , New York at 10.16.1.

Western blot analysis generally involves the preparation of protein samples, electrophoresis of protein samples in polyacrylamide gels (eg, 8% -20% SDS-PAGE depending on the molecular weight of the antigen), polyacrylamide gels of protein samples Transfer to membranes such as nitrocellulose, PVDF or nylon, blocking membranes in blocking solution (eg PBS containing 3% BSA or non-fat milk), membranes in wash buffer (eg PBS-Tween 20) Washing the membrane, washing the membrane in the washing buffer, diluting in the buffer, applying the antibody of the invention or the albumin fusion protein (diluted in blocking buffer) to the membrane (Eg horseradish peroxidase or alkaline phosphatase) or radiation Applying a second antibody (eg recognizing an albumin fusion protein, eg anti-human serum albumin antibody) conjugated to an active molecule (eg ³² P or ¹²⁵ I), washing the membrane in wash buffer, and Detecting the presence. Those skilled in the art know that parameters can be modified to increase the detected signal and to reduce background noise. For further discussion on Western blot protocols, see, eg, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc. , New York at 10.8.1.

  ELISA prepares antigen, coats well of 96-well microtiter plate with antigen, wash away antigen that did not bind to well, enzymatic substrate (eg horseradish peroxidase or alkaline phosphatase) Adding an antibody of the invention conjugated to a detectable compound such as or an albumin fusion protein (including at least one fragment or variant of an antibody that binds to a therapeutic protein) to the well and incubating for a period of time; Washing away unbound or non-specifically bound albumin fusion protein and detecting the presence of antibody or albumin fusion protein specifically bound to the antigen coating the well. In an ELISA, the antibody or albumin fusion protein need not be conjugated to a detectable compound; instead, a second antibody conjugated to the detectable compound (recognizing the antibody or albumin fusion protein, respectively) may be added to the well. . Furthermore, instead of coating the well with the antigen, the antibody or albumin fusion protein may be coated to the well. In this case, the detectable molecule can be an antigen conjugated to a detectable compound such as an enzymatic substrate (eg, horseradish peroxidase or alkaline phosphatase). Those skilled in the art know that parameters can be modified to increase the detected signal, as well as various other ELISAs known in the art. For further discussion on ELISA, see Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc. , New York at 11.2.1.

The binding affinity of an albumin fusion protein to a protein, antigen or epitope, and the off-rate of the antibody- or albumin fusion protein-protein / antigen / epitope interaction can be detected by competitive binding assays. One example of a competitive binding assay is the incubation of labeled antigen (eg, ³ H or ¹²⁵ I) with an antibody or albumin fusion protein of the present invention in the presence of increasing amounts of unlabeled antigen, and labeling. Detection of antibodies bound to the conjugated antigen. The affinity of an antibody or albumin fusion protein of the invention for a particular protein, antigen or epitope, and binding off-rate can be determined from the data by Scatchard plot analysis. Competition with a second protein that binds to the same protein, antigen or epitope as an antibody or albumin fusion protein can also be detected using a radioimmunoassay. In this case, the protein, antigen or epitope is bound to the labeled compound ( ³ H or ¹²⁵ I) in the presence of an increasing amount of unlabeled second protein that binds to the same protein, antigen or epitope as the albumin fusion protein of the invention. Incubate with a conjugated antibody or albumin fusion protein of the invention.

In a preferred embodiment, a BIAcore kinetic assay is used to determine the binding on and off rates of an antibody or albumin fusion protein of the invention for a protein, antigen or epitope. BIAcore kinetic analysis includes antibodies, albumin fusion proteins, or specific polypeptides, antigens or antigens from a chip with a specific polypeptide, antigen or epitope, antibody or albumin fusion protein, respectively, immobilized on their surface. Analyzing epitope binding and dissociation is included.

Therapeutic use

  The present invention further includes an antibody of the invention, or at least one fragment or variant of an antibody that binds to a therapeutic protein, for treating one or more of the disclosed diseases, conditions or conditions. Directed to antibody-based therapy comprising administering to a subject, preferably a mammal, most preferably a human patient. Therapeutic compounds of the present invention include, but are not limited to, antibodies of the present invention (including fragments, analogs and derivatives as described herein), fragments thereof, similar as described herein, and the like. Bodies and derivatives, and nucleic acid encoding antibodies of the invention (including anti-idiotype antibodies), albumin fusion proteins of the invention comprising at least one fragment or variant of an antibody that binds to a therapeutic protein, and so on Nucleic acids encoding various albumin fusion proteins are included. An albumin fusion protein of the invention comprising at least one fragment or variant of an antibody of the invention, or an antibody that binds to a therapeutic protein, includes, but is not limited to any one or more of the albumin fusion proteins described herein. It can be used to treat, inhibit or prevent a disease, disorder or condition associated with aberrant expression and / or activity of a therapeutic protein, including a disease, disorder or condition. Treatment and / or prevention of diseases, illnesses or conditions associated with abnormal expression and / or activity of therapeutic proteins includes, but is not limited to, alleviating symptoms associated with those diseases, illnesses or conditions. It is. An albumin fusion protein of the invention comprising at least one fragment or variant of an antibody of the invention, or an antibody that binds to a therapeutic protein, as known in the art or described herein Can be provided in a pharmacologically acceptable composition.

  In certain and preferred embodiments, the invention includes, but is not limited to, neurological disease, immune system disease, muscle disease, regenerative disease, gastrointestinal disease, lung disease, cardiovascular disease, renal disease, proliferative disease, and Antibodies of the invention to treat one or more diseases, conditions or conditions, including cancerous diseases, conditions, and / or as described elsewhere herein Or an antibody, comprising administering an albumin fusion protein of the invention comprising at least one fragment or variant of an antibody that binds to a therapeutic protein to an animal, preferably a mammal, and most preferably a human patient. Oriented treatment based. Therapeutic compounds of the present invention include, but are not limited to, antibodies of the invention (eg, antibodies directed to full-length proteins expressed on the cell surface of mammalian cells, (fragments thereof as described herein) Including, but not limited to, antibodies directed to therapeutic protein and nucleic acid epitopes encoding the antibodies of the present invention (including analogs and derivatives and anti-idiotype antibodies). Can be used to treat, inhibit or prevent a disease, disorder or condition associated with aberrant expression and / or activity of a therapeutic protein, including one or more diseases, disorders or conditions described in the literature The treatment and / or prevention of a disease, disorder or condition associated with abnormal expression and / or activity of a therapeutic protein includes, but is not limited to, Alleviating symptoms associated with a disease, disease or condition comprising an albumin fusion protein of the invention comprising at least one fragment or variant of an antibody of the invention, or an antibody that binds to a therapeutic protein. It can be provided in a pharmacologically acceptable composition as is known in the art or as described herein.

  A summary of methods in which an albumin fusion protein of the invention comprising at least one fragment or variant of an antibody of the invention, or an antibody that binds to a therapeutic protein, can be used therapeutically includes locally in the body, or It involves the therapeutic protein binding systemically or by the direct cytotoxicity of the antibody as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. With the techniques provided herein, one of ordinary skill in the art will be able to at least one of the antibodies of the present invention or antibodies that bind to a therapeutic protein for diagnostic, monitoring or therapeutic purposes without undue experimentation. One will know how to utilize an albumin fusion protein of the invention comprising one fragment or variant.

  An albumin fusion protein of the present invention comprising at least one fragment or variant of an antibody of the present invention, or an antibody that binds to a therapeutic protein may be combined with other monoclonal or chimeric antibodies, or for example, effector cells that interact with the antibody It may be advantageously used in combination with lymphokines (such as IL-2, IL-3 and IL-7) or hematopoietic growth factors which are utilized to increase the number or activity.

  An albumin fusion protein of the invention comprising at least one fragment or variant of an antibody of the invention, or an antibody that binds to a therapeutic protein, can be used alone or in other types of treatment (eg, radiation therapy, chemotherapy, hormones). Therapy, immunotherapy and anti-cancer drugs). In general, administration of species-derived or species-reactive (in the case of antibodies) products that are the same species as the patient is preferred. Accordingly, in a preferred embodiment, human antibodies, fragments, derivatives, analogs or nucleic acids are administered to human patients for treatment or prevention.

Both for immunoassays directed to polynucleotides or polypeptides comprising these fragments of the present invention, and for the treatment of diseases associated therewith, therapeutic proteins, fragments or regions thereof (or albumin fusion proteins of such antibodies) It is preferred to use anti-affinity and / or potent in vivo inhibition and / or neutralizing antibodies against the relevant). Such an antibody, fragment or region preferably has an affinity for a polynucleotide or polypeptide of the invention comprising the fragment. Preferred binding affinities include those with a dissociation constant of 5 × 10 ⁻² M, 10 ⁻² M, 5 × 10 ⁻³ M, 10 ⁻³ M, 5 × 10 ⁻⁴ M, 10 ⁻⁴ M or less or Kd. More preferred binding affinities are 5 × 10 ⁻⁵ M, 10 ⁻⁵ M, 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M, 10 ⁷ M, 5 × 10 ⁻⁸ M, or 10 ⁻⁸ M or less dissociation constant. Or the thing of Kd is included. More preferred binding affinities include 5 × 10 ⁻⁹ M, 10 ⁻⁹ M, 5 × 10 ⁻¹⁰ M, 10 ⁻¹⁰ M, 5 × 10 ⁻¹¹ M, 10 ⁻¹¹ M, 5 × 10 ⁻¹² M, 10 ⁻¹² M, 5 × 10. Dissociation constants or those with a Kd of ⁻¹³ M, 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5 × 10 ⁻¹⁵ M, or 10 ⁻¹⁵ M are included.

Gene therapy

  In certain embodiments, a nucleic acid comprising a sequence encoding an antibody that binds to an albumin fusion protein comprising a therapeutic protein or at least one fragment or variant of an antibody that binds the therapeutic protein is obtained by a method of gene therapy. Administered to treat, inhibit or prevent diseases or conditions associated with abnormal expression and / or activity of therapeutic proteins. Gene therapy refers to a therapy performed by administering a nucleic acid that is expressed or expressible to a subject. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any method for gene therapy available in the art can be used in accordance with the present invention. Exemplary methods are described in further detail elsewhere herein.

Demonstration of therapeutic or prophylactic activity

The compounds or pharmacological compositions of the invention are preferably tested in vitro and then in vivo for the desired therapeutic or prophylactic activity prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmacological composition include the effect of the compound on cell lines or patient tissue samples. The effect of a compound or composition on cell lines and / or tissue samples can be determined using techniques known to those skilled in the art including, but not limited to, rosette formation assays and cell lysis assays. In in vitro assays that can be used to determine which administration of a particular compound is suggested according to the present invention, a patient tissue sample is grown in culture and exposed or administered to the compound. In vitro cell culture assays that measure the effect of such compounds on tissue samples are included.

Therapeutic / prophylactic administration and compositions

  The present invention provides methods of treatment, inhibition and prevention by administration to a subject of an effective amount of a compound or pharmacological composition of the present invention. In a preferred embodiment, the compound is essentially purified (eg, essentially free of substrates that limit its effect or produce unwanted side effects). The subject is preferably an animal, preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs and the like, most preferably a human.

  Formulations and methods of administration that are available when the compound comprises a nucleic acid or immunoglobulin are described above, and more suitable formulations and routes of administration can be selected from those described hereinbelow.

  See, for example, liposomes, microparticles, microcapsules, recombinant cells capable of expressing a compound, encapsulation in receptor-mediated endocytosis (Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987). A variety of delivery systems are known and can be used to administer the compounds of the invention, including the construction of nucleic acids as part of retroviruses or other vectors. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The compound or composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucosal linings (eg oral mucosa, vaginal and intestinal mucosa, etc.) May be administered together with the active substance. Administration can be systemic or local. Furthermore, it may be desirable to introduce the pharmacological compound or composition of the invention into the central nervous system by any suitable route, including injections including intraventricular and subarachnoid. Intraventricular injection can be facilitated by an intraventricular catheter connected to a reservoir, such as, for example, an Ommaya reservoir. Pulmonary administration can also be utilized, for example, by use of inhalation or nebulizers and formulations with aerosolized drugs.

  In certain embodiments, it may be desirable to administer the pharmacological compound or composition of the invention locally to the area in need of treatment, such as, but not limited to, local injection during surgery, such as post-surgical Topical application in combination with a bandage, by injection, by catheter method, by suppository method or by implant method, said implant comprising a membrane, such as a shear membrane or fiber, porous, Non-porous or gelatinous material. Preferably, care should be taken to use materials herein that do not absorb protein when administering proteins, including antibodies.

  In other embodiments, the compound or composition can be delivered into excipients, particularly liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Infectious Diseases. and Cancer, Lopez-Berstein and Fiddler (eds.), Liss, New York, pp. 353-365 (1989); see Lopez-Berstein, ibid., pp. 317-327. )

  In yet other embodiments, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); et al., N. Engl. J. Med. 321: 574 (1989)). In other embodiments, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); See, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983) et al. , Sci nce 228: 190 (1985); During et al, Ann Neurol 25:..... 351 (1989); Howard et al, J.Neurosurg 71: 105 (1989)). In yet other embodiments, the controlled release system may be placed in close proximity to the therapeutic target, eg, the brain, thus requiring a whole body / time segment (eg, Goodson, in Medical Applications of Controlled Release). , Supra, vol. 2, pp. 115-138 (1984)).

  Other controlled release systems are discussed in an overview by Langer (Science 249: 1527-1533 (1990)).

  In certain embodiments where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid is constructed as part of a suitable nucleic acid expression vector, eg, retroviral vectors (US Pat. No. 4,980,286). See)) or by direct injection or by the use of microparticle irradiation (eg gene gun, Biolistic, Dupont) or coating with lipids or cell surface receptors or transfection agents, or entering the nucleus By linking to known hemeobox-like peptides (see, eg, Joliot et al., Proc. Natl. Acad. Sci. USA 88: 1864-1868 (1991)) and the like So that By, in order to promote expression of its encoded protein, it can be administered by in vivo. Alternatively, the nucleic acid can be introduced into the cell and incorporated into the host cell DNA for expression by homologous recombination.

  The present invention also provides a pharmacological composition. Such compositions include a therapeutically effective amount of the compound, and a pharmacologically acceptable carrier. In certain embodiments, the phrase “pharmacologically acceptable” is approved by the Pharmaceutical Safety Agency for use in animals, more particularly humans, or the US Pharmacopoeia or other commonly used It means what is listed in the recognized pharmacopoeia. The phrase “carrier” means a diluent, adjuvant, excipient or vehicle administered with a therapeutic agent. Such pharmacological carriers can be sterile liquids such as water and hot water, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmacological composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmacological excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, Propylene, glycol, water, ethanol and the like are included. If desired, the composition can also include minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmacological grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Examples of suitable pharmacological compositions are described in E.I. W. It is described in “Remington's Pharmaceutical Sciences” by Martin. Such compositions comprise a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should be appropriate for the mode of administration.

  In a preferred embodiment, the composition is formulated according to routine procedures as a pharmacological composition adapted for intravenous administration to humans. Typically, an intravenous composition is a solution in a sterile isotonic aqueous buffer. Where necessary, the composition may also include a soluble agent and a local anesthetic such as lignocaine to eliminate pain at the site of the injection. In general, the components are administered separately or in unit dosage, such as a dry lyophilized powder or a water-free concentrate in a sealed container such as an ampoule or sachette indicating the amount of active agent Provided either mixed together in form. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmacological grade water or saline. Where the compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

  The compounds of the invention can be formulated as natural or salt forms. Pharmacologically acceptable salts include those derived from hydrochloric acid, phosphoric acid, acetic acid, oxaloacetic acid, tartaric acid, and the like, and sodium, potassium, ammonium, calcium, iron, hydroxide, isopropylamine, triethylamine, 2 -Anions such as those formed with ethylaminoethanol, histidine, procaine and the like are included.

  The amount of a compound of the present invention that can be effective in the treatment, inhibition and prevention of diseases or conditions associated with abnormal expression and / or activity of therapeutic proteins can be determined by standard clinical techniques. In addition, in vitro assays may optionally be used to help identify optimal dosage ranges. The actual dose used in the formulation will also depend on the route of administration, the severity of the disease or condition and should be determined according to the practitioner's judgment and the status of each patient. Effective doses may be extrapolated from / response curves derived from in vitro or animal model test systems.

For antibodies, the dose / time administered to a patient is typically 0.1 mg / kg patient weight to 100 mg / kg patient weight. Preferably, the dose administered to the patient is 0.1 mg / kg patient weight to 20 mg / kg patient weight, more preferably 1 mg / kg patient weight to 10 mg / kg patient weight. Generally, human antibodies have a longer lifespan in the human body than antibodies from other species due to an immune response against the foreign polypeptide. Thus, lower / times human antibodies and less frequent administration is often possible. Furthermore, the dosage and frequency of administration of the antibodies of the invention can be reduced by enhancing antibody uptake and tissue invasion (eg, into the brain) by modifications such as lipidation.

Diagnostic and imaging

  Treating labeled antibodies and derivatives and analogs thereof that bind to a therapeutic protein (or fragment or variant thereof) (including albumin fusion proteins comprising at least one fragment or variant of an antibody that binds to the therapeutic protein); Can be used for diagnostic purposes to detect, diagnose or monitor diseases, illnesses and / or conditions associated with aberrant expression and / or activity of genetic proteins. The invention comprises (a) assaying the expression of a therapeutic protein in individual cells or body fluids using one or more antibodies specific for the polypeptide subject, and (b) standard gene expression. Comparing the level of the gene expression with the level of gene expression, thereby detecting an abnormal expression of the therapeutic protein, thereby increasing the assayed therapeutic protein expression level compared to the standard expression level Or a decrease suggests abnormal expression.

  The present invention provides for the use of an albumin fusion protein comprising (a) one or more antibodies specific for a polypeptide subject, or at least one fragment or variant of an antibody specific for a therapeutic protein. Providing a diagnostic assay for diagnosing a disease comprising assaying expression of a therapeutic protein in a cell or body fluid, and (b) comparing the level of gene expression with a standard gene expression level; Thereby, an increase or decrease in the assayed therapeutic protein expression level compared to the standard expression level is indicative of a particular disease. For cancer, the presence of a relatively large amount of transcript in biopsy tissue from an individual may suggest a predisposition to disease development, or a method for detecting disease before the occurrence of actual clinical symptoms Can be provided. This type of more definitive diagnosis may allow health professionals to take advantage of earlier preventive measures or aggressive treatment, thereby preventing cancer development or further progression.

  Using an albumin fusion protein comprising at least one fragment or variant of an antibody of the invention, or an antibody specific for a therapeutic protein, using classical immunohistological methods known to those skilled in the art, Protein levels in biological samples can be assayed (eg, Jalkanen et al., J. Cell. Biol. 101: 976-985 (1985); Jalkanen et al., J. Cell. Biol. 105: 3087-). 3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme immunoassay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In) and Included are radioisotopes such as technetium (99Tc), luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and biotin.

  One aspect of the present invention is the detection and diagnosis of diseases or conditions associated with abnormal expression of therapeutic proteins in animals, preferably mammals, most preferably humans. In one embodiment, for diagnosis, a) administering to a subject (eg, parenterally, subcutaneously or intraperitoneally) an effective amount of a labeled molecule that specifically binds to the subject polypeptide; b) Wait for a time interval after administration, preferably in the part in the subject where the therapeutic protein is expressed, to concentrate the labeled molecules (and to clear unbound labeled molecules to background levels) C) determining the background level, and d) detecting the labeled molecule in the subject, so that the detection of the labeled molecule above the background level Suggests having a specific disease or condition associated with abnormal expression of a therapeutic protein. The background level can be determined by a variety of methods including comparing the amount of labeled molecule detected to a standard value already determined for a particular system.

  It is understood in the art that the size of the subject and the imaging system used can determine the amount of imaging site needed to produce a diagnostic image. In the case of a radioisotope site, for a human subject, the amount of radioactivity injected is usually in the range of about 5-20 millicuries of 99mTc. An albumin fusion protein comprising at least one fragment or variant of an antibody that binds to a labeled antibody, antibody fragment, or therapeutic protein is then preferably accumulated locally in the cell containing the specific therapeutic protein. In vivo tumor imaging W. Burchiel et al. , "Immunopharmacokinetics of Radiolabeled Antibodies and Therology. The Chapter of Infra-red." eds., Masson Publishing Inc. (1982)).

  Depending on various variables, including the type of label used and the mode of administration, to allow the labeled molecule to accumulate preferably at sites in the subject, and unbound labeled molecules to background levels The time interval after administration to clear is 6 to 48 hours, or 6 to 24 hours, or 6 to 12 hours. In other embodiments, the time interval after administration is 5 to 20 days, or 5 to 10 days.

  In one embodiment, the disease or disease is monitored by repeating a method for diagnosing the disease or disease, for example, 1 month after the initial diagnosis, 6 months after the initial diagnosis, or 1 year after the initial diagnosis.

  The presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One skilled in the art may be able to determine an appropriate method for detecting a particular label. Methods and instruments that may be used in the diagnostic methods of the present invention include, but are not limited to, computer tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and sonography. included.

In a particular embodiment, the molecule is labeled with a radioisotope and detected in the patient using a radioresponsive surgical instrument (Thurston et al., US Pat. No. 5,441,050). In other embodiments, the molecule is labeled with a fluorescent compound and detected in the patient using a fluorescence responsive scanning instrument. In other embodiments, the molecule is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI). Antibodies that specifically detect albumin fusion proteins, as well as albumin or therapeutic proteins, are a preferred embodiment. These can be used to detect albumin fusion proteins as described throughout this specification.

kit

  The present invention provides a kit that can be used in the above method. In one embodiment, the kit comprises an antibody, preferably a purified antibody, in one or more containers. In certain embodiments, the kit of the invention comprises an essentially isolated polypeptide comprising an epitope that is specifically immunoreactive with the antibody contained within the kit. Preferably, the kit of the present invention further comprises a control antibody that does not react with the polypeptide of interest. In other specific embodiments, the kits of the invention comprise a method for detecting binding of an antibody to a polypeptide of interest (eg, an antibody comprising a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, Or may be conjugated to a detectable substrate, such as a second antibody that recognizes a first antibody that can be conjugated to the detectable substrate).

  In another particular embodiment of the invention, the kit is a diagnostic kit for use in screening of sera containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. Such a kit can include a control antibody that does not react with the polypeptide of interest. Such a kit can include an essentially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such kits include a method for detecting binding of the antigen to an antibody (eg, the antibody is conjugated to a fluorescent compound such as fluorescein or rhodamine that can be detected by flow cytometry. Is good). In certain embodiments, the kit can include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigens of the kit can also be bound to a solid support.

  In a more specific embodiment, the detection means of the kit includes a solid support to which the polypeptide antigen binds. Such a kit can also include an unbound reporter-labeled anti-human antigen. In this embodiment, the binding of the antibody to the polypeptide antigen can be detected by the binding of the reporter-labeled antigen.

  In a further embodiment, the present invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the present invention. The diagnostic kit includes an essentially isolated antibody that is specifically immunoreactive with a polypeptide or polynucleotide antigen, and a method for detecting binding of the polynucleotide or polypeptide antigen to the antibody. . In one embodiment, the antibody binds to a solid support. In certain embodiments, the antibody can be a monoclonal antibody. The detection means of the kit can include a second, labeled monoclonal antibody. Alternatively or additionally, the detection means can include a labeled, competing antibody.

  In one diagnostic form, test serum is reacted with a solid phase reagent having a surface-bound antibody obtained by the method of the present invention. After binding of the specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent reacts with the reporter-labeled anti-human antibody and is proportional to the amount of bound anti-antigen antibody on the solid support The reporter binds to the reagent. The reagent is washed again to remove unbound labeled antibody and the amount of reporter bound to the reagent is measured. Typically, the reporter is an enzyme that is detected by incubating the solid phase in the presence of a suitable fluorescent, luminescent or colored substrate (Sigma, St. Louis, MO).

  The solid surface reagent in the above assay is prepared by known techniques for binding protein material to solid support material such as polymerized beads, dipstick, 96-well plate or filter material. These attachment methods generally involve non-specific adsorption of proteins to a solid, or typically on a solid support, such as activated carboxyl, hydroxyl or aldehyde groups via free amino groups. Includes covalent attachment of proteins to chemically reactive groups. Alternatively, streptavidin-coated plates can be used with biotinylated antigen.

Accordingly, the present invention provides an assay system or kit for performing this diagnostic method. The kit generally includes a support comprising a surface-bound recombinant antibody and a receptor-labeled anti-human antibody for detecting the surface-bound anti-antigen antibody.

Albumin fusion protein

  The present invention relates generally to albumin fusion proteins and methods for treating, preventing or reducing a disease or condition. As used herein, an “albumin fusion protein” is at least one therapeutic protein (or fragment or variant thereof) of at least one molecule of albumin (or fragment or variant thereof). Means a protein formed by the fusion of molecules. Albumin fusion proteins of the invention include at least one fragment or variant of a therapeutic protein and at least one fragment or variant of human serum albumin that bind to each other or to each other, preferably by genetic fusion (Ie, an albumin fusion allows a polynucleotide encoding all or a portion of a therapeutic protein to bind in-frame to a polynucleotide encoding all or a portion of albumin). The therapeutic protein and the albumin protein, part of the albumin fusion protein may be referred to as a “part”, “region” or “site” of the albumin fusion protein, respectively.

  In a preferred embodiment, the present invention provides an albumin fusion protein encoded by a polynucleotide or albumin fusion construct described in Table 1 or Table 2. Polynucleotides encoding these albumin fusion proteins are also encompassed by the present invention.

  Preferred albumin fusion proteins of the present invention include, but are not limited to, at least albumin (or a fragment or variant thereof) bound in frame to at least one polynucleotide encoding at least one molecule of a therapeutic protein. A nucleic acid molecule comprising or consisting of a polynucleotide encoding one molecule, at least one of the therapeutic proteins (or fragments or variants thereof) produced as described in Table 1, Table 2 or Examples A nucleic acid molecule comprising or consisting of at least one molecule of albumin (or a fragment or variant thereof) linked in frame to at least one polynucleotide encoding one molecule, or further For example one or more of the following Elements, (1) functional self-replicating vectors (including but not limited to shuttle vectors, expression vectors, integration vectors, and / or replication systems), (2) regions for initiation of transcription (eg, regulatable or A therapeutic protein (or fragment thereof) comprising an inducible promoter, a promoter region, such as a structural promoter), (3) a region for termination of transcription, (4) a leader sequence, and (5) a selectable marker A polynucleotide encoding at least one molecule of albumin (or a fragment or variant thereof) linked in frame to at least one polynucleotide encoding at least one molecule of Or a nucleic acid molecule consisting of

  In one embodiment, the present invention provides an albumin fusion protein comprising or consisting of a therapeutic protein (eg, as described in Table 1) and serum albumin protein. In other embodiments, the present invention provides albumin fusion proteins comprising or consisting of biologically active and / or therapeutically active fragments of therapeutic proteins and serum albumin proteins. In other embodiments, the present invention provides albumin fusion proteins comprising or consisting of biologically active and / or therapeutically active variants of therapeutic proteins and serum albumin proteins. In a preferred embodiment, the albumin protein component of the albumin fusion protein is the mature portion of serum albumin.

  In a further embodiment, the present invention provides an albumin fusion protein comprising or consisting of a therapeutic protein and a biologically active and / or therapeutically active fragment of serum albumin. In a further embodiment, the present invention provides an albumin fusion protein comprising or consisting of a therapeutic protein and a biologically active and / or therapeutically active variant of serum albumin. In a preferred embodiment, the therapeutic protein portion of the albumin fusion protein is the mature portion of the therapeutic protein.

  In further embodiments, the present invention provides biologically active and / or therapeutically active fragments of therapeutic proteins and biologically active and / or therapeutically active variants of serum albumin. An albumin fusion protein comprising or consisting of a body is provided. In a preferred embodiment, the present invention provides an albumin fusion protein comprising or consisting of a mature portion of a therapeutic protein and a mature portion of serum albumin.

  Preferably, the albumin fusion protein comprises HA as the N-terminal portion and therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising HA as the C-terminal portion and therapeutic protein as the N-terminal portion may also be used.

  In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminus and C-terminus of albumin. In a preferred embodiment, the therapeutic proteins fused at the N- and C-termini are the same therapeutic protein. In other preferred embodiments, the therapeutic proteins fused at the N- and C-termini are different therapeutic proteins. In other preferred embodiments, the therapeutic proteins fused at the N- and C-termini are the same or related diseases, diseases (eg, as listed in “Preferred indications Y” in Table 1). Or a different therapeutic protein that can be used to treat or prevent a condition. In other preferred embodiments, therapeutic proteins fused at the N- and C-termini are commonly generated in patients, commonly occurring simultaneously, simultaneously, in succession, or commonly associated with patients. May be used to treat, reduce or prevent a disease or condition known in the art to occur (eg, as listed in “Preferred indications Y” in Table 1). It is a therapeutic protein.

  The albumin fusion protein of the present invention comprises one, two, three, four or more N- or C-termini of the albumin fusion protein of the present invention, and / or N- and albumin or variants thereof. And / or a protein comprising the therapeutic protein X or a variant thereof fused to the C-terminus. The therapeutic protein X or variant thereof includes, but is not limited to, a “head to head” orientation (eg, the N-terminus of one molecule of therapeutic protein X is another molecule of therapeutic protein X). Or the “head-to-tail” direction (eg, the C-terminus of one molecule of therapeutic protein X is at the N-terminus of another molecule of therapeutic protein X). Can be in any number of directions, including

  In one embodiment, one, two or more repeat-directed therapeutic protein X polypeptides (or fragments or variants thereof) are present at the N- or C-terminus of the albumin fusion protein of the invention, and Fusion to the N- and / or C-terminus of albumin or variants thereof.

  The albumin fusion protein of the present invention further comprises the protein X or its fusion fused to the N- or C-terminus of the albumin fusion protein of the present invention and / or to the N- and / or C-terminus of albumin or a variant thereof. It includes a protein comprising one, two, three, four or more molecules of fragments, the molecules being linked via a peptide linker. Examples include peptide linkers described in US Pat. No. 5,073,627 (incorporated herein by reference). Albumin fusion proteins comprising multiple therapeutic protein X polypeptides separated by peptide linkers may be produced using conventional recombinant DNA techniques. Linkers are particularly important when small peptides are fused to large HSA molecules. The peptide itself can be a linker by fusing tandem copies of the peptide, and other known linkers can be used. Constructs containing linkers are described in Table 2 or appear when testing SEQ ID NO: Y.

  Furthermore, the albumin fusion proteins of the present invention also provide therapeutic protein X or a variant thereof in the method that allows the formation of intermolecular and / or intramolecular multiplexed forms and the N-terminus of albumin or a variant thereof and It may also be produced by fusing to the C-terminus. In one embodiment of the invention, the albumin fusion protein may be in single or multiple forms (ie, dimers, trimers, tetramers and higher order multimers). In a further embodiment of the invention, the therapeutic protein portion of the albumin fusion protein can be in a single or multiple form (ie, dimer, trimer, tetramer and higher order multimer). In certain embodiments, the therapeutic protein portion of the albumin fusion protein is multi-form (ie, dimer, trimer, tetramer and higher order multimer), and the albumin protein portion is multi-form.

  In addition to albumin fusion proteins in which the albumin moiety is fused to the N-terminus and / or C-terminus of the therapeutic protein, the albumin fusion proteins of the present invention also comprise a therapeutic protein or peptide of interest (eg, in Table 1). An antibody that binds therapeutic protein X as disclosed, or therapeutic protein or fragment or variant thereof) may be produced by insertion into an internal region of HA. For example, within the protein sequence of the HA molecule, there are numerous loops or turns between the end and beginning of the α-helix, which are stabilized by disulfide bonds. For the most part, the loop extends away from the body of the molecule as determined from the plasma structure of HA (PDB identifiers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E-1E7I and 1UOR). These loops are useful for therapeutically active peptides, especially those required for structure to be functional, or for the insertion or internal fusion of therapeutic proteins, and specific biological activities Give rise to essentially an albumin molecule with

  The loop in the human albumin structure into which the peptide or polypeptide is inserted to produce the albumin fusion protein of the present invention includes Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176, His247-Glu252, Glu266-Glu277, Glu280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and Lys560-Thr566 are included. In a further preferred embodiment, the peptide or polypeptide is inserted into Val54-Asn61, Gln170-Ala176, and / or Lys560-Thr566 of mature human albumin (SEQ ID NO: 1).

  The peptide to be inserted may be derived from either the phage display or synthetic peptide library screened for a particular biological activity, or from the active portion of the molecule with the desired function. Furthermore, it is possible to generate a random peptide library within a specific loop by inserting a randomized peptide into a specific loop of the HA molecule, in which all possible amino acid combinations Is represented.

Such a library can be produced on HA or a domain fragment of HA by one of the following methods.
Random mutagenesis of amino acids within one or more peptide loops of HA or HA domain fragments. One or more or all residues in the loop can be mutated in this manner.
Replacement of one or more loops of HA or HA domain fragment (ie, internal fusion) of randomized peptide (s) of length Xn, where X is an amino acid and n is the number of residues Or insert into.
N-, C- or N- and C-terminal peptide / protein fusions in addition to (a) and / or (b).

  HA or HA domain fragments can also be made multifunctional by passing peptides derived from different screens of different loops against different targets within the same HA or HA domain fragment.

  In a preferred embodiment, the peptide inserted into the loop of human serum albumin is a peptide fragment or peptide variant of the therapeutic protein disclosed in Table 1. More particularly, the invention relates to a length of at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, inserted into a loop of human serum albumin. Albumin fusion proteins comprising peptide fragments or peptide variants with at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids. The present invention also includes at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least fused to the N-terminus of human serum albumin. Including albumin fusion proteins comprising peptide fragments or peptide variants at 30, at least 35, or at least 40 amino acids. The present invention also includes at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least fused to the C-terminus of human serum albumin. Including albumin fusion proteins comprising peptide fragments or peptide variants at 30, at least 35, or at least 40 amino acids. For example, the short peptides described in Tables 1 and 2 (eg, therapeutic Y) can be inserted into the albumin loop.

  In general, the albumin fusion proteins of the invention can have one HA-derived region and one therapeutic protein-derived region. However, multiple regions of each protein may be used to make the albumin fusion protein of the invention. Similarly, one or more therapeutic proteins may be used to make an albumin fusion protein of the invention. For example, the therapeutic protein is fused to both the N- and C-terminal ends of HA. In such a conformation, the therapeutic protein moiety can be the same or different therapeutic protein molecules. The structure of the bifunctional albumin fusion protein can be represented as X-HA-Y or Y-HA-X.

  For example, anti-BLys ™ scFv-HA-IFNa-2b fusions may be prepared to modulate the immune response to IFNa-2b by anti-BLyS ™ scFv. Alternatively, bi- (or multi-) functional / round HA-fusions, eg, HA-anti-BLyS ™ scFv fusions or other HA-, at various ratios depending on function, half-life, etc. HA-IFNa-2b fusions mixed with the fusion may be made.

  Bi- or other functional albumin fusion proteins may be prepared to target the therapeutic protein moiety to the target organ or cell type via the protein or peptide at the opposite end of the HA.

  As an alternative to known therapeutic molecule fusions, the peptide may be HA, or typically 6, 8, 12, 20 or 25 or Xn, where X is an amino acid (aa) and n is the residue (Equal to a number), which can be obtained by screening libraries constructed as random or amino acid HA fusions to the N-, C- or N- and C-termini, representing all possible amino acid combinations. . A particular advantage of this approach is that peptides can be screened in situ on HA molecules and that the properties of the peptides are thus derived essentially by any other method that adheres to the HA. It can be selected other than by modification as in the case.

  Furthermore, the albumin fusion proteins of the present invention provide greater physical separation between sites, and thus, for example, to maximize the accessibility of a therapeutic protein moiety for binding to its cognate receptor. A linker peptide may be included between the fusion moieties. The linker peptide may consist of amino acids that are flexible or more robust.

  The linker sequence may be cleaved by a protease or chemically to produce a growth hormone related site. Preferably, the protease is e.g. Those produced naturally by the host, such as the cerevisiae protease kex2 or equivalent protease.

  Thus, as described above, the albumin fusion protein of the invention has the following formula R1-L-R2, R2-L-R1, or R1-L-R2-L-R1, wherein R1 is It is at least one therapeutic protein, peptide or polypeptide sequence and need not be the same therapeutic protein, L is a linker and R2 is a serum albumin sequence.

  In a preferred embodiment, an albumin fusion protein of the invention comprising a therapeutic protein has a higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability is typically the case where the therapeutic protein is administered in vivo and carried into the bloodstream, and the kidney or liver that ultimately removes the therapeutic protein from the bloodstream and the therapeutic protein from the body. Means the period of time between degradation and purification within an organ such as Plasma stability is calculated with respect to the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

  In a preferred embodiment, an albumin fusion protein of the invention comprising a therapeutic protein has an extended lifetime compared to the lifetime of the same therapeutic protein when not fused to albumin. Lifetime typically refers to the time interval during which therapeutic activity of a therapeutic protein is stable in solution, or in other stored formulations, without unnecessary loss of its therapeutic activity. Many therapeutic proteins are very unstable in their unfused state. As described below, the typical lifetime of these therapeutic proteins is clearly extended upon incorporation into the albumin fusion proteins of the present invention.

  An albumin fusion protein of the invention with an “extended” or “extended” lifetime exhibits greater therapeutic activity compared to a standard adapted to the same storage and handling conditions. The standard may be a non-fused full length therapeutic protein. If the therapeutic protein portion of an albumin fusion protein is an analog, variant, or altered from its full sequence, or does not contain it, prolong the therapeutic activity, It may be compared to analogs, mutants, altered peptides, or non-fused equivalents of incomplete sequences. By way of example, the albumin fusion protein of the invention, when compared at that time point, when applied to the same storage and handling conditions as a standard, is about 100% or more of the standard therapeutic activity, or the standard therapeutic activity About 105%, 110%, 120%, 130%, 150%, or 200% or more.

Lifespan is also assessed for therapeutic activity that is maintained after storage and is normalized to therapeutic activity when storage is initiated. As demonstrated by prolonged or extended therapeutic activity, an albumin fusion protein of the invention having an extended or extended lifetime has about 50% of the therapeutic activity of an equivalent non-fused therapeutic protein when applied to the same conditions. As such, about 60%, 70%, 80% or 90% or more of the therapeutic activity may be maintained.

Fusion protein expression

  The fusion proteins of the invention may be produced as recombinant molecules by secretion from yeast, microorganisms such as bacteria, or human or animal cell lines. Preferably the polypeptide is secreted from the host cell.

  Particular embodiments of the present invention include signal sequences that are effective for directing secretion in yeast, especially signal sequences derived from yeast (especially those that are homologous to the yeast host), and the first aspect of the invention A DNA construct encoding the fusion molecule is included, where there is no yeast-derived prosequence between the signal and the mature polypeptide.

  The Saccharomyces cerevisiae invertase signal is a preferred example of a yeast-derived signal sequence.

  Poznansky et al., Wherein separately prepared polypeptides are joined by chemical cross-linking. (FEBS Lett. 239: 18 (1988)).

  The invention also includes cells transduced to express an albumin fusion protein of the invention, preferably yeast cells. Compared to the transduced host cells themselves, the present invention also contemplates cultures of those cells in nutrient medium, preferably from a monoclonal (clonally homogeneous) culture, or from a monoclonal antibody solution. To do. If the polypeptide is secreted, the medium will contain the polypeptide with the cells or without the cells if filtered or centrifuged. Many expression systems are known including bacteria (eg E. coli and Bacillus subtilis), yeast (eg Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris), filamentous fungi (eg Aspergillus), plant cells, animal cells and insect cells. Yes, you may use it.

Preferred yeast strains that can be used in the production of albumin fusion proteins are D88, DXY1 and BXP10. D88 [leu2-3, leu2-122, can1, pr1, ubc4] is a derivative of the parent strain AH22his ⁺ (also see as DB1, see for example Sleep et al. Biotechnology 8: 42-46 (1990)). is there. This strain contains a leu2 mutation that allows auxotrophic secretion of a 2 micron based plasmid containing the LEU2 gene. D88 also shows derepression of PRB1 in glucose excess. The PRB1 promoter is normally regulated by two cytokines that monitor glucose levels and growth stages. The promoter is activated in wild type yeast upon glucose derepression and entry into the stationary phase. Strain D88 shows derepression by glucose, but maintains induction upon entry into the stationary phase. The PRA1 gene encodes the yeast vacuolar protease, YscA endoprotease A, which is localized in the ER. The UBC4 gene is in the ubiquinone pathway and is involved in short live and above protein targeting for ubiquitin-dependent degradation. This isolation of the ubc4 mutant increases the copy number of the expression plasmid in the cell and causes an increase in the level of expression of the desired protein expressed from the plasmid (for example, all of which are hereby incorporated by reference). See incorporated international patent specification WO 99/00504).

  DXY1, a derivative of D88, has the following genotype [leu2-3, leu2-122, can1, pr1, ubc4, ura3 :: yap3]. In addition to the mutation isolated in D88, this strain also has a knockout of YAP3 protease. This protease causes cleavage of almost dibasic residues (RR, RK, KR, KK), but also facilitates cleavage at a single basic residue in the protein. Isolation of this yap3 mutation results in higher levels of full-length HSA production (eg, US Pat. No. 5,965,386 and Kerry-Williams, all of which are incorporated herein by reference). et al., Yeast 14: 161-169 (1998)).

  BXP10 has the following expression systems: leu2-3, leu2-122, can1, pr1, ubc4, ura3, yap3 :: URA3, lys2, hsp150 :: LYS2, pmt1 :: URA3. In addition to the mutations isolated in DXY1, this strain also has a PMT1 gene and HSP150 knockout. The PMT1 gene is an evolutionarily conserved member of the dotilyl-phosphate-D-mannose protein O-mannosyltransferase (Pmts). The transmembrane topology of Pmt1p suggests that it is an integral membrane protein of the endoplasmic reticulum that has a role in O-linked glycosylation. This mutation serves to reduce / eliminate O-linked glycosylation of the HSA fusion (see, eg, International Patent No. WO 00/44772, which is incorporated by reference herein in its entirety). Studies have shown that Hsp150 protein is poorly separated from rHA by ion exchange chromatography. Mutations in the SHP150 gene remove contaminants that may prove difficult to remove by standard purification techniques. See, for example, US Pat. No. 5,783,423, which is hereby incorporated by reference in its entirety.

  The desired protein is produced by conventional methods, for example from cloning sequences inserted in the host chromosome or on a free plasmid. Yeast is transduced with the coding sequence for the desired protein by any useful method, such as electroporation. A method for transduction of yeast by electroporation is described by Becker & Guarente (1990) Methods Enzymol. 194, 182.

  Cells that have been successfully transduced, ie, cells that contain a coding construct with the DNA of the present invention, can be identified by well-known techniques. For example, cells as a result of the introduction of the expression construct can be grown to produce the desired polypeptide. Cells are harvested, lysed, and their DNA content is determined by Southern (1975) J. MoI. Mol. Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208 can be tested for the presence of DNA using methods such as those described in US Pat. Alternatively, the presence of the protein in the supernatant can be detected using an antibody.

  Useful yeast plasmid vectors include pRS403-406 and pRS413-416, and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, 7RP1, LEU2 and URA3, plasmid pRS413-4 (Yeast centromede plasmid (Yeast plastromes plasmid). Ycps)).

  Preferred vectors for making albumin fusion proteins for expression in yeast include pPPC0005, pScCHSA, pScNHSA and pC4: HSA described in detail in Example 1. FIG. 2 shows a map of the pPPC0005 plasmid that can be used as a base vector in which a polynucleotide encoding a therapeutic protein can be cloned to form a HA-fusion. This is because PRB1 S.I. It includes a cerevisiae promoter (PRB1p), a fusion leader sequence (FL), a DNA encoding HA (rHA) and an ADH1S cerevisiae terminator sequence. The sequence of the fusion leader sequence is the first 19 amino acids (SEQ ID NO: 3) of the signal peptide of human serum albumin and the mating factor alpha 1 promoter (SLDKR, all of which are incorporated by reference, EP-A -387 319)).

  Plasmids pPPC0005, pScCHSA, pScNHSA and pC4: HSA were deposited on April 11, 2001 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Pt. 3276, PTA-3279 and PTA-3277 were obtained. Another vector useful for expressing albumin fusion proteins in yeast, pSAC35, is incorporated by reference in Sleep et al. BioTechnology 8:42 (1990).

A yeast promoter that can be used to express an albumin fusion protein is the MET25 promoter. See, for example, Dominik Mumburg, Rol Muller and Martin Funk. Nucleic Acids Research, 1994, Vol. 22, no. 25, pp. See 5767-5768. The Met25 promoter is 383 bases long (bases -382 to -1), and the genes expressed by this promoter are also known as Met15, Met17 and YLR303W. A preferred embodiment uses the following sequence, the Not1 site used for cloning is underlined at the 5 ′ end of the following sequence, and the ATG start codon is underlined at the 3 ′ end: It is.
GCGGCCGC CGGATGCAAGGGTTCGAATCCCTTAGCTCTCATTATTTTTTGCTTTTTCTCTTGAGGTCACATGATCGCAAAATGGCAAATGGCACGTGAAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGTTAGTCAAGGCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAAGTGTCGAAAAGGTGGCACCTTGTCCAATTGAACACGCTCGATGAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTTTCCTTTTTCTTGCTCTCTTGTCTTTTCATCTACTATTTCCT TCGTGTAATACAGGGTCGTCCAGATACATAGATACAATTCTATACCCCCCATCCATACA ATG (SEQ ID NO: 5)

Additional promoter can be used to express the albumin fusion protein in yeast, the following a) cbh1 promoter TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATATAA AGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCACCTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTGTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCACGGCAAGGGAAACCA CGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGGTGATCCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATACAACCAAGGG CAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGTCACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCAAAAGATTGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGTT CGAATAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGGCATC (SEQ ID NO: 113)

cysD promoter AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTCTTAGCCGCATGAGATTCCTACCATTTATGTCCTATCGTTCAGGGTCCTATTTGGACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATTCACGCAATTACGATAGTATTTGGCTCTTTTCGTTTGGCCCAGGTCAATTCGGGTAAGACGCGATCACGCCATTGTGGCCGCCGGCGTTGTGCTGCTGCTATTCCCCGCATATAAACAACCCCTCCACCAGTTCGTTGGGCTTTG from b) Aspergillus nidulans (Aspergillus nidulans) GAATGCTGTACTCTATTTCAAGTTGTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATATCAAAGATATCAAAGCCATC (SEQ ID NO: 114)

Modified cbh1 promoter TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGC with an array of c) below GGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCACCTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTGTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCACGGCAAGGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCA TGCAGCATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGGTGATCCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATACAACCAAGGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGA GTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGTCACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCAAAAGATTGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGTTTCGAATAGAAAGAGAAGCTTAGCCTGCAGCCTCTTAT GAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGGCA C (SEQ ID NO: 115)

cysD promoter AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTCTTAGCCGCATGAGATTCCTACCATTTATGTCCTATCGTTCAGGGTCCTATTTGGACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATTCACGCAATTACGATAGTATTTGGCTCTTTTCGTTTGGCCCAGGTCAATTCGGGTAAGACGCGATCACGCCATTGTGGCCGCCGGCGCTGCAGCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGC from Aspergillus nidulans (Aspergillus nidulans) having the sequence of d) below AAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTACGCCGGCGTTGTGCTGCTGCTATTCCCCGCATATAAACAACCCCTCCACCAGTTCGTTGGGCTTTGCGAATGCTGTACTCTATTTCAAGTTGTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATATCAAAGATATCAAAGCCATC (SEQ ID NO: 116)
Is included.

  Various methods have been developed to operably link DNA to vectors via complementary sticky ends. For example, complementary homopolymer sections can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined between complementary homopolymer tails by hydrogen bonding to form a recombinant DNA molecule.

  Synthetic linkers containing one or more restriction sites provide another way of joining a DNA segment to a vector. Bacteriophage T4 DNA polymerase or E. coli, which is an enzyme that removes the raised gamma-single-stranded ends with their 3 ′ 5′-exonucleoactivity from DNA segments generated by endonuclease restriction digestion Treat with DNA polymerase I to fill the embedded 3 'end with their polymerization activity.

  The combination of these activities thus creates a blunt end DNA segment. The blunt-ended segment is then incubated with a large molar excess of linker molecule in the presence of an enzyme capable of catalyzing the ligation of blunt-ended DNA molecules, such as flat bacteriophage T4 DNA ligase. Thus, the reaction product becomes a DNA segment containing a multiplexed linker sequence at its end. These DNA segments are then cleaved with appropriate restriction enzymes and ligated into an expression vector cleaved with an enzyme that produces termini compatible with those DNA segments.

  Synthetic linkers containing various restriction endonuclease sites are commercially available from a number of sources, including International Biotechnologies Inc, New Haven, CT, USA.

  For example, where HA variants are to be prepared, a desirable method for modifying DNA according to the present invention is described by Saiki et al. (1988) Science 239, 487-491, using the polymerase chain reaction. In this method, the DNA to be enzymatically amplified is flanked by two special oligonucleotide primers that are themselves incorporated into the amplified DNA. Special primers can include restriction endonuclease recognition sites that can be used to clone into expression vectors using methods known in the art.

  Exemplary genera of yeasts contemplated as useful in the practice of the present invention as hosts for expressing albumin fusion proteins are Pichia (Hansenula), Saccharomyces, Kluyveromyces. (Kluyveromyces), Candida (Corida), Torulopsis (Torulaspora), Schizosaccharomyces (Nitros), Citeromyces (Citeromyces), Pachisolem (Citeromyces) Rhodosporidium), Leucospodium Leucosporidium), Botsurioasukusu (Botryoascus), Sporidiobolus (Sporidiobolus), and the like Endomikopushisu (Endomycopsis). Preferred genera are Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia and Torlaspora. Saccharomyces spp. Examples of S. cerevisiae, S. cerevisiae Italix and S. italicus S. rouxii.

  Kluyveromyces spp. Examples of K.K. Fragilis, K. fragilis Lactis and K. lactis. K. marxianus. Suitable torus laspora species are T. pylori. T. delbrueckii. Pichia (Hansenula) spp. An example of P.I. In P. angusta (formerly H. Polymorpha, P. anomala (formerly H. anomala), and P. pastoris) There are methods for transduction of S. cerevisiae, European Patent No. 251 744, European Patent No. 258 067 and International Patent No. 90/01063, all of which are hereby incorporated by reference. Is generally doctrine.

  Preferred exemplary species of Saccharomyces include S. cerevisiae. Cerevisiae, S. et al. Italics, S.M. Included are S. diastaticus, and Zygosaccharomyces rouxii. Preferred exemplary species of Kluyveromyces include K. cerevisiae. Fragilis and K. Includes lactis. Preferred exemplary species of Hansenula include H. H. polymorpha (currently Pichia angusta), H. anomala (currently Pichia anomala), and Pichia capsula (and Piccia capsula). Preferred exemplary species include P. pastoris Preferred exemplary species of Aspergillus include A. niger and A. nidulans. Preferred exemplary species of Yarrowia include Y. lipolytica Many preferred yeast species are available from ATCC, such as the following preferred yeast species: Saccharomyces cerevisiae Hansen, Teleomorpha species (BY4743) yap3 mutant (ATCC Accession No. 4022731), Saccharomyces cerevisiae Hansen, Teleomorpha species BY4743 hsp150 mutant (available from ATCC) ATCC Accession No. 4021266), Saccharomyces cerevisiae Hansen, Teleomorpha sp. BY4743 pmt1 mutant (ATCC Accession No. 4023792), Saccharomyces cerevisiae Hansen, Teleomorpha (ATCC Accession Nos. 44773, 44774 and 6295) re, Saccharomyces di wil van der Walt (ATCC Contract) No. 62987), Dombrowskis van der Walt (ATCC Accession No. 76492), Hansenula Polymorpha de Morais et Maia, Tehison AT et al. Deposited as Teleomorpha. 26012), Aspergillus niger van Tieghem, Anamorpha (ATCC Accession No. 9029), Aspergillus niger van Tieghem, Anamorpha (ATCC Accession No. 16404), Aspergillus nidulans (Eidam), Winter AT No. 48756) and Yarovia Lipo Atlantica (Wickerham et al. ) Van der Walt et von Arx, Teleomorpha (ATCC Accession No. 201847).

  S. Suitable promoters for cerevisiae include PGK1 gene, GAL1 or GAL10 gene, CYCI, PHO5, TRPI, ADHI, ADH2, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, formofructokinase , Triose phosphate isomerase, morphoglucose isomerase, glucokinase, alpha-mating factor pheromone, those related to genes for [mating factor pheromone], PRBI promoter, GUT2 promoter, GODI promoter, and 5 'regulatory region, etc. Hybrid promoters involved in hybridizing to the 5 'regulatory region of the promoter of or the upstream activation site (see, for example, the promotion of EP-A-258 067) Over) are included.

  A convenient regulatable promoter for use in Schizosaccharomyces pombe is described by Maundrell (1990) J. Mol. Biol. Chem. A thiamine-repressible promoter from the nmt gene as described by H.265, 10857-10864, and a glucose repressive jbpl gene promoter as described by Hoffman & Winston (1990) Genetics 124, 807-816.

  Methods for transducing Pichia for expression of foreign genes are described, for example, in Cregg et al. (1993), a variety of Phillips patents (eg, US Pat. No. 4,857,467, which is incorporated herein by reference), Phia expression kits are described in Invitrogen, BV, Commercially available from Leek, Netherlands, and Invitrogen Corp., San Diego, California. Suitable promoters include AOX1 and AOX2. Gleeson et al. (1986) J. Am. Gen. Microbiol. 132, 3459-3465 contain information on Hansenula vectors and transduction, suitable promoters are MOX1 and FMD1, while European Patent No. 361 991, Fleer et al. (1991) and other publications from Rhone-Poulenc Roller are available from Kluyveromyces spp. Teaches how to express foreign proteins therein, and the preferred promoter is PGK1.

  The transcription termination signal is preferably the 3 'flanking sequence of a eukaryotic gene, including appropriate signals for transcription termination and polyadenylation. A suitable 3 'flanking sequence may be, for example, a gene naturally linked to the expression control sequences used, i.e. may correspond to a promoter. Alternatively, they may be different, in which case S.P. A termination signal for the S. cerevisiae ADH1 gene is preferred.

The desired albumin fusion protein may be first expressed with a secretory leader sequence and may be any leader effective in the selected yeast. Useful leaders in yeast include any of the following:
a) MPIF-signal sequence (eg amino acids 1 to 21 of GenBank Accession No. AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO: 6)
b) stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO: 7)
c) Pre-pro region of the HSA signal sequence (eg MKWVTFISLLFLSSAYSRRGVFRR, SEQ ID NO: 8)
d) Pre-region of HSA signal sequence (eg MKWVTFISLLFLSFSYS, SEQ ID NO: 9) or eg MKWVSFISLLFLSFSYS (SEQ ID NO: 10) variants thereof e) Invertase signal sequence (eg MLLQAFLFLLAGFAAKISA, SEQ ID NO: 11)
f) Yeast mating factor alpha signal sequence (eg, MRFPSIFTAVLAFAASSALALAVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSTNSTNGLLFINTTIGSIVEGTDNGSTDNGD
g) K.K. Lactis killer toxin leader sequence h) hybrid signal sequence (eg MKWVSFISLLFFSSAYSRSLEKR, SEQ ID NO: 13)
i) HSA / MFa-1 hybrid signal sequence (also known as HSA / kex2) (eg MKWVSFISLLFLSSAYSRSLDKR, SEQ ID NO: 14)
j) K. Lacti Killer / MFa-1 fusion leader sequence (eg, MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO: 15)
k) Immunoglobulin Ig signal sequence (eg, MGWSCIILFLVATATGVHS, SEQ ID NO: 16)
l) Fibrin B precursor signal sequence (eg, MERAAPSRRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO: 17)
m) Clusterin precursor signal sequence (eg, MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO: 18)
n) Insulin-like growth factor-binding 4 signal sequence (eg MLPLCLVAALLLAAGPGPSLG, SEQ ID NO: 19)
o) For example, MKWVSFISLLFLSSAYSRGVFRR (SEQ ID NO: 20),
MKWVTFISLLFLFAGVLG (SEQ ID NO: 21),
MKWVTFISLLFLFSGVLG (SEQ ID NO: 22),
MKWVTFISLLFLFGGVLG (SEQ ID NO: 23),
Modified HSA leader HSA # 64-MKWVTFISLLFLFAGVSG (SEQ ID NO: 24),
Modified HSA leader HSA # 66-MKWVTFISLLFLFGGVSG (SEQ ID NO: 25),
Modified HSA (A14) leader-MKWVTFISLLFLFAGVSG (SEQ ID NO: 26),
Modified HSA (S14) leader (also known as modified HSA # 65)-MKWVTFISLLFLFSGVSG (SEQ ID NO: 27),
Modified HSA (G14) leader-MKWVTFISLLFLFGGVSG (SEQ ID NO: 28), or MKWVTFISLLFLFGGVLLGDLHKS (SEQ ID NO: 29)
P) Consensus signal sequence (MPTWAWWWLFLVLLLALWAPARG, SEQ ID NO: 30)
q) Acid phosphatase (PH05) leader (eg MFKSVVYSILAASLANA SEQ ID NO: 31)
r) Pre-sequence of MFoz-1 s) Pre-sequence of O-glucanase (BGL2)
t) Killer toxin leader u) Pre-sequence of killer toxin
v) K. Lactis killer toxin prepro (29 amino acids, pre 26 amino acids and pro 13 amino acids)
w) S.M. Diastatics glucoamylase II secretion leader sequence x) Calisbergen cis α-galactosidase (MEL1) secretion leader sequence y) Candida glucoamylase leader sequence z) Hybrid leader aa) gp67 signal disclosed in EP-A-387319 (incorporated by reference) Sequence (in combination with a baculovirus expression system) (eg, amino acids 1-19 of GenBank accession number AAA72759) or bb) natural leader cc of therapeutic protein X) Japanese Patent No. 62-096086 (Granted as 911036516, Reference S., which is incorporated by reference). S. cerevisiae invertase (SUC2) leader, or dd) inulinase-MKLAYSLLPLAGVSAVINYKR (SEQ ID NO: 33).
ee) Modified TA57 propeptide leader mutant # 1-MKLKTVRSAVLSSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFAATQTNSGLDVVGLISMAKR (SEQ ID NO: 34)
ff) Modified TA57 Propeptide Leader Mutant # 2-MKLKTVRSAVLSSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLVVGLISMAEEEPEPKR (SEQ ID NO: 35)
gg) Consensus signal peptide-MWWWLWWWLLLLLLLWPMVWA (SEQ ID NO: 111)
hh) Modified HSA / kex2 signal sequence-MKWVSFISLLFLFSASAGSGSLDKR (SEQ ID NO: 112)
ii) Consensus signal peptide # 2-MRPTWAWWWLFLVLLLALWAPARG (SEQ ID NO: 105)

Further methods of production by recombination and synthesis of albumin fusion proteins

  The invention also relates to vectors, host cells, and production of albumin fusion proteins by synthetic and recombinant techniques comprising a polynucleotide encoding an albumin fusion protein of the invention. The vector may be, for example, a phage, plasmid, virus or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally can only occur in complementary host cells.

  A polynucleotide encoding an albumin fusion protein of the invention may be linked to a vector containing a selectable marker for growth in a host. In general, plasmid vectors are introduced during precipitation, such as calcium phosphate precipitation, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell and then transduced into the host cell.

  The polynucleotide insert is operably linked to a suitable promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and the promoters of retroviral LTRs, to name a few. Should. Other suitable promoters are known to those skilled in the art. Expression constructs can further include sites for transcription initiation, termination, and in the transcription region, ribosome binding sites for translation. The coding portion of the transcript expressed by the construct preferably includes a translation start codon and a termination codon (UAA, UGA or UAG) at the start, located appropriately at the end of the polypeptide to be translated. .

  As suggested, the expression vector preferably includes at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or amypicillin resistance genes for culture in E. coli and other bacteria. It is. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cell bacterial cells, yeast cells (eg, Saccharomyces cerevisiae or Pichia pastoris (ATCC accession number Fungi cells such as 2011178)), insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells, and plant cells. Suitable culture media and conditions for the above host cells are known in the art.

  Preferred vectors for use in bacteria are pQE70, pQE60 and pQE-9 available from QIAGEN, Inc., available from Stratagene Cloning Systems, Inc. Examples include pBluescript vector, Pagescript vector, pNH8A, pNH16a, pNH18A, pNH46A, and prc99a, pKK223-3, pKK540-3, pDR540, pDR540 available from Pharmacia Biotech, Inc. Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene, and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to, pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1 , PPIC3.5K, pPIC9K, and PAO815 (all are available from Invitrogen, Cartbad, CA). Other suitable vectors will be readily apparent to those skilled in the art.

  In one embodiment, a polynucleotide encoding an albumin fusion protein of the invention directs the localization of the protein of the invention to a specific compartment of a prokaryotic or eukaryotic cell and / or prokaryotic or eukaryotic. It may be fused to a signal sequence that directs secretion of the protein of the invention from a nuclear cell. For example, in E. coli, it may be desired to direct protein expression to the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) that the albumin fusion protein of the invention may be fused to direct the expression of the polypeptide to the bacterial periplasmic section include, but are not limited to, the pelB signal Sequence, maltose binding protein (MBP) signal sequence, MBP, ompA signal sequence, periplasmic E. coli heat labile enterotoxin B-subunit signal sequence, and alkaline phosphatase signal sequence. Various vectors, such as the pMAL vector series (particularly the pMAL-p series) available from New England Biolabs, are commercially available for the construction of fusion proteins directed to protein localization. In certain embodiments, the polynucleotide albumin fusion proteins of the invention may be fused to a pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria. Good. See US Pat. Nos. 5,576,195 and 5,846,818, all of which are hereby incorporated by reference.

Examples of signal peptides that may be fused to the albumin fusion protein of the present invention to direct its secretion in mammalian cells include, but are not limited to:
a) MPIF-1 signal sequence (eg, amino acids 1-21 of GenBank accession number AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO: 6)
b) stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO: 7)
c) Pre-pro region of the HSA signal sequence (eg MKWVTFISLLFLSSAYSRRGVFRR, SEQ ID NO: 8)
d) Pre-region of HSA signal sequence (eg MKWVTFISLLFLFFSSAYS, SEQ ID NO: 9) or a variant thereof such as MKWVSFISLLFLFFSAYS, (SEQ ID NO: 10) e) Invertase signal sequence (eg MLLQAFLFLLAGFAAKISA, SEQ ID NO: 11)
f) Yeast mating factor alpha signal sequence (eg, MRFPSIFTAVLAFAASSALALAVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSTNSTNGLLFINTTIGSIVEGTDNGSTDNGD
g) K.K. Lactis killer toxin leader sequence h) hybrid signal sequence (eg MKWVSFISLLFFSSAYSRSLEKR, SEQ ID NO: 13)
i) HSA / MFα-1 hybrid signal sequence (also known as HSA / kex2) (eg MKWVSFISLLFLSSAYSRSLDKR, SEQ ID NO: 14)
j) K. Lacti Killer / MFa-1 fusion leader sequence (eg, MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO: 15)
k) Immunoglobulin Ig signal sequence (eg, MGWSCIILFLVATATGVHS, SEQ ID NO: 16)
l) Fibrin B precursor signal sequence (eg, MERAAPSRRR VPLPLLLLGGLALLAAGVDA, SEQ ID NO: 17)
m) Clusterin precursor signal sequence (eg, MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO: 18)
n) Insulin-like growth factor-binding 4 signal sequence (eg MLPLCLVAALLLAAGPGPSLG, SEQ ID NO: 19)
o) For example, MKWVSFISLLFLSSAYSRGVFRR (SEQ ID NO: 20),
MKWVTFISLLFLFAGVLG (SEQ ID NO: 21),
MKWVTFISLLFLFSGVLG (SEQ ID NO: 22),
MKWVTFISLLFLFGGVLG (SEQ ID NO: 23),
Modified HSA leader HSA # 64-MKWVTFISLLFLFAGVSG (SEQ ID NO: 24),
Modified HSA leader HSA # 66-MKWVTFISLLFLFGGVSG (SEQ ID NO: 25),
Modified HSA (A14) leader-MKWVTFISLLFLFAGVSG (SEQ ID NO: 26),
Modified HSA (S14) leader (also known as modified HSA # 65)-MKWVTFISLLFLFSGVSG (SEQ ID NO: 27),
Modified HSA (G14) leader-MKWVTFISLLFLFGGVSG (SEQ ID NO: 28), or MKWVTFISLLFLFGGVLLGDLHKS (SEQ ID NO: 29)
P) Consensus signal sequence (MPTWAWWWLFLVLLLALWAPARG, SEQ ID NO: 30)
q) Acid phosphatase (PH05) leader (eg MFKSVVYSILAASLANA SEQ ID NO: 31)
r) Pre-sequence of MFoz-1 s) Pre-sequence of O-glucanase (BGL2)
t) Killer toxin leader u) Pre-sequence of killer toxin
v) K. Lactis killer toxin prepro (29 amino acids, pre 26 amino acids and pro 13 amino acids)
w) S.M. Diastatics glucoamylase Il secretion leader sequence x) Charisbergen cis α-galactosidase (MEL1) secretion leader sequence y) Candida glucoamylase leader sequence z) Hybrid leader aa) gp67 signal disclosed in EP-A-387319 (incorporated by reference) Sequence (in combination with a baculovirus expression system) (eg, amino acids 1-19 of GenBank accession number AAA72759) or bb) natural leader cc of therapeutic protein X) Japanese Patent No. 62-096086 (Granted as 911036516, Reference S., which is incorporated by reference). S. cerevisiae invertase (SUC2) leader, or dd) inulinase-MKLAYSLLPLAGVSAVINYKR (SEQ ID NO: 33).
ee) Modified TA57 propeptide leader mutant # 1-MKLKTVRSAVLSSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFAATQTNSGLDVVGLISMAKR (SEQ ID NO: 34)
ff) Modified TA57 propeptide leader mutant # 2-MKLKTVRSAVLSSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLVVGLISMAEEEPEPKR (SEQ ID NO: 35)
gg) Consensus signal peptide-MWWWLWWWLLLLLLLWPMVWA (SEQ ID NO: 111)
jj) Modified HSA / kex2 signal sequence-MKWVSFISLLFLFSASAGSGSLDKR (SEQ ID NO: 112)
kk) Consensus signal peptide # 2-MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 105)

  In a preferred embodiment, the modified HSA / Kex2 signal sequence (SEQ ID NO: 112) is a fusion protein comprising albumin and a therapeutic protein as described herein, and each of which is hereby incorporated by reference in its entirety. International Patent No. WO 93/15199, International Patent No. 97/24445, International Patent No. 03/60071, International Patent No. 03/59934, and International Patent No. PCT / US04 / 01369. Fusion to the amino terminus of albumin fusion proteins, including albumin fusion proteins. Modified HSA / Kex2 signal sequences are described, for example, in Sleep et al., Both of which are incorporated herein by reference in their entirety. , Biotechnology 1990, vol. 8, pp. 42-46 and US Pat. No. 5,302,697, based on the HSA / Kex2 signal sequence (SEQ ID NO: 14). The modified HSA / Kex2 leader sequence disclosed herein includes a non-conservative amino acid substitution (Arg to Gly) at residue 19 of the parent signal peptide. The modified HSA / Kex2 signal peptide is found to produce an unexpectedly better expression yield and / or more disruption effect of the albumin fusion protein when expressed in yeast than the unmodified HSA / Kex2 signal sequence. It was. Variants of modified HSA / Kex2 signal peptides are also included in the present invention. In particular, the Gly residue at position 19 of SEQ ID NO: 112 can be replaced with a Pro residue. Other conservative substitution variants of the modified HSA / Kex2 signal sequence are also contemplated. The modified HSA / Kex2 signal sequence of SEQ ID NO: 112, as well as conservative substitution variants thereof, are also included in the present invention.

  Vectors using glutamine synthase (GS) or DHFR as selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of a glutamine synthase-based vector is the availability of cell lines that are negative for glutamine synthase (eg, the murine myeloma cell line, NSO). The glutamine synthase expression system can also function in glutamine synthase expressing cells (eg, Chinese hamster oocytes (CHO) cells) by providing additional inhibitors to prevent foreign gene function. The glutamine synthase expression system and components thereof are incorporated herein by reference in their entirety, International Patent Nos. WO87 / 04462, WO86 / 05807, WO89 / 01036, WO89 / 10404. And in WO 91/06657. Furthermore, a glutamine synthase expression vector can be obtained from Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using the GS expression system in murine myeloma cells has been described by Bebbington et al., Which is incorporated herein by reference. , Bio / technology 10: 169 (1992) and Biblia and Robinson Biotechnology. Prog. 11: 1 (1995).

  The present invention also relates to host cells comprising the above-described vector constructs described herein, and further using one or more heterologous regulatory regions (eg, promoters and / or enhancers) using techniques known in the art. And a host cell comprising a nucleotide sequence of the invention operably linked to. The host cell can be a higher order eukaryotic cell such as a mammalian cell (eg, a human derived cell), or a lower order eukaryotic cell such as a yeast cell, or the host cell can be a bacterial cell. It can be a prokaryotic cell. The host strain may be selected to modulate the expression of the inserted gene sequence or to modify and process the gene product in the specific fashion desired. Expression from a particular promoter is increased in the presence of a particular inducer and can thus control the expression of a genetically modified polypeptide. Furthermore, different host cells have characteristic and special mechanisms for the translation and post-translational processing and modification (eg phosphorylation, cleavage) of proteins. Appropriate cell lines can be selected to ensure the desired modification and processing of the expressed foreign protein.

  Introduction of the nucleic acids and nucleic acid constructs of the present invention into host cells can be performed by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic live lipid mediated transfection, electroporation, transduction, infection or other methods. . Such a method is described in Davis et al. , Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the invention may indeed be expressed by host cells that lack a recombinant vector.

  In addition to containing host cells containing the vector constructs discussed in the present invention, the present invention also provides coding sequences corresponding to therapeutic proteins (eg, corresponding to therapeutic proteins). May be replaced with an albumin fusion protein corresponding to a therapeutic protein) and / or to contain genetic material (eg, a heterologous protein such as an albumin fusion protein of the invention corresponding to a therapeutic protein). Also included are first, second and immortalized host cells derived from genetically engineered vertebrates, particularly mammals, which may contain polynucleotide sequences. Genetic material operably linked to the endogenous polynucleotide may activate, alter and / or amplify the endogenous polynucleotide.

  In addition, using techniques known in the art, heterologous polynucleotides (eg, polynucleotides encoding albumin fusion proteins, or fragments or variants thereof) and / or heterologous regulatory regions (eg, promoters and / or Or enhancers) may be operably linked to the endogenous polynucleotide sequence encoding the therapeutic protein via homologous recombination (eg, with disclosures that are each incorporated by reference respectively). U.S. Pat. No. 5,641,670, International Patent Specification No. WO 96/29411, International Patent Specification No. WO 94/12650, Koller et al., Proc, issued June 24, 1997. Natl.Acad.Sci.USA 86: 893 2-8935 (1989), and Zijlstra et al., Nature 342: 435-438 (1989)).

  The albumin fusion protein of the present invention comprises ammonium sulfate or ethanol precipitation, acid extraction, anion and cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, hydrophobic charge It can be recovered and purified from recombinant cell culture media by well-known methods, including interaction chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

  In a preferred embodiment, the albumin fusion protein of the present invention is, but not limited to, Q-Sepharose, DEAE Sepharose, Poros HQ, Poros DEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Purify using anion exchange chromatography, including DEAE column chromatography.

  In certain embodiments, the albumin fusion proteins of the present invention include, but are not limited to, SP-Sepharose, CM Sepharose, Poros HS, Poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S and CM, Fractogel S and CM columns And cation exchange chromatography containing their equivalents and equivalents.

  In certain embodiments, the albumin fusion protein of the present invention is, but is not limited to, phenyl, butyl, methyl, octyl, hexyl-sepharose, porosphenyl, butyl, methyl, octyl, hexyl, Toyopearl phenyl, butyl, methyl, octyl Purify using hydrophobic interaction chromatography, including hexyl Resource / Source phenyl, butyl, methyl, octyl, hexyl, Fractogel phenyl, butyl, methyl, octyl, hexyl columns and their equivalents and equivalents.

  In certain embodiments, the albumin fusion proteins of the invention are purified using size exclusion chromatography, including but not limited to Sepharose S100, S200, S300, and Superdex resin and their equivalents and equivalents. .

  In certain embodiments, the albumin fusion proteins of the present invention include affinity, including but not limited to peptide affinity and antibody affinity columns that are selective for either Mimatic Dye affinity, HSA or “fusion target” molecules. Purify using chromatography.

  In a preferred embodiment, the albumin fusion protein of the invention is purified using one or more chromatography listed above. In another preferred embodiment, the albumin fusion protein of the present invention comprises one or more of the following chromatography columns: Q Sepharose FF column, SP Sepharose FF column, Q Sepharose high performance column, Blue Sepharose FF column, Blue column. Purify using a Phenyl Sepharose FF column, DEAE Sepharose FF or methyl column.

  Further, the albumin fusion protein of the present invention may be purified using the process described in PCT International Patent Specification No. WO 00/44772, all of which are incorporated herein by reference. Good. One skilled in the art can easily modify the process described therein for use in the purification of albumin fusion proteins of the invention.

  The albumin fusion proteins of the present invention are recovered from the products of chemical synthesis procedures and products produced by recombinant techniques from prokaryotic or eukaryotic hosts including, for example, bacteria, yeast, higher plants, insects and mammalian cells. May be. Depending on the host utilized in the recombinant production procedure, the polypeptide of the invention may or may not be glycosylated. Furthermore, the albumin fusion proteins of the present invention may also include an internally modified methionine residue in some cases as a result of host-mediated processes. Thus, it is well known that the N-terminal methionine encoded by the translation initiation codon is generally efficiently removed from any protein after translation in all eukaryotic cells. The N-terminal methionine on most proteins is also efficiently removed in most prokaryotes, and this prokaryotic for some proteins depends on the nature of the amino acid to which the N-terminal methionine is covalently attached. The biological removal process is inefficient.

In one embodiment, yeast Pichia pastoris is used to express the albumin fusion protein of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast that can metabolize methanol as its sole carbon source. Main stages methanol metabolic pathway is the oxidation of methanol to formaldehyde using O _2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris, in part, because relative O ₂ is relatively low affinity alcohol oxidase must produce high levels of alcohol Oxidase. As a result, one promoter region of two alcohol oxidase genes (AOX1) is very active during growth culture depending on methanol as the main carbon source. Alcohol oxidase produced from the AOX1 gene in the presence of methanol contains up to approximately 30% total soluble protein in Pichia pastoris. Ellis, S.M. B. , Et al. , Mol. Cell. Biol. 5: 1111-21 (1985); Koutz, P .; J, et al. Yeast 5: 167-77 (1989); Tschop, J. et al. F. , Et al. , Nucl. Acids Res. 15: 3859-76 (1987). Thus, heterologous coding sequences, such as, for example, the polynucleotides of the present invention, are expressed at otherwise high levels in Pichia yeast growth in the presence of methanol under the transcriptional control of all or part of the AOX1 regulatory sequences.

  In one example, the plasmid vector pPIC9K was used essentially in “Pichia Protocols: Methods in Molecular Biology”, D.M. R. Higgins and J.H. Cregg, eds. The DNA encoding the albumin fusion protein of the present invention is expressed in a Pichia yeast system, as listed herein, as described in The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of the polypeptide of the present invention thanks to the strong AOX1 promoter linked to a Pichia pastoris alkaline phosphatase (PHO) secretion signal peptide (ie leader) located upstream of the multiple cloning site. It is.

  As one skilled in the art will readily appreciate, the proposed expression construct will provide appropriate localized signals for transcription, translation, secretion (if desired), including in-frame AUG if necessary. And many other yeast vectors such as pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815. Can be used in place of pPIC9K.

  In other embodiments, the invention provides for high level expression of heterologous coding sequences, such as polynucleotides encoding albumin fusion proteins of the invention, into expression vectors such as pGAPZ or pGAPZalpha. The cloning of the heterologous polynucleotide and the growth of the yeast culture in the absence of methanol.

  Furthermore, the albumin fusion proteins of the present invention can be chemically synthesized using techniques known in the art (see, eg, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310: 105-111 (1984)). For example, a polypeptide corresponding to a fragment of the polypeptide can be synthesized by using a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, the D-isomers of common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu E-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t -Designer amino acids such as butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, b-methylamino acids, Ca-methylamino acids, Na-methylamino acids and common amino acid analogs. Further, the amino acid can be D (dextrorotatory) or L (left-handed).

  The invention can be used during or after translation, for example by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linking to antibody molecules or other cellular ligands, etc. Included are albumin fusion proteins of the invention that are modified differently. Any number of chemical modifications include, but are not limited to, cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, specific chemical cleavage with NaBH4, acetylation, formylation, oxidation, reduction, metabolism in the presence of tunicamycin You may implement by a well-known technique including an automatic synthesis etc.

  Further post-translational modifications included by the present invention include, for example, N-linked or O-linked sugar chains, treatment of the N-terminal or C-terminal end, attachment of chemical moieties to the amino acid backbone, N-linked or O-linked Includes chemical modification of the sugar chain and addition or deletion of the N-terminal methionine residue as a result of prokaryotic host cell expression. The albumin fusion protein may also be modified with a detectable label, such as an enzymatic, fluorescent, isotonic or affinity label, to allow detection and isolation of the protein.

Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, and examples of suitable artificial group complexes include streptavidin / biotin and avidin / biotin. Examples of fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, examples of luminescent materials include luminol, and bioluminescence Examples of substances include luciferase, luciferin and aequorin, and examples of suitable radioactive substances include iodine ( ¹²¹ I, ¹²³ I, ¹²⁵ I, ¹³¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S). , Tritium ³ H), indium ^{(111 In, 112 In, 113m} In, 115m In) technetium ^{(99 Tc,} 99m ^Tc), Sariumu ⁽²⁰¹ Ti), gallium ^{(68 Ga,} 67 ^Ga), palladium ⁽¹⁰³ Pd), Moribudeniumu ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, and ⁹⁷ Ru are included.

In certain embodiments, the albumin fusion protein or fragment or variant thereof of the present invention binds to a radioactive metal ion including, but not limited to, ¹⁷⁷ Lu, ⁹⁰ Y, ¹⁶⁶ Ho, and 153 Sm to the polypeptide. Combines with a macrocyclic chelator. In a preferred embodiment, the emissive metal ion associated with the macrocyclic chelator is ¹¹¹ In. In another preferred embodiment, the radioactive metal ion associated with the macrocyclic chelator is ⁹⁰ Y. In a particular embodiment, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid (DOTA). In another specific embodiment, DOTA binds to an antibody of the invention or fragment thereof via a linker molecule. Examples of linker molecules useful for conjugating DOTA to polypeptides are generally known in the art and are described, for example, in DeNardo et al. , Clin Cancer Res. 4 (10): 2483-90 (1998); Peterson et al. , Bioconjug. Chem. 10 (4): 553-7 (1999), and Zimmerman et al, Nucl. Med. Biol. 26 (8): 943-50 (1999).

  As mentioned, the albumin fusion protein of the invention may be modified either by natural processes such as post-translational processing or by chemical modification techniques well known in the art. It will be understood that the same type of modification may be present in the same or varying degrees at various sites within the polypeptide. The polypeptides of the invention may be branched, for example as a result of ubiquinoneation, and may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may be the result of post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin supply linkage, hemosite covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphotidylinositol Covalent bond, crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cysteine formation, pyroglutamic acid formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination -RNA-mediated addition of amino acids to proteins, such as methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitination (E.g., PROTEINS − STRUCTURE AND MOLECULAR PROPERITES, 2nd Ed., T.E. York, pgs.1-12 (1983); Seifter et al., Meth.Enzymol.182: 626-646 (1990); Rattan et al., Ann.NY Acad. Sci. 663: 48-62 ( 1992)).

  The albumin fusion proteins of the present invention, and antibodies that bind to therapeutic proteins or fragments or variants thereof can be fused to marker sequences such as peptides to facilitate purification. In a preferred embodiment, the marker amino acid sequence is available in many commercially available, such as hexa tags, such as the tags provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91111). -A histidine peptide. For example, Gentz et al. , Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), hexa-histidine provides convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, “HA” tags (Wilson et al., Cell 37: 767 (1984)) and “corresponding to epitopes derived from influenza hemagglutinin protein” "flag" tag is included.

  Furthermore, the albumin fusion proteins of the present invention may be conjugated to therapeutic moieties such as cytotoxins such as cytostatic or cytocidal agents, therapeutic agents or radioactive metals such as alpha-radiants such as 213Bi. . A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, methine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, corcitine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, misramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propanolol and puromycin, and analogs or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechloretamine, thioepachlorambucil, melphalan, Carmustine (BSNU) and Lomustine (CCNU), cyclosofamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatinum (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) And doxorubicin), antibiotics (eg dactinomycin (formerly actinomycin), bleomycin, misramycin and ansoramicin (AMC) )) And antimitotic drugs (eg vincristine and vinblastine).

  The conjugates of the invention can be used to modify the biological response and the therapeutic agent or drug moiety is not to be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, Pseudomonas etotoxin, or diphtheria toxin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminol Proteins such as gen activators, apoptotic drugs such as TNF-alpha, TNF-beta, AIMI (see International Patent Specification No. WO 97/33899), AIM II (International Patent Specification No. WO 97/34911) ), Fas ligand (Takahashi et al., Int. Immunol., 6: 1567-1574 (1994)), VEGI (see International Patent Specification No. WO 99/23105), eg, Angiostati Or anti-angiogenic agents, such as lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 ( "IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF") or other growth factors. Techniques for conjugating such therapeutic moieties to physiological response modifiers such as proteins (eg, albumin fusion proteins) are well known in the art.

  The antibody may also be bound to a solid support and is particularly useful for immunoassays or purification of polypeptides bound to, bound to, or linked to an albumin fusion protein of the invention. Such solid supports include but are not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

  Albumin fusion proteins administered with or without a conjugated therapeutic site, alone or in combination with cytotoxic factor (s) and / or cytokine (s) can be used as therapeutics.

  In embodiments where the albumin fusion protein of the present invention includes only the VH domain of an antibody that binds to the therapeutic protein, it is necessary to co-express the fusion protein with the VL domain of the same antibody that binds to the therapeutic protein. And / or desirably, the VH-albumin fusion protein and the VL protein are linked after transcription (either covalently or non-covalently).

  In embodiments where the albumin fusion protein of the present invention includes only the VL domain of an antibody that binds to the therapeutic protein, it is necessary to co-express the fusion protein with the VH domain of the same antibody that binds to the therapeutic protein. And / or desirably, the VL-albumin fusion protein and the VH protein are linked post-transcriptionally (either covalently or non-covalently).

  Some therapeutic antibodies are bispecific antibodies, meaning that an antibody that binds to a therapeutic protein is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. To make an albumin fusion protein corresponding to a therapeutic protein, it is possible to make an albumin fusion protein with an scFv fragment fused to both the N- and C-termini of the albumin protein site. More particularly, the scFv fused to the N-terminus of albumin represents one of the original antibody heavy / light (VH / VL) pairs that bind to the therapeutic protein, and the scFv fused to the C-terminus of albumin , Corresponding to other heavy / light (VH / VL) pairs of native antibodies that bind to therapeutic proteins.

  Also provided by the present invention are chemically modified derivatives of the albumin fusion proteins of the present invention that may provide additional benefits, such as increased solubility, stability and circulation time of the polypeptide, or decreased immunogenicity. (See U.S. Pat. No. 4,179,337). The chemical moiety for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. Albumin fusion proteins may be modified at random locations within the molecule or at predetermined locations within the molecule and may comprise one, two, three or more binding chemical sites.

  The polymer may be of any molecular weight and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is about 1 kDa to about 100 kDa for ease of handling and manufacturing (the phrase “about” means that some molecules are heavier than the standard molecular weight in the preparation of polyethylene glycol. Or suggest a slightly lighter). Desired therapeutic profile (eg, desired duration of sustained release, effects in biological activity, ease of handling, degree or lack of antigenicity, and other known effects of polyethylene glycol on therapeutic proteins or analogs) Depending on the, other sizes may be used. For example, polyethylene glycol is about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000. 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16 , 500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000 50,000, 55,000, 60,000, 65,000, 70,000 75,000,80,000,85,000,90,000,95,000 or may have an average molecular weight of 100,000kDa,.

  As mentioned above, polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in US Pat. No. 5,643,575, Morpurgo et al., The disclosures of each of which are hereby incorporated by reference. , Appl. Biochem. Biotechnol. 56: 59-72 (1996); Vorobjev et al. , Nucleosides Nucleotides 18: 2745-2750 (1999); and Calisetti et al. , Bioconjug. Chem. 10: 638-646 (1999).

  Polyethylene glycol molecules (or other chemical moieties) should be attached to the protein taking into account effects on the functional or antigenic domains of the protein. Numerous attachment methods are available to those skilled in the art, such as, for example, the method disclosed in European Patent No. 0401384 (PEG attachment to G-CSF), which is incorporated by reference. Also, Malik et al., Who reported PEGylation of GM-CSF using tresyl chloride. , Exp. Hematol. 20: 1028-1035 (1992). For example, polyethylene glycol may be covalently bonded through an amino acid residue through a reactive group, such as a free amino or carboxyl group. A reactive group is one to which an activated polyethylene glycol molecule binds. Amino acid residues having a free dance group include lysine residues and N-terminal amino acid residues, and those having a free carboxyl group include aspartic acid residues and glutamic acid residues, and C-terminal amino acid residues. Groups can be included. Sulfohydryl groups can also be used as reactive groups for attaching polyethylene glycol molecules. For therapeutic purposes, attachment at the amino group is preferred, such as attachment at the N-terminus or lysine group.

  As pointed out above, the polyethylene glycol may be bound to the protein via a bond to any number of amino acid residues. For example, polyethylene glycol can be linked to a protein via a covalent bond to a lysine, histidine, aspartic acid, glutamic acid or cysteine residue. One or more reaction chemistry is directed to a specific amino acid residue of a protein (eg, lysine, histidine, aspartic acid, glutamic acid or cysteine) or one or more types of amino acid residues of a protein (eg, lysine, Histidine, aspartic acid, glutamic acid, cysteine, and combinations thereof) may be utilized to bind polyethylene glycol.

  Proteins that are chemically modified at the N-terminus may be particularly desirable. Using polyethylene glycol as an example of this composition, various polyethylene glycol molecules (by molecular weight, branching, etc.), ratio of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mixture, pegylation reaction carried out And a method for obtaining a selected N-terminal PEGylated protein. Methods for obtaining N-terminal PEGylation modulators (ie, separating this site from other mono-pegylated sites, if necessary) can be obtained from N-terminal PEGylated material from a population of PEGylated protein molecules. Can be by purification. Selective proteins modified with N-terminal modifications utilize different reactivity of different types of primary amino groups (lysine versus N-terminal) reduction available for derivatization in specific proteins It can be achieved by alkylation. Under appropriate reaction conditions, selective derivatization of the protein at the N-terminus with an essentially carboxyl group-containing polymer is achieved.

  As suggested above, pegylation of the albumin fusion protein of the invention can be accomplished by any number of methods. For example, polyethylene glycol may be attached to the albumin fusion protein directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are disclosed by Delgado et al., Each of which is incorporated herein by reference. , Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992); Francis et al. , Intern. J. et al. of Hematol. 68: 1-18 (1998), U.S. Pat. No. 4,002,531, U.S. Pat. No. 5,349,052, International Patents WO 95/06058 and WO 98/32466.

One system for attaching polyethylene glycol directly to amino acid residues of a protein without an intervening linker is produced by modification of monmethoxypolyethylene glycol (MPEG) using tresyl chloride (ClSO ₂ CH ₂ CF ₃ ). Use tresylated MPEG. During tresylated MPEG of proteins, polyethylene glycol is directly attached to the amino group of the protein. Accordingly, the present invention includes protein-polyethylene glycol conjugates produced by reacting the protein of the present invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethanesulfonyl group.

  Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, US Pat. No. 5,612,460, the entire disclosure incorporated herein by reference, discloses urethane linkers for linking polyethylene glycol to proteins. A protein-polyethylene glycol conjugate in which polyethylene glycol is attached to the protein by a linker is also MPEG, MPEG-2,4,5- It can be produced by reaction of proteins with compounds such as trichloropenyl carboxylic acid, MPEG-p-nitrophenol carboxylic acid, and various MPEG-succinic acid derivatives. A number of additional polyethylene glycol derivatives and chemical reactions for attaching polyethylene glycol to proteins are described in International Patent No. WO 98/32466, the entire disclosure incorporated herein by reference. Yes. Included within the scope of the present invention are PEGylated protein products produced using the chemical reactions listed herein.

  The number of polyethylene glycol sites that bind to each albumin fusion protein of the invention (ie, the degree of substitution) may also vary. For example, the PEGylated proteins of the present invention bind on average to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. May be. Similarly, the average degree of substitution is 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12 per protein. 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19 or 18-20 within the range of polyethylene glycol molecules. Methods for determining the degree of substitution are described, for example, in Delgado et al. , Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992).

  Polypeptides of the invention can be, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography It can be recovered from chemical synthesis and recombinant cell culture medium and purified by standard methods, including hydrophobic charge interaction chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification. Well-known techniques for refolding proteins may be used to reshape the active conformation when the polypeptide is denatured during isolation and / or purification.

The presence and amount of the albumin fusion protein of the invention may be determined using an ELISA, a well-known immunoassay known in the art. One ELISA protocol that may be useful for detecting / quantifying the albumin fusion protein of the invention includes coating the ELISA plate with the albumin fusion protein of the invention, blocking the plate to prevent non-specific binding. Washing the ELISA plate, adding a solution containing the albumin fusion protein of the invention (at one or more different concentrations), as described herein or as known in the art And) adding a secondary anti-therapeutic protein specific antibody conjugated to a detectable label and detecting the presence of the secondary antibody. In other versions of this protocol, ELISA plates can be coated with an anti-therapeutic protein specific antibody and the labeled secondary reagent can be an anti-human albumin specific antibody.

Use of polynucleotides

  Each polynucleotide identified herein can be used as a reagent in a number of ways. The following description should be considered as an example and utilizes known techniques.

  The polynucleotide of the present invention is useful for producing the albumin fusion protein of the present invention. As described in more detail below, the polynucleotides of the invention (encoding albumin fusion proteins) are used in recombinant DNA methods useful in genetic modification to produce albumin fusion proteins of the invention. Cells, cell lines or tissues expressing an albumin fusion protein encoded by a polynucleotide encoding can be produced.

The polynucleotides of the invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism with a defective gene in order to correct the genetic defect. The polynucleotides disclosed in the present invention provide a method for targeting such genetic defects in a very accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new feature in the host cell. Further non-limiting examples of gene therapy methods encompassed by the present invention are more fully described elsewhere herein (see, eg, item “Gene Therapy” and Examples 61 and 62).

Use of polypeptides

  Each polypeptide identified herein can be used in a number of ways. The following description should be considered as an example and utilizes known techniques.

  Albumin fusion proteins of the present invention can be expressed in tissue (s) (eg, immunohistochemical assays such as ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29: 577-580 (1981)), or cell types. Useful for providing immunological probes for different identification of class (es) (eg immunocytochemical assays).

Albumin fusion proteins are obtained by classical immunohistological methods known to those skilled in the art (eg, Jalkanen, et al., J. Cell. Biol. 101: 976-985 (1985); Jalkanen, et al., J. Cell. Biol.105: 3087-3096 (1987)) can be used to assay the level of a polypeptide in a biological sample. Other methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Known in suitable assay labels the art and include enzyme labels, such as glucose oxidase, iodine ^{(121 I, 123 I, 125} I, 131 I), carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), tritium ( ³ H), indium ^{(111 In, 112 In, 113m} In, 115m In) technetium ^{(99 Tc,} 99m ^Tc), Sariumu ⁽²⁰¹ Ti), gallium ^{(68 Ga,} 67 ^Ga), palladium ⁽¹⁰³ Pd), Moribudeniumu ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, radioisotopes such as ^{142 Pr, 105 Rh, 97 Ru} , Luminescent labels, such as Minoru, and fluorescein and fluorescent labels, such as rhodamine, and a biotin.

  The albumin fusion proteins of the invention can also be detected in vivo by imaging. Labels or markers for in vivo imaging of proteins include those detectable by X-radiography, nuclear magnetic resonance (NMR) or electron spin resonance (ESR). For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not clearly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, and labeling of nutrients provided to cell lines expressing the albumin fusion protein of the invention Can be incorporated into an albumin fusion protein.

Radioisotopes (eg, ¹³¹ I, ¹¹² In, ^99m Tc, ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹¹ In, ¹¹² In, ^{113 m} In, ^{115 m} In) Technetium ( ⁹⁹ Tc, ^{99 m} Tc), Salium ( ²⁰¹ Ti), Gallium ( ⁶⁸ Ga, ⁶⁷ Ga), Palladium ( ¹⁰³ Pd), Molybdenium ( ⁹⁹ Mo), Xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Detected by Ru), radiopaque substrate, or nuclear magnetic resonance Ability suitable detectable (e.g. parenteral, subcutaneous or intraperitoneal) to a mammal to be tested for imaging sites labeled albumin fusion proteins of the immune system diseases such as substances introduced. It will be appreciated that the size of the object and the imaging system used will determine the amount of imaging site needed to provide a diagnostic image. In the case of a radioisotope site, for a human subject, the amount of radioactivity injected will typically range from about 5 to 20 millicuries of ^99m Tc. The labeled albumin fusion protein is then preferably in the body in which one or more receptors, ligands or substrates (corresponding to those of the therapeutic protein used to make the albumin fusion protein of the invention) are localized. Accumulate in a location (eg, organ, cell, extracellular space or matrix). Alternatively, if the albumin fusion protein comprises at least one fragment or variant of a therapeutic antibody, the labeled albumin fusion protein is preferably a therapeutic antibody (used to make the albumin fusion protein of the invention). Polypeptides / eptotops corresponding to those bound by have accumulated in locations in the body where they are localized (eg organs, cells, extracellular space or matrix). In vivo tumor imaging W. Burchiel et al. , “Immunopharmacokinetics of Radiolabeled Antibodies and Therology. The Chapter of the Biochem. , Masson Publishing Inc. (1982)). The protocol described therein can be easily modified by those skilled in the art for use with the albumin fusion proteins of the present invention.

  In one embodiment, the invention provides an albumin fusion protein of the invention conjugated to a heterologous polypeptide or nucleic acid (eg, a polypeptide and / or antibody encoded by a polynucleotide encoding an albumin fusion protein of the invention). ) Is provided for the specific delivery of albumin fusion proteins of the present invention to cells. In one example, the present invention provides a method for delivering a therapeutic protein to a targeted cell. In other examples, the invention can be integrated into a target cell, single-stranded nucleic acid (eg, antisense or ribozyme) or double-stranded nucleic acid (eg, integrated into the genome of the cell, or replicated episomally) And a method is provided for delivering DNA) that is replicable.

  In another embodiment, the present invention provides a method for the specific destruction of cells by administering an albumin fusion protein of the invention conjugated with a toxin or cytotoxic prodrug.

By "toxin", an endogenous cytotoxic effector system, radioisotope, holotoxin, modified toxin, toxin catalytic subunit, or in a cell or cell surface that causes cell death under defined conditions By one or more molecules that bind and activate any molecule or enzyme not normally present above is meant. Toxins that can be used in accordance with the methods of the present invention include, but are not limited to, radioisotopes known in the art, such as innate or induced endogenous cytotoxic effector systems, thymidine kinases, endonucleases, Like RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, xeronine, pokeweed antiviral protein, alpha-sarcin and cholera toxin-binding antibodies (or complement binding including parts thereof) Compounds. “Toxins” also include cytostatic or cytocidal agents, therapeutic agents or radioactive metal ions, eg alpha-radiants such as ²¹³ Bi, or eg ¹⁰³ Pd, ¹³³ Xe, ¹³¹ I, ⁶⁸ Ge, ^{57 Co, 65 Zn, 85 Sr,} 32 P, 35 S, 90 Y, 153 Sm, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, 90 Yttrium, 117 Tin, 186 ^{rhenium, 166} holmium, And other radioisotopes such as ¹⁸⁸ rhenium, luminescent labels such as luminol, fluorescent labels such as fluorescein and rhodamine, and biotin. In certain embodiments, the invention provides for the specific destruction of cells (eg, tumor cell destruction) by administering a polypeptide of the invention or an antibody of the invention conjugated to the radioisotope 90Y. Provide a method. In another specific embodiment, the present invention provides for the specific destruction of cells (eg, the destruction of tumor cells) by administering a polypeptide of the invention or an antibody of the invention conjugated to the radioisotope ¹¹¹ In. Provide a way for. In a further specific embodiment, the present invention relates to the specific destruction of cells (eg tumor cell destruction) by administering a polypeptide of the invention or an antibody of the invention conjugated to a radioisotope ¹³¹ I. Providing a method for

  Techniques known in the art can be applied to the lab polypeptides of the present invention. Such techniques include, but are not limited to, the use of bifunctional conjugates (eg, US Pat. No. 5,756,065, each of which is incorporated by reference in its entirety). 5,714,631, 5,696,239, 5,652,361, 5,505,931, 5,489,425, 5,435,990, 5,428,139, 5,342,604, 5,274; 119, 4,994,560, and 5,808,003).

  The albumin fusion proteins of the present invention are useful for diagnosis, treatment, prevention and / or prognosis of various diseases in mammals, preferably humans. Such diseases include, but are not limited to, those described herein under the following item “Bionuclear Activity”.

  Accordingly, the present invention comprises (a) assaying the expression level of a particular polypeptide in an individual cell or body fluid using the albumin fusion protein of the present invention, and (b) the assayed polypeptide expression level, A method for diagnosing a disease is provided, comprising comparing to a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level relative to the standard expression level is indicative of the disease. For cancer, the presence of a relatively large amount of transcript in biopsy tissue from an individual may suggest a predisposition to disease development, or a method for detecting disease before the occurrence of actual clinical symptoms Can be provided. This type of more definitive diagnosis may allow health professionals to take advantage of early preventive measures or aggressive treatment, thereby preventing the development or further progression of cancer.

  Furthermore, the albumin fusion protein of the present invention can be used, for example, for neurological diseases, immune system diseases, muscle diseases, regeneration diseases, gastrointestinal diseases, lung diseases, cardiovascular diseases, kidney diseases, proliferative diseases, and / or cancerous diseases and conditions. Can be used to treat or prevent diseases or conditions such as For example, to replace a patient with a lack of or decreased level of a polypeptide (eg, insulin), to compensate for a lack or level of a different polypeptide (eg, hemoglobin S, SOD, catalase, DNA repair protein relative to hemoglobin B) To inhibit the activity of the polypeptide (for example by binding to the receptor), to activate the activity of the polypeptide (for example by binding to the receptor), A polypeptide of the invention can be administered to reduce activity (eg, soluble TNF receptor in reducing inflammation) or to cause a desired response (eg, inhibit vascular growth, enhance immune response to proliferating cells or tissues) It is.

  In particular, albumin fusion proteins comprising at least one fragment or variant of a therapeutic antibody can also be used to treat a disease (as described above and as described elsewhere herein). is there. For example, administration of an albumin fusion protein comprising at least one fragment or variant of a therapeutic antibody binds and / or neutralizes the polypeptide to which the therapeutic antibody used to make the albumin fusion protein specifically binds. And / or can reduce overproduction of polypeptides to which the therapeutic antibody used to make the albumin fusion protein specifically binds. Similarly, administration of an albumin fusion protein comprising at least one fragment or variant of a therapeutic antibody may be used to make an albumin fusion protein by binding to a polypeptide bound to a membrane (receptor). It is possible to activate a polypeptide to which an antibody specifically binds.

Finally, the albumin fusion proteins of the invention can be used as molecular mass markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those skilled in the art. The albumin fusion proteins of the invention can also be used to produce antibodies, thereby providing therapeutic methods from recombinant cells or in biological samples as a way to assess host cell transduction. It may be used to measure proteins, albumin proteins, and / or albumin fusion proteins of the invention. In addition, the albumin fusion proteins of the present invention can be used to test the biological activities described herein.

Diagnostic assay

  The compounds of the present invention are useful for the diagnosis, treatment, prevention and / or prognosis of various diseases in mammals, preferably humans. Such diseases include, but are not limited to, the corresponding columns of Table 1 and items “immune activity”, “blood related diseases”, “hyperproliferative diseases”, “kidney diseases” above, “Cardiovascular disease”, “respiratory disease”, “anti-angiogenic activity”, “disease at the cellular level”, “wound healing and epithelial proliferation”, “neurological activity and neurological disease”, “endocrine disease” , "Regenerative system diseases", "infectious diseases", "regeneration", and / or "gastrointestinal diseases", those described for each therapeutic protein.

  For many diseases, there is an essentially altered (increased or decreased) level at the gene level compared to the “standard” gene expression level, ie, the expression level in tissues or body fluids from individuals without the disease. Can be detected in tissues, cells or body fluids (eg serum, plasma, urine, semen, synovial fluid or cerebrospinal fluid) taken from individuals with such diseases. Accordingly, the present invention provides diagnostic methods that are useful during disease gait diagnosis, measuring the expression levels of genes encoding polypeptides in tissues, cells or body fluids from individuals, and standard Comparing the gene expression level with the measured gene expression level, whereby an increase or decrease in gene expression level compared to the standard is indicative of the disease. These diagnostic assays may be performed in vivo or in vitro, such as on a blood sample, biopsy tissue or autopsy tissue.

  The present invention is also useful as a prognostic indicator, whereby patients showing enhanced or decreased gene expression may experience worse clinical results.

  “Assaying the expression level of a gene encoding a polypeptide” can be direct (eg, by determination or estimation of absolute protein levels or mRNA levels) or relative (eg, polypeptides in a second biological sample). The level of a particular polypeptide (eg, a polypeptide corresponding to a therapeutic protein disclosed in Table 1) in the first biological sample, either by comparing the level or mRNA level, or It is intended to qualitatively and quantitatively measure or estimate the level of mRNA encoding the polypeptide of the invention. Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, wherein the standard is obtained from an individual without a disease. Determined by averaging levels from individual populations obtained from biological samples or without disease. As understood in the art, once a standard polypeptide level or mRAN level is known, it can be used repeatedly as a standard for comparison.

  By “biological sample” any biological sample obtained from an individual, cell line, tissue culture fluid or other source containing a polypeptide or mRNA of the invention (including parts thereof) Intended. As indicated, biological samples include body fluids (such as serum, plasma, urine, synovial fluid and cerebrospinal fluid), and tissue sources found to express the full length of polypeptide or mRNA or fragments thereof. Is included. Tissue biopsies and body fluids from mammals are well known in the art. When the biological sample is to contain mRNA, tissue biopsy is the preferred source.

  Total cellular RNA was obtained from Chomczynski and Sacchi, Anal. Biochem. 162: 156-159 (1987) can be isolated from a biological sample using any suitable technique such as the single stage guanidinium-thiocyanate-phenol-chloroform method. The level of mRNA encoding the polypeptide of the invention is then assayed using any suitable method. These include Northern blot analysis, S1 nuclease mapping, polymerase chain reaction (PCR), reverse transcription combined with polymerase chain reaction (RT-PCR), and reverse transcription combined with ligase chain reaction (RT-LCR).

  The present invention also includes polypeptides that are bound by, bound to or linked to the albumin fusion proteins of the present invention in biological samples (eg, cells and tissues), including measurement of normal and abnormal levels of the polypeptide. Relates to diagnostic assays, such as quantitative and diagnostic assays for detecting levels. Thus, for example, a diagnostic assay in accordance with the present invention for detecting aberrant expression of a polypeptide bound to, bound to or linked to an albumin fusion protein of the present invention as compared to a normal control tissue sample May be used to detect the presence of a tumor. Assay techniques that can be used to measure levels of a polypeptide bound, bound to, or linked to an albumin fusion protein of the invention in a sample derived from a host are well known to those of skill in the art. . Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays. Assaying polypeptide levels in a biological sample is performed using any method known in the art.

Assays for polypeptide levels in a biological sample can be performed using a variety of techniques. For example, polypeptide expression in tissues can be determined using classical immunohistological methods (Jalkanen et al., J. Cell. Biol. 101: 976-985 (1985); Jalkanen, M., et al., J. Cell Biol.105: 3087-3096 (1987)). Other methods that are useful for detecting polypeptide gene expression include immunoassays such as enzyme immunoassays (ELISA) and radioimmunoassays (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, and iodine, such as glucose oxidase ^{(125 I, 121 I),} carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), tritium ⁽³ H) , Radioisotopes such as indium ( ¹¹² In) and technetium ( ^99m Tc), and fluorescent labels such as fluorescein and rhodamine, and biotin.

  The tissue or cell type to be analyzed generally includes what is known or expected to express the gene of interest (eg, cancer). The protein isolation methods used herein are described, for example, in Harlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: Laboratory”, all of which are incorporated herein by reference. Manuals (Antibodies: A Laboratory Manual) ”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Isolated cells can be derived from cell culture media or from patients. Analysis of cells taken from the culture can be a necessary step in assessing the cells that can be used as part of cell-based gene therapy techniques, or to test the effects of compounds on gene expression.

  For example, an albumin fusion protein may be used to quantitatively or qualitatively detect the presence of a polypeptide that binds to, binds to, or links to an albumin fusion protein of the invention. This can be done, for example, by immunofluorescence techniques using fluorescently labeled albumin fusion proteins coupled with light microscopy, flow cytometry or fluorescence detection.

  In preferred embodiments, at least one of the antibodies that specifically bind to at least one therapeutic protein disclosed herein (eg, the therapeutic proteins disclosed in Table 1) or those known in the art. An albumin fusion protein containing one fragment or variant may be used to quantitatively or qualitatively detect the presence of the gene product or a conserved variant or peptide fragment thereof. This can be done, for example, by immunofluorescence techniques using fluorescently labeled albumin fusion proteins coupled with light microscopy, flow cytometry or fluorescence detection.

  The albumin fusion proteins of the present invention can be further subjected to immunofluorescence, immunoelectron microscopy, or non-immunological assays for in situ detection of polypeptides that bind to, bind to or link to the albumin fusion proteins of the present invention. It may be used histologically. In situ detection may be achieved by removing a histological section from a patient and applying a labeled antibody or polypeptide of the invention thereto. The albumin fusion protein is preferably applied by overlaying a labeled albumin fusion protein on the biological sample. Through the use of such a procedure, it is possible to determine not only the presence of a polypeptide that binds to, or binds to, the albumin fusion protein, but also its distribution in the test tissue. Using the present invention, those skilled in the art will readily appreciate that any of a wide variety of histological methods (such as staining procedures) can be modified to achieve such in situ detection. .

  Immunoassays and non-immunoassays that detect polypeptides that bind to, bind to, or link to albumin fusion proteins typically are biological fluids, tissue extracts, freshly harvested cells, or cell cultures Incubating lysates of cells incubated in liquid in the presence of detectably labeled antibodies capable of binding to the gene product or conserved variants or peptide fragments thereof, and are well known in the art Detection of bound antibody by any of a number of techniques.

  The biological sample may be brought into contact with and on a solid support or carrier such as nitrocellulose, or other solid support capable of immobilizing cells, cell particles or soluble proteins. It may be fixed. The support is then washed with a suitable buffer and subsequently treated with detectably labeled albumin fusion protein of the invention. The solid support is then washed twice with buffer to remove unbound antibody or polypeptide. Any antibody is essentially labeled. The amount of bound label on the solid support may then be detected by conventional methods.

  A “solid phase support or carrier” can bind a polypeptide (eg, a polypeptide that is bound or linked to an albumin fusion protein, or an albumin fusion protein of the invention). Any support is contemplated. Well known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gaboros and magnetite. The characteristics of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have a structural conformation that is virtually any possible as long as the bound molecule is capable of binding to the polypeptide. Thus, the support conformation can be a sphere, as in a bead, or a cylinder, as in the inner surface of a test tube, or the outer surface of a rod. Alternatively, the surface may be planar, such as a sheet, a test strap, etc. Preferred supports include polystyrene beads. One skilled in the art knows many other suitable carriers for binding antigens or antibodies, or can elucidate the same through the use of routine experimentation.

  The binding activity of the lot of albumin fusion protein may be measured according to well-known methods. One of ordinary skill in the art can determine effective and optimal assay conditions for each measurement by using routine experimentation.

  In addition to assaying polypeptide levels in biological samples obtained from individuals, polypeptides can be detected in vivo by imaging. For example, in one embodiment of the invention, the albumin fusion protein of the invention is used to image a disease or neoplastic cell.

  Labels or markers for in vivo imaging of albumin fusion proteins of the present invention include those detectable by X-radiography, NMR, MRI, CAT-scan or ESR. For X-radiography, suitable labels include radioactive isotopes such as barium or cesium that emit detectable radiation but are not clearly harmful to the subject. Suitable labels for NMR and ESR include detectable features such as deuterium that can be incorporated into albumin fusion proteins by labeling nutrients of modified cell lines (or bacteria or yeast strains). Includes those with spin.

  Furthermore, the albumin fusion protein of the present invention whose presence can be detected can be administered. For example, an albumin fusion protein of the invention labeled with a radio-opaque substrate or other suitable compound can be administered and visualized in vivo as discussed above for labeled antibodies. In addition, such polypeptides can be used for in vitro diagnostic procedures.

.
Polypeptide-specific antibody or antibody labeled with a suitable detectable imaging moiety, such as a radionuclide (eg ¹³¹ I, ¹¹² In, ^99m Tc), a radio-opaque substrate, or a substance detectable by nuclear magnetic resonance Fragments are introduced into the mammal to be tested for disease (eg, parenterally, subcutaneously or intraperitoneally). It will be appreciated that the size of the subject and the imaging system used will determine the amount of imaging site needed to produce a diagnostic image. In the case of a radioisotope site, for a human subject, the amount of radioactivity injected is usually in the range of about 5-20 millicuries of ^99m Tc. The labeled albumin fusion protein then accumulates specifically in the body, including the polypeptide and other substances that bind to the albumin fusion protein of the present invention. In vivo tumor imaging W. Burchiel et al. , "Immunopharmacokinetics of Radiolabeled Antibodies and Therology. The Chapter of Infra-Er., The Radiocheme of the Radio-labeled Antibodies and Their Fragments". eds., Masson Publishing Inc. (1982)).

  One method for detectably labeling an albumin fusion protein of the invention is by linking the protein to a reporter enzyme and using the linked product in a manner similar to an enzyme immunoassay (EIA). Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons MD: 1-7, Microbiological Associates , J. et al. Clin. Pathol. 31: 507-520 (1978); Butler, J. et al. E. , Meth. Enzymol. 73: 482-523 (1981); Maggio, E .; (Ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL, Ishikawa, E .; et al. (Eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). A reporter enzyme that binds to the antibody reacts with a suitable substrate, preferably a chromogenic substrate, in a manner that produces a chemical moiety that can be detected, for example, by a spectrophotometer, a fluorimeter, or by a visualization method. Reporter enzymes that can be used to detectably label antibodies include, but are not limited to, maleate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase , Triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Furthermore, detection can be performed by colorimetric methods using a chromogenic substrate for the reporter enzyme. Detection may also be performed by visual comparison of the extent of the enzymatic reaction of the substrate compared to a similarly prepared standard.

  Albumin fusion proteins may also be radiolabeled and used in any of a variety of other immunoassays. For example, albumin fusion proteins can be used in radioimmunoassay (RIA) by radioactive labeling of albumin fusion proteins (see, eg, Weintraub, B., incorporated herein by reference). , Principles of Radioimmunosassays, Seventh Training Course on Radioligand Assay Technologies, The Endocrine Society, March, 1986). The radioactive isotope can be detected by methods including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.

  In addition, chelator molecules are known in the art and can be used to label albumin fusion proteins. A chelator molecule can bind to the albumin fusion protein of the invention and facilitate labeling the protein with a metal ion including a radionuclide or a fluorescent label. For example, Subramanian, R.A. and Meares, C.I. F. , "Bifunctional Chelating Agents for Radiometal-labeled Monocology Ab. Saji, H .; , "Radiolabeled imaging and targeted delivery of therapeutic agents: Targeted delivery of radiolabeled imaging and therapeutic agents: bifunctional radiopharmaceuticals." Crit. Rev. Ther. Drug Carrier Syst. 16: 209-244 (1999); Srivastava S .; C. and Mease R.M. C. , "Progress in research on ligands, nuclides and technologies for labeling monoclonals.", Int. J. et al. Rad. Appl. Instrum. B 18: 589-603 (1991); and Liu, S .; and Edwards, D.A. S. , "Bifunctional chelators for therapeutic lanthanide radiopharmaceuticals." Bioconjug. Chem. 12: 7-34 (2001). Any chelator capable of covalent binding to the albumin fusion protein may be used in accordance with the present invention. The chelator may further comprise a linker site that links the chelating site to the albumin fusion protein.

  In one embodiment, the albumin fusion protein of the invention comprises diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid (DPTA), an analog of DPTA and an analog and derivative of DPTA. It binds to such acyclic chelators. As a non-limiting example, the chelator is 2- (p-isothiocyanatobenzyl) -6-methyldiethylenetriaminepentaacetic acid (1B4M-DPTA, also known as MX-DTPA), 2-methyl-6- (rho-nitro Benzyl] -1,4,7-triazaheptane-N, N, N ′, N ″, N ″ -pentaacetic acid (nitro-1B4M-DTPA or nitro-MX-DTPA), 2- (p- Isothiocyanatobenzyl) -cyclohexyldiethylenetriaminepentaacetic acid (CHX-DTPA) or N- [2-amino-3- (rho-nitrophenyl) propyl] -trans-cyclohexane-1,2-diamine-N, N ′, N '' -Pentaacetic acid (nitro-CHX-A-DTPA).

  In another embodiment, the albumin fusion protein of the invention comprises 6,6 ″ -bis [[N, N, N ″, N ″ -tetra (carboxymethyl) amino] methyl] -4 ′-(3 Bind to acyclic terperidines such as -amino-4-methoxyphenyl) -2,2 ', 6', 2 "-terperidine (TMT-amine).

  In a particular embodiment, the macrocyclic chelator that binds to the albumin fusion protein of the invention is 1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid ( DOTA). In another specific embodiment, DOTA binds to the albumin fusion protein via a linker molecule. Examples of linker molecules useful for coupling DOTA to polypeptides are generally known in the art and are described, for example, by DeNardo et al., Which is incorporated by reference in its entirety. , Clin. Cancer Res. 4 (10): 2483-90, 1998; Peterson et al. , Bioconjug. Chem. 10 (4): 553-7, 1999, and Zimmerman et al. , Nucl. Med. Biol. 26 (8): 943-50, 1999. In addition, US Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents that can be conjugated to antibodies, and methods of making and using them, are all referenced. It is incorporated by literature. While US Pat. Nos. 5,652,361 and 5,756,065 focus on conjugating a chelating agent to an antibody, one skilled in the art will recognize chelating agents as other polypeptides. Can be easily adapted to the method disclosed therein.

  A bifunctional chelator based on a macrocyclic ligand, wherein the conjugation is via an activating arm or functional group attached to the carbon skeleton of the ligand; Moi et al. , J. et al. Amer. Chem. Soc. 49: 2639 (1989) (2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N, N ', N ", N" "-tetraacetic acid), S.P. V. Deshbande et al. , J. et al. Nucl. Med. 31: 473 (1990); Ruser et al. , Bioconj. Chem. 1: 345 (1990), C.I. J. et al. Broan et al. , J. et al. C. S. Chem. Comm. 23: 1739 (1990), and C.I. J. et al. Anderson et al. , J. et al. Nucl. Med. 36: 850 (1995).

  In one embodiment, a macrocyclic chelator, such as a polyaza macrocyclic chelator, containing any one or more carboxy, amino, hydroxamate, phosphonic acid or phosphate groups is included in the albumin fusion protein of the invention. Join. In other embodiments, the chelator is a chelator selected from DOTA, analogs of DOTA, and derivatives of DOTA.

  In one embodiment, suitable chelator molecules that can bind to the albumin fusion protein of the invention include DOXA (1-oxa-4,7,10-triazacyclododecane triacetic acid), NOTA (1,4,7 -Triazacyclononanetriacetic acid), TETA (1,4,8,11-tetraazacyclotetradecanetetraacetic acid), and THT (4 '-(3-amino-4-methoxy-phenyl) -6,6 " -Bis (N ′, N′-dicarboxymethyl-N-methylhydrazino) -2,2 ′: 6 ′, 2 ″ -terpyridine), and analogs and derivatives thereof. See, for example, Ohmono et al. , J. et al. Med. Chem. 35: 157-162 (1992); Kung et al. , J. et al. Nucl. Med. 25: 326-332 (1984); Jurisson et al. Chem. Rev. 93: 1137-1156 (1993) and US Pat. No. 5,367,080. Other suitable chelators include US Pat. Nos. 4,647,447, 4,687,659, 4,885,363, European Patent No. EP-A-71564, International Patent No. WO 89/00557. And the chelating agents disclosed in European Patent No. EP-A-232751.

In other embodiments, suitable macrocyclic carboxylic acid chelators that can be used in the present invention include:
1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid (DOTA)
1,4,8,12-tetraazacyclopentadecane-N, N ′, N ″, N ′ ″-tetraacetic acid (15N4)
1,4,7-triazacyclononane-N, N ′, N ″ -triacetic acid (9N3),
1,5,9-triazacyclododecane-N, N ′, N ″ -triacetic acid (12N3) and 6-bromoacetamido-benzyl-1,4,8,11-tetraazacyclotetradecane-N, N ', N'',N'''-tetraacetic acid (BAT)
Is included.

  A preferred chelator capable of binding to the albumin fusion protein of the present invention is α- (5-isothiocyanato-2-methoxyphenyl) -1,4,7,10-tetraazacyclododecane-also known as MeO-DOTA-NCS. 1,4,7,10-tetraacetic acid. A salt or ester of α- (5-isothiocyanato-2-methoxyphenyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid may also be used.

An albumin fusion protein to which a chelator as described is covalently bound may be labeled (via the chelator's regulatory sites) with a radionuclide that is suitable for therapeutic, diagnostic or therapeutic and diagnostic purposes. . Examples of suitable metals include Ag, At, Au, Bi, Cu, Ga, Ho, In, Lu, Pb, Pd, Pm, Pr, Rb, Re, Rh, Sc, Sr, Tc, Tl, Y, And Yb. Examples of radionuclides used for diagnostic purposes are Fe, Gd, ¹¹¹ In, ⁶⁷ Ga, or ⁶⁸ Ga. In other embodiments, the radionuclide used for diagnostic purposes is ¹¹¹ In or ⁶⁷ Ga. Examples of radionuclides used for therapeutic purposes are ¹⁶⁶ Ho, ¹⁶⁵ Dy, ⁹⁰ Y, ^{115 m} In, ⁵² Fe, or ⁷² Ga. In one embodiment, the radionuclide used for diagnostic purposes is ¹⁶⁶ Ho, ⁹⁰ Y, ^{115 m} In, ⁵² Fe, or ⁷² Ga. Examples of radionuclides used for both therapeutic and diagnostic purposes include ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁷⁵ Yb, or ⁴⁷ Sc. In one embodiment, the radionuclide includes ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁷⁵ Yb, or ¹⁵⁹ Gd.

Preferred metal radionuclides include ⁹⁰ Y, ^99m Tc, ¹¹¹ In, ⁴⁷ Sc, ⁶⁷ Ga, ⁵¹ Cr, ^{177 m} Sn, ⁶⁷ Cu, ¹⁶⁷ Tm, ⁹⁷ Ru, ¹⁸⁸ Re, ¹⁷⁷ Lu, ¹⁹⁹ Au, ⁴⁷ Sc, ⁶⁷ Ga, ⁵¹ Cr, ^177m Sn, ⁶⁷ Cu, ¹⁶⁷ Tm, ⁹⁵ Ru, ¹⁸⁸ Re, ¹⁷⁷ Lu, ¹⁹⁹ Au, ²⁰³ Pb and ¹⁴¹ Ce are included.

In certain embodiments, an albumin fusion protein of the invention to which a chelator is covalently bound is labeled with a metal ion selected from the group consisting of ⁹⁰ Y, ¹¹¹ In, ¹⁷⁷ Lu, ¹⁶⁶ Ho, ²¹⁵ Bi, and ²²⁵ Ac. Also good.

In addition, g-emitter radionuclides such as ^99m Tc, ¹¹¹ In, ⁶⁷ Ga, and ¹⁶⁹ Yb are allowed or under investigation for diagnostic imaging, while ⁶⁷ Cu, ¹¹¹ Ag, ¹⁸⁶ Re, Β-emitters such as and ⁹⁰ Y are useful for applications in tumor therapy. Still other useful radionuclides include γ-emitters such as ^99m Tc, ¹¹¹ In, ⁶⁷ Ga, and ¹⁶⁹ Yb, β-emitters such as ⁶⁷ Cu, ¹¹¹ Ag, ¹⁸⁶ Re, and ⁹⁰ Y, And ²¹¹ At, ²¹² Bi, ¹⁷⁷ Lu, ⁸⁶ Rb, ¹⁰⁵ Rh, ¹⁵³ Sm, ¹⁹⁸ Au, ¹⁴⁹ Pm, ⁸⁵ Sr, ¹⁴² Pr, ²¹⁴ Pb, ¹⁰⁹ Pd, ¹⁶⁶ Ho, ²⁰⁸ Tl, and ⁴⁴ Sc Other radionuclides are included. The albumin fusion protein of the present invention to which the chelator is covalently bonded may be labeled with the above radionuclide.

  In other embodiments, the albumin fusion protein to which the chelator is covalently bound is an ion of a transition and lanthanide metal, such as a metal having atomic number 21-29, 42, 43, 44 or 57-71, especially Cr, V, Paramagnetic metal ions including ions of Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu It may be labeled. Paramagnetic metals used in compositions for magnetic resonance imaging include elements having atomic numbers 22-29, 42, 44 and 58-70.

  In other embodiments, an albumin fusion protein of the invention to which a chelator is covalently bonded is a fluorescent metal comprising a lanthanide, in particular La, Ce, Pr, Nd, Pm, Sm, Eu (eg 152 Eu), Gd, Tb, Dy. , Ho, Er, Tm, Yb, and Lu.

  In other embodiments, the albumin fusion proteins of the invention to which the chelator is covalently bound may be labeled with a heavy metal-containing reporter that may include Mo, Bi, Si, and W.

  It is also possible to label the albumin fusion protein with a fluorescent compound. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected by fluorescence. The most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoelsulin, phycocyanin, allophycocyanin, orthoaldehyde and fluorescamine.

  Albumin fusion proteins can also be detectably labeled with fluorescent emitter metals such as 152Eu, or other lanthanide series. These metals can be bound to the antibody using a group of metal chelates such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

  Albumin fusion proteins can also be detectably labeled by linking to a chemiluminescent compound. The presence of the chemiluminescent-tagged albumin fusion protein is then measured by detecting the presence of luminescence that occurs during the course of the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, cellomatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Similarly, a bioluminescent compound may be used to label the albumin fusion protein of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems, where catalytic proteins increase the efficiency of chemiluminescent reactions. The presence of the bioluminescent protein is measured by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.

Transgenic organism

  Transgenic organisms that express the albumin fusion proteins of the present invention are also included in the present invention. Transgenic organisms are generally genetically modified organisms into which recombinant, foreign or cloned genetic material has been transported. Such genetic material is often referred to as a transgene. The transgene nucleic acid sequence may include one or more transcriptional regulatory sequences and other nucleic acid sequences such as introns, which are necessary for optimal expression and secretion of the encoded protein. possible. A transgene directs the expression of the encoded protein in a manner that facilitates its recovery from the organism or from the product produced by the organism, for example from the milk, blood, urine, egg, hair or seed of the organism. Can be designed to The transgene may consist of nucleic acid sequences derived from the genome of the same species as the target animal or from a different species. The transgene may be integrated either at a genomic locus where a particular nucleic acid sequence is not normally found, or at the normal locus of the transgene.

  The phrase “germ cell line transgenic organism” means a transgenic organism in which genetic changes or genetic information is introduced into germline cells, thereby transmitting genetic information to the offspring. Given the ability of transgenic organisms. Such a progeny is indeed a transgenic organism if it contains some or all of its changes or genetic information. The change or genetic information may be foreign to the species of organism to which the recipient belongs, may be foreign only to a particular individual recipient, or genetics already owned by the recipient It may be information. In the last-mentioned case, the altered or introduced gene may be expressed differently than the Tennobu gene.

  The transgenic organism may be a transgenic animal or a transgenic plant. Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, eg, US Pat. No. 4,736). , 866, U.S. Patent No. 5,602,307, Mullins et al. (1993) Hypertension 22 (4): 630-633; Brenin et al. (1997) Surg. Oncol. 6 (2) 99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1 97) that the reference). The method of introducing a nucleic acid fragment into a recombinant competent mammalian cell can be by any method that favors co-transduction of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals, including the disclosures in US Pat. No. 5,489,743 and US Pat. No. 5,602,307, are readily available to those skilled in the art.

  A number of recombinant or transgenic mice have been produced that express activated oncogene sequences (US Pat. No. 4,736,866), those that express simian SV40 T-antigen (US Pat. 728,915), those lacking expression of interferon regulatory factor 1 (IRF-1) (US Pat. No. 5,731,490), those showing dopamine dysfunction (US Pat. No. 5,723,719), One that expresses at least one human gene involved in blood pressure control (US Pat. No. 5,731,489), one that exhibits a higher similarity to the condition present in naturally occurring Alzheimer's disease (US Pat. 720,936), those with reduced capacity to mediate cell adhesion (US Pat. No. 5,602,307), bovine growth hormone (Clutter et al. (1996) Genetics 143 (4): 1753-1760) or capable of producing a fully human antibody response (McCarthy (1997) The Lancet 349 (9049): 405). It is.

  Although mice and rats are still the animals of choice for most transgenic experiments, in some instances it is preferred or necessary to use other animal species. Transgenic procedures have been successfully used in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, eg, Kim et al. Dev. 46 (4): 515-526; Houdebine (1995) Reprod. Nutr. Dev. 35 (6): 609-617; Petters (1994) Reprod. Fertil. ): 643-645; Schnieke et al. (1997) Science 278 (5346): 2130-2133; and Amoah (1997) J. Animal Science 75 (2): 578-585. checking ...

  The transgene-encoded protein of the invention may be placed under the control of a promoter that is preferably activated in mammalian endothelial cells to direct secretion into the milk of the transgenic animal. Promoters that control genes encoding milk proteins are preferred, eg, promoters for casein, beta-lactoglobulin, whey protein, or lactalbumin (eg, DiTullio (1992) BioTechnology 10: 74-77; Clark (1989) BioTechnology 7: 487-492; Gorton et al. (1987) BioTechnology 5: 1183-1187; and Soulier et al. (1992) FEBS Letts. 297: 13). Selected transgenic animals, such as goats, cows, camels or sheep, produce large amounts of milk and have a long lactating period.

The albumin fusion proteins of the invention can be expressed in transgenic plants, such as plants in which a DNA transgene is inserted into the nucleus or plasmid genome. Plant transduction procedures used to introduce foreign nucleic acids into plant cells or protoplasts are known in the art. See, generally, Methods in Enzymology Vol. 153 ("Recombinant DNA Part D") 1987, Wu and Grossman Eds. , Academic Press and European Patent Specification EP 695554. US Pat. No. 5,283,184, US Pat. No. 5,482,852, and European patent specifications, which further incorporate methods for the production of genetically engineered plants, all of which are incorporated by reference. Document EP 693 554.

Pharmacological and therapeutic compositions

  The albumin fusion proteins of the invention or fragments thereof may be administered by any conventional method including parenteral (eg, subcutaneous or intramuscular) injection or intravenous infusion. Treatment may consist of a single dose or multiple doses over time.

  While it is possible for an albumin fusion protein of the invention to be administered alone, it is preferable to present it as a pharmacological formulation with one or more acceptable carriers. The carrier (s) must be “acceptable” in the sense that it is compatible with the albumin fusion protein and not harmful to the recipient. Typically, the carrier is sterile or pyrogen-free water or saline. The albumin fusion proteins of the present invention are formulated in aqueous carriers such as sterile, pyrogen-free water, saline, or other isotonic solutions, particularly for extending their life in solution. Fits well. For example, the pharmacological compositions of the present invention may be well formulated in aqueous form, for example, a few weeks or months or longer before being formulated.

  For example, a formulation comprising albumin fusion protein may be prepared taking into account the extended lifetime of albumin fusion protein in an aqueous formulation. As discussed above, the life span of many of these therapeutic proteins is significantly increased or prolonged after fusion to HA.

  In examples where aerosol administration is appropriate, the albumin fusion proteins of the invention can be formulated as an aerosol using standard procedures. The phrase “aerosol” includes a suspension phase with any gas of an albumin fusion protein of the invention that can be aspirated into the bronchi or nostril. In particular, aerosols include suspensions with droplet gas of the albumin fusion protein of the invention, such as can be produced in a metered inhaler or nebulizer, or in a mist spray. Aerosols also include dry powder compositions of the compounds of the invention suspended in air or other carrier gases, and may be delivered by inhalation, for example, from an inhaler device. Ganderton & Jones, Drug Delivery to the Respiratory Tract, Elis Horwood (19 87); Gonda (1990) Critical Reviews in Therapeutic Three (1992) Pharmacol. Toxicol. See Methods 27: 143-159.

  The formulations of the present invention are also typically partially non-immunogenic for utilization of the composition of albumin fusion proteins derived from the appropriate species. For example, for human use, both the therapeutic protein and the albumin portion of an albumin fusion protein are typically human. In some instances, if any component is of non-human origin, that component will appear to the human immune system where the specific epitope should be human in nature rather than foreign. May be humanized by substitution of key amino acids.

  The formulation may exist conventionally in unit dosage forms and may be prepared by any method well known in the pharmacy area. Such methods include subjecting the albumin fusion protein to binding with a carrier that builds one or more accessory components. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, finely divided solid carriers or both, and then, if necessary, shaping the product.

  Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, and suspensions, which may include antioxidants, buffers, antimicrobial agents, and solutes that provide the appropriate formulation for the intended recipient. Aqueous and non-aqueous sterile suspensions may be included, which may contain agents and thickeners. The formulation may be present in unit dose or multi-dose containers, such as sealed ampoules, vials or syringes, and only requires the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. It may be stored in a lyophilized state (1 lyophilized). Extemporaneous injection solutions and suspensions may be prepared from sterile powders. The dosage formulation is at a lower molar concentration, or at a lower dosage compared to a non-fusion standard formulation for therapeutic proteins, which gives an extended serum half-life exhibited by many albumin fusion proteins of the invention. And may include a therapeutic protein moiety.

  By way of example, if the albumin fusion protein of the invention includes one of the proteins listed in the “Therapeutic Protein X” column of Table 1 as one or more therapeutic protein regions, Can be calculated based on the strength of the albumin fusion protein compared to that of the therapeutic protein alone, while taking into account the serum half-life and lifetime of the extended albumin fusion protein compared to that of the therapeutic protein . For example, if the therapeutic protein is typically administered at 0.3-30.0 IU / kg / week, or 0.9-12.0 IU / kg / week, one year or more, 3 Or it is given in 7 divided times. In an albumin fusion protein consisting of full-length HA fused to a therapeutic protein, an equivalent dose for the unit will show a larger amount of drug, but it is possible to reduce the frequency of administration, for example twice a week or one It can be reduced to once a week or less.

  The formulations or compositions of the present invention may be packaged with an instruction manual or package insert that refers to the extended lifetime of the albumin fusion protein composition, or may be included in a kit containing them. For example, such instructions or package inserts may handle recommended storage conditions, such as time, temperature and light, taking into account the extended or extended half-life of the albumin fusion protein of the invention. . Such instructions or package inserts may also be used for albumin fusion proteins of the present invention, such as ease of storage for formulations that may require use in the art, controlled out-of-hospital, clinic or office conditions. Certain benefits may also be handled. As described above, the formulations of the present invention may be in aqueous form and stored under sub-ideal conditions without a clear loss of therapeutic activity.

  The albumin fusion protein of the present invention can also be included in a dietary supplement. For example, certain albumin fusion proteins of the invention may be administered into natural products, including milk or dairy products from transgenic mammals that express albumin fusion proteins. Such compositions can also include plants or plant products obtained from transgenic plants that express albumin fusion proteins. Albumin fusion proteins can also be provided in powder or tablet form, with or without other known additives, carriers, fillers and diluents. Nutritional supplements are available from Scott Hegenhart, Food Product Design, Dec. 1993.

  The invention also provides a subject with an effective amount of an albumin fusion protein of the invention, or a polynucleotide encoding an albumin fusion protein of the invention (“albumin fusion”, in a pharmacologically acceptable carrier. Also provided are methods of treatment and / or prevention of a disease or condition (such as one or more diseases or conditions disclosed herein) by administering a "polynucleotide".

  Albumin fusion proteins and / or polynucleotides are subject to individual patient clinical status (especially the side effects of treatment with albumin fusion proteins and / or polynucleotides only), delivery site, method of administration, schedule of administration, and practitioner. It can be formulated and administered in a manner consistent with good medical practice, taking into account other known factors. An “effective amount” for purposes herein is thus determined by such considerations.

  As a general proposition, a total pharmacologically effective amount of albumin fusion protein administered parenterally at a time, as noted above, is subject to therapeutic discretion, but is approximately 1 ug / kg patient weight / day to 10 mg / kg patient weight / day. More preferably, the dosage is at least 0.01 mg / kg / day, and most preferably for humans between about 0.01-1 mg / kg / day for hormones. When given continuously, albumin fusion proteins are typically administered at a dosage rate of about 1 ug / kg / hour to about 50 ug / kg / hour, either by 1-4 injections per day, or by continuous subcutaneous infusion. However, it is administered using, for example, a mini-pump. Intravenous bag solutions may also be used. The length of treatment required to observe the change, and the post-treatment sensation to the response that occurs, appears to vary depending on the effect desired.

  As mentioned above, the albumin fusion protein of the present invention has a higher plasma stability compared to the therapeutic protein portion (or fragment or variant thereof) alone. Increased plasma stability should take into account the effective dosage of albumin fusion protein to be administered per dose, and the dose dosing schedule. In particular, due to the higher plasma stability, the albumin fusion protein can be administered at a lower dose with the same frequency of administration, or the albumin fusion protein can be administered with fewer doses. Preferably, the higher stability allows the albumin fusion protein of the invention to be administered less frequently at lower doses. More preferably, the albumin fusion protein can be administered once every two weeks. Even more preferably, the albumin fusion protein can be administered once every 3, 4, 5 or more weeks depending on the pharmacokinetics of the albumin fusion protein. For example, as discussed above, the pharmacokinetics of the IFN-alpha-HSA fusion protein supports a dosing regimen of 2-4 weeks or more, and even at intervals of every 4 weeks or even more than 4 weeks. Is done.

  The effective amount of albumin fusion protein to be administered per dose can also be expressed as the total formulated albumin fusion protein concentration given per dose. In one embodiment, the total formulated albumin fusion protein concentration administered to the patient per dose is in the range of about 10 ug / dose to about 2000 ug / dose. More preferably, the total concentration is in the range of about 100 ug / dose to about 1000 ug / dose, alternatively about 1000 ug / dose to about 1200 ug / dose, or about 900 ug / dose to about 1800 ug / dose.

  In certain embodiments, for example, the invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) Of IFN-alpha-HSA fusion protein is administered at a total formulation concentration of about 90 ug / dose to about 2000 ug / dose. In a more preferred embodiment of the invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) The IFN-alpha-HSA fusion protein is administered at a total formulation concentration of about 900 ug / dose to about 2000 ug / dose, about 900 ug / dose to about 1200 ug / dose, about 900 ug / dose to about 1800 ug / dose, and most preferably about 1200 ug / dose. Administer at a total prescription concentration of from about 1800 ug / dose. In a further preferred embodiment of the invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) IFN-alpha-HSA fusion protein administered at a total formulation concentration of 600 ug / dose, 720 ug / dose, 800 ug / dose, 900 ug / dose, 1000 ug / dose, 1200 ug / dose, 1500 ug / dose, 1800 ug / dose or 2000 ug / dose To do. In a further embodiment, the IFN of the present invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296). -The total formulation / time of alpha-HSA fusion protein is administered either alone or in combination with an antiviral compound such as ribavirin. In a more preferred embodiment, the total prescription concentration (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) The IFN-alpha-HSA fusion protein of the invention is administered in combination with one or more antiviral compounds, including but not limited to ribavirin.

  In a further embodiment, the total prescription concentration (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296). The IFN-alpha-HSA fusion protein of the invention, either at a total formulation concentration of about 90 ug / dose to about 2000 ug / dose, either alone or in combination with an effective amount of an antiviral compound such as ribavirin. In HCV treated naive patients. In a more preferred embodiment of the invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) The IFN-alpha-HSA fusion protein is administered at a total formulation concentration of about 900 ug / dose to about 2000 ug / dose, about 900 ug / dose to about 1200 ug / dose, about 900 ug / dose to about 1800 ug / dose, and most preferably about 1200 ug / dose. Administered to naïve patients with HCV, either alone or in combination with an effective amount of an antiviral compound such as ribavirin, at a total formulation concentration of about 1800 ug / dose. In a further preferred embodiment of the invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) IFN-alpha-HSA fusion protein at a total formulation concentration of 600 ug / dose, 720 ug / dose, 800 ug / dose, 900 ug / dose, 1000 ug / dose, 1200 ug / dose, 1500 ug / dose, 1800 ug / dose or 2000 ug / dose Administered to naïve patients with HCV, either alone or in combination with an effective amount of an antiviral compound such as ribavirin.

In a further embodiment, the IFN-alpha-HSA fusion protein of the invention is administered at one or more anti-bodies comprising an effective amount of, for example, ribavirin, at a total formulation concentration of about 90 ug / dose to about 2000 ug / dose. Administered to a naive patient treated with HCV in combination with a viral compound. In a more preferred embodiment of the invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) The IFN-alpha-HSA fusion protein is administered at a total formulation concentration of about 900 ug / dose to about 2000 ug / dose, about 900 ug / dose to about 1200 ug / dose, about 900 ug / dose to about 1800 ug / dose, and most preferably about 1200 ug / dose. Administered to an HCV-treated naïve patient in combination with one or more antiviral compounds, including, for example, ribavirin, at a total prescribed concentration of about 1800 ug / dose. In a further preferred embodiment of the invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) IFN-alpha-HSA fusion protein at a total formulation concentration of 600 ug / dose, 720 ug / dose, 800 ug / dose, 900 ug / dose, 1000 ug / dose, 1200 ug / dose, 1500 ug / dose, 1800 ug / dose or 2000 ug / dose An effective amount, eg, in combination with one or more antiviral compounds, including ribavirin, is administered to a naive patient treated with HCV.
Give.

  In a further embodiment, the IFN of the present invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296). The alpha-HSA fusion protein of HCV, either alone or in combination with an effective amount of an antiviral compound such as ribavirin, at a total formulation concentration of about 90 ug / dose to about 2000 ug / dose. Administer to treated patients. In a more preferred embodiment of the invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) The IFN-alpha-HSA fusion protein is administered at a total formulation concentration of about 900 ug / dose to about 2000 ug / dose, about 900 ug / dose to about 1200 ug / dose, about 900 ug / dose to about 1800 ug / dose, and most preferably about 1200 ug / dose. Administered to patients with HCV treatment experience at a total prescribed concentration of about 1800 ug / dose, either alone or in combination with an effective amount of an antiviral compound such as ribavirin. In a further preferred embodiment of the invention (produced by CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296) IFN-alpha-HSA fusion protein at a total formulation concentration of 600 ug / dose, 720 ug / dose, 800 ug / dose, 900 ug / dose, 1000 ug / dose, 1200 ug / dose, 1500 ug / dose, 1800 ug / dose or 2000 ug / dose Administered to patients experiencing HCV treatment, either alone or in combination with an effective amount of an antiviral compound such as ribavirin.

  In a further embodiment, (the IFN-alpha-HSA fusion protein of the invention comprises one or more effective amounts of, for example, ribavirin, at a total formulation concentration of about 90 ug / dose to about 2000 ug / dose. In combination with antiviral compounds, it is administered to patients who have experienced HCV treatment.In a more preferred embodiment, (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290 , 4291, 4292, 4295, or 4296) of an IFN-alpha-HSA fusion protein of the invention (from about 900 ug / dose to about 2000 ug / dose, from about 900 ug / dose to about 1200 ug / dose, about 900 ug / dose) A total prescription concentration of about 1800 ug / dose, And most preferably patients who have been treated for HCV in an effective amount, eg, in combination with one or more antiviral compounds, including ribavirin, at a total prescription concentration of about 1200 ug / dose to about 1800 ug / dose In a more preferred embodiment, it is produced by (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296. ) The IFN-alpha-HSA fusion protein of the present invention is administered in a total of 600 ug / dose, 720 ug / dose, 800 ug / dose, 900 ug / dose, 1000 ug / dose, 1200 ug / dose, 1500 ug / dose, 1800 ug / dose or 2000 ug / dose. Effective at prescription concentration The amounts, including for example ribavirin, in combination with one or more antiviral compound is administered to a treatment-experienced patient of HCV.

  The total prescribed concentration of albumin fusion protein and the time interval between administration of the albumin fusion protein can vary depending on the desired effect and the particular therapeutic protein administered. In one embodiment, the total prescribed albumin fusion protein concentration administered to the patient at one time is in the range of about 10 ug / dose to about 2000 ug / dose, once a week, once every two weeks, every three weeks. It is administered once, once every four weeks, or once every longer period. More preferably, the total concentration is in the range of about 100 ug / dose to about 1000 ug / dose, once a week, once every two weeks, once every three weeks, once every four weeks, or more. Of about 1000 ug / dose to about 1200 ug / dose, or about 900 ug / dose to about 1800 ug / dose, once a week, once every two weeks, three times It is administered once a week, once every four weeks, or once every longer period.

  In certain embodiments, the IFN-alpha-HSA fusion proteins (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, Or produced by 4296) once every 2, 3, 4 or 5 weeks at a total formulation concentration of about 90 ug / dose to about 2000 ug / dose. In a more preferred embodiment, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. Or produced by 4296), once every 1, 2, 3, 4 or 5 weeks, from about 900 ug / dose to about 2000 ug / dose, or once every 1, 2, 3, 4 or 5 weeks, Administered at a total formulation concentration of about 900 ug / dose to about 1200 ug / dose, or once every 1, 2, 3, 4 or 5 weeks, about 900 ug / dose to about 1800 ug / dose, most preferably 2, 3 Administered once every 4 or 5 weeks at a total prescription concentration of about 1200 ug / dose to about 1800 ug / dose . The IFN-alpha-HSA fusion proteins of the invention are produced by (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or 4296. Once every 2, 3, 4 or 5 weeks, about 600 ug / dose, or once every 2, 3, 4 or 5 weeks, about 800 ug / dose, or every 2, 3, 4 or 5 weeks Once, every 900 ug / time, once every 2, 3, 4 or 5 weeks, about 1000 ug / time, once every 2, 3, 4 or 5 weeks, about 1200 ug / time, or 2, Once every 3, 4 or 5 weeks, about 1500 ug / dose, or once every 2, 3, 4 or 5 weeks, about 1600 g / dose, or once every two, three, four or five weeks, from about 1800 ug / dose, or two, three, four, or once every five weeks, is administered in a total formulation concentration of about 2000 ug / dose. In a more preferred embodiment, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. , Or produced by 4296) once every two weeks, about 900 ug / time, more preferably once every two weeks, about 1200 ug / time, once every four weeks, about 1200 ug / time, or 4 Once a week, administered at a total formulation concentration of about 1800 ug / dose. In further embodiments, the IFN-alpha-HSA fusion proteins of the invention (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, or The total formulation concentration (produced by 4296) is administered either alone or in combination with an antiviral compound, such as ribavirin. In further preferred embodiments, the IFN-alpha-HSA fusion proteins of the present invention (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, Or produced by 4296) is administered in combination with one or more antiviral compounds, including, for example, ribavirin.

  In certain embodiments, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. , Or produced by 4296), either alone or in combination with an antiviral compound such as ribavirin, once every 2, 3, 4 or 5 weeks, from about 90 ug / dose to about 2000 ug / dose Is administered to treated naive HCV patients at a total prescribed concentration of In a more preferred embodiment, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. , Or produced by 4296), either alone or in combination with antiviral compounds including ribavirin, once every 1, 2, 3, 4 or 5 weeks, from about 900 ug / dose to about 2000 ug / d Administered to treated naïve HCV patients at a total prescription concentration of about 900 ug / dose to about 1200 ug / dose once, or once every 1, 2, 3, 4 or 5 weeks, or 1, 2, 3, 4 or Once every 5 weeks, about 900 ug / time to about 1800 ug / time, most preferred Is once every 1, 2, 3, 4 or 5 weeks, in a total formulation concentration of about 1200 ug / dose to about 1800 ug / dose, administered in the treatment naïve HCV patients. In a further embodiment, the IFN-alpha-HSA fusion protein of the invention has (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, Or produced by 4296), either alone or in combination with an antiviral compound such as ribavirin, once every 1, 2, 3, 4 or 5 weeks, about 600 ug / dose, or 1, Once every 2, 3, 4 or 5 weeks, at about 800 ug / dose total formulation concentration, or once every 1, 2, 3, 4 or 5 weeks, about 900 ug / dose, alternatively 1, 2, 3, Once every 4 or 5 weeks, about 1000 ug / time, or once every 1, 2, 3, 4 or 5 weeks, 1200 ug / dose, or once every 1, 2, 3, 4 or 5 weeks, about 1500 ug / dose, or once every 1, 2, 3, 4 or 5 weeks, about 1600 ug / dose, or 1, 2 Administer to treated naive HCV patients once every 3, 4 or 5 weeks, about 1800 ug / dose, or once every 1, 2, 3, 4 or 5 weeks at a total prescription concentration of about 2000 ug / dose Is done.

  In a preferred specific embodiment, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, From about 90 ug / dose to about 2000 ug / dose once every 2, 3, 4 or 5 weeks in combination with one or more antiviral compounds including, for example, ribavirin) Is administered to treated naive HCV patients at a total prescribed concentration of In a more preferred embodiment, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. , Or produced by 4296), for example, once every 1, 2, 3, 4 or 5 weeks in combination with one or more antiviral compounds including ribavirin, from about 900 ug / dose to about 2000 ug / Administered to treated naïve HCV patients at a total prescription concentration of about 900 ug / dose to about 1200 ug / dose once, or once every 1, 2, 3, 4 or 5 weeks, or 1, 2, 3, 4 or Once every 5 weeks, about 900 ug / time to about 1800 ug / time, most preferred Details, once every 1, 2, 3, 4 or 5 weeks, in a total formulation concentration of about 1200 ug / dose to about 1800 ug / dose, administered in the treatment naïve HCV patients. In a further embodiment, the IFN-alpha-HSA fusion protein of the invention has (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, Or in combination with one or more antiviral compounds including, for example, ribavirin, once every 1, 2, 3, 4 or 5 weeks, about 600 ug / dose, or 1, 2 Once every 3, 4 or 5 weeks, at about 800 ug / dose total formulation concentration, or once every 1, 2, 3, 4 or 5 weeks, about 900 ug / dose, alternatively 1, 2, 3, 4 Or once every 5 weeks, about 1000 ug / time, or once every 1, 2, 3, 4 or 5 weeks About 1200 ug / dose, once every 1, 2, 3, 4 or 5 weeks, about 1500 ug / dose, once every 1, 2, 3, 4 or 5 weeks, about 1600 ug / dose, or 1, Treated naïve HCV patients once every 2, 3, 4 or 5 weeks, at a total prescription concentration of about 1800 ug / dose, or once every 1, 2, 3, 4 or 5 weeks, about 2000 ug / dose Be administered.

  In a more preferred embodiment, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. , Or produced by 4296), either alone or in combination with an antiviral compound such as ribavirin, once every 2 weeks, 900 ug / time, more preferably every 2 weeks, once every 1200 ug / time, every 4 weeks At a total prescribed concentration of 1200 ug / dose once, or 1800 ug / dose once every 4 weeks, to treated naive HCV patients. In the most preferred embodiment, the IFN-alpha-HSA fusion proteins of the present invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. Or in combination with one or more antiviral compounds including ribavirin, for example, 900 ug / dose every 2 weeks, more preferably 1200 ug / dose once every 2 weeks, 4 weeks Treated naïve HCV patients at a total prescription concentration of 1200 ug / dose once every or 1800 ug / dose once every 4 weeks.

  In a more specific embodiment, the IFN-alpha-HSA fusion proteins of the present invention (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292 , 4295, or 4296), either alone or in combination with an antiviral compound such as ribavirin, once every 2, 3, 4 or 5 weeks, from about 90 ug / dose to about 2000 ug Administered to treated HCV patients at a total prescribed concentration / time. In a more preferred embodiment, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. , Or produced by 4296), either alone or in combination with an antiviral compound such as ribavirin, once every 1, 2, 3, 4 or 5 weeks, from about 900 ug / dose to about 2000 ug. Or once every 1, 2, 3, 4 or 5 weeks at a total prescription concentration of about 900 ug / dose to about 1200 ug / dose to treated HCV patients, or 1, 2, 3, 4 Or once every 5 weeks, about 900 ug / time to about 1800 ug / time, most preferably , Once every 1, 2, 3, 4 or 5 weeks, in total prescriptions concentration of about 1200ug / dose to about 1800ug / dose, is administered in treatment experienced HCV patients. In a further embodiment, the IFN-alpha-HSA fusion protein of the invention has (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, Or produced by 4296), either alone or in combination with an antiviral compound such as ribavirin, once every 1, 2, 3, 4 or 5 weeks, about 600 ug / dose, or 1, Once every 2, 3, 4 or 5 weeks, at about 800 ug / dose total formulation concentration, or once every 1, 2, 3, 4 or 5 weeks, about 900 ug / dose, alternatively 1, 2, 3, Once every 4 or 5 weeks, about 1000 ug / time, or once every 1, 2, 3, 4 or 5 weeks, 1200 ug / dose, or once every 1, 2, 3, 4 or 5 weeks, about 1500 ug / dose, or once every 1, 2, 3, 4 or 5 weeks, about 1600 ug / dose, or 1, 2 Administered to treated HCV patients once every 3, 4 or 5 weeks, approximately 1800 ug / dose, or once every 1, 2, 3, 4 or 5 weeks at a total prescription concentration of about 2000 ug / dose Is done.

  In a more specific embodiment, the IFN-alpha-HSA fusion proteins of the present invention (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292 , 4295, or 4296), for example, once every 2, 3, 4 or 5 weeks in combination with one or more antiviral compounds including ribavirin, from about 90 ug / dose to about 2000 ug / The total prescribed concentration of the dose is administered to the treated HCV patient. In a more preferred embodiment, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. , Or produced by 4296), for example, once every 1, 2, 3, 4 or 5 weeks in combination with one or more antiviral compounds including ribavirin, from about 900 ug / dose to about 2000 ug / Administered to treated HCV patients at a total prescription concentration of about 900 ug / dose to about 1200 ug / dose once, or once every 1, 2, 3, 4 or 5 weeks, or 1, 2, 3, 4 or Once every 5 weeks, about 900 ug / time to about 1800 ug / time, most preferred Is, once every 1, 2, 3, 4 or 5 weeks, in total prescriptions concentration of about 1200ug / dose to about 1800ug / dose, is administered in treatment experienced HCV patients. In a further embodiment, the IFN-alpha-HSA fusion protein of the invention has (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295, Or in combination with one or more antiviral compounds including, for example, ribavirin, once every 1, 2, 3, 4 or 5 weeks, about 600 ug / dose, or 1, 2 Once every 3, 4, or 5 weeks, at a total prescription concentration of about 800 ug / dose, or once every 1, 2, 3, 4 or 5 weeks, about 900 ug / dose, or 1, 2, 3, Once every 4 or 5 weeks, approximately 1000 ug / dose, or once every 1, 2, 3, 4 or 5 weeks About 1200 ug / dose, once every 1, 2, 3, 4 or 5 weeks, about 1500 ug / dose, once every 1, 2, 3, 4 or 5 weeks, about 1600 ug / dose, or 1 Patients treated with HCV at a total prescription concentration of about 1800 ug / dose once every 2, 3, 4 or 5 weeks, or about once every 1, 2, 3, 4 or 5 weeks at about 2000 ug / dose To be administered.

  In a more preferred embodiment, the IFN-alpha-HSA fusion proteins of the invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. , Or produced by 4296), either alone or in combination with an antiviral compound such as ribavirin, once every 2 weeks, 900 ug / time, more preferably every 2 weeks, once every 1200 ug / time, every 4 weeks At a total prescribed concentration of 1200 ug / dose once, or 1800 ug / dose once every four weeks. In the most preferred embodiment, the IFN-alpha-HSA fusion proteins of the present invention are (CIDs 2249, 2343, 2366, 2381, 2382, 2410, 3165, 3422, 3423, 3424, 3476, 3960, 4290, 4291, 4292, 4295. Or in combination with one or more antiviral compounds including ribavirin, for example, 900 ug / dose every 2 weeks, more preferably 1200 ug / dose every 2 weeks, 4 weeks It is administered to treated HCV patients at a total prescription concentration of 1200 ug / dose once every, or 1800 ug / dose once every 4 weeks.

  Albumin fusion proteins and / or polynucleotides can be oral, rectal, parenteral, intracapsular, intravaginal, intraperitoneal, topical (such as by powder, ointment, gel, drop or transdermal patch), buccal, or oral or It can be administered as a nasal spray. “Pharmaceutically acceptable carrier” means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating agent or any formulation aid. As used herein, the phrase “parenteral” means intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

  The albumin proteins and / or polynucleotides of the present invention are also preferably administered by a sustained release system. Examples of sustained release albumin fusion proteins and / or polynucleotides include oral, rectal, parenteral, intracapsular, intravaginal, intraperitoneal, topical (such as by powder, ointment, gel, drop or transdermal patch), buccal Or as an oral or nasal spray. “Pharmaceutically acceptable carrier” means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating agent or any formulation aid. As used herein, the phrase “parenteral” means intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. Further examples of sustained-release albumin proteins and / or polynucleotides include suitable multiplexing materials (eg, in the form of molded articles, such as films or microcapsules, eg, semipermeable polymer matrices), (eg, Suitable hydrophobic materials (such as acceptable emulsions in oil), or ion exchange resins, and poorly soluble derivatives (such as, for example, sparingly soluble salts).

  The radiation maintenance matrix includes polylactide (US Pat. No. 3,773,919, European Patent 58,481), a copolymer of L-glutamic acid and gamma-ethyl-L-glutamic acid (Sidman et al., Biopolymers 22: 547). -556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and Langer, Chem. Tech. 12: 98-105. (1982)), ethylene vinyl acetate (Langer et al., Id.) Or poly-D-(-)-3-hydroxybutyric acid (European Patent No. 133,988).

  Release sustained albumin fusion proteins and / or polynucleotides also include liposomal trap albumin fusion proteins and / or polynucleotides of the present invention (generally, Langer, Science 249: 1527-1533 (1990); Treat et al. , In Liposomes in the Therapeutic of Infectious Disease and Cancer, Lopez-Berstein and Fiddler (eds.), Liss, New York, pp. 317-327 and 336-335. Liposomes containing albumin fusion protein and / or polynucleotide are prepared by methods known per se. German Patent DE 3,218,121, Epstein et al. , Proc. Natl. Acad. Sci. (USA) 82: 3688-3692 (1985); Hwang et al. , Proc. Natl. Acad. Sci. (USA) 77: 4030-4034 (1980); European Patent No. 52,322, European Patent No. EP 36,676, European Patent No. EP 88,046, European Patent No. EP 143,949, European Patent No. EP 142,641, Japanese Patent No. 83-118008, US Pat. Nos. 4,485,045 and 4,544,545, and European Patent No. EP 102,324. Liposomes are originally of small (about 200-800 angstrom) monolayer type with a lipid content greater than about 30 mol percent cholesterol, and the selected ratio is optimally adapted for therapeutic use.

  In yet a further embodiment, the albumin fusion proteins and / or polynucleotides of the invention are delivered by the method of a pump (Langer, supra; Safeton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); see Saudek et al., N. Engl. J. Med. 321: 574 (1989)).

  Other controlled release systems are discussed in an overview by Langer (Science 249: 1527-1533 (1990).

  For parenteral administration, in one embodiment, the albumin fusion protein and / or polynucleotide is generally delivered to the recipient in a pharmacologically acceptable carrier, ie used / dose and concentration. Formulated by mixing in the desired purity in a unit dose injection form (solution, suspension or emulsion) with what is non-toxic and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and compounds that are known to be harmful to therapeutics.

  In general, formulations are prepared by contacting the albumin fusion protein and / or polynucleotide with a liquid carrier or a finely divided solid carrier, or both, homogeneously and intimately. Then, if necessary, the product is molded into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carriers include water, saline, Ringer's solution, and dextrose solution. Non-aqueous excipients such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

  The carrier preferably contains minor amounts of additives such as substrates that enhance isotonicity and chemical stability. Such substances are non-toxic to recipients at the dosages and concentrations used, and buffers such as phosphate, citric acid, succinic acid, acetic acid and other organic acids or their salts, ascorbic acid Antioxidants such as, low molecular weight (less than about 10 residues) polypeptides, eg polyarginine or tripeptides, proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, glycine, glutamic acid , Amino acids such as aspartic acid or arginine, monosaccharides, disaccharides, and hydrocarbons including cellulose or derivatives thereof, glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, sodium Counterion, and / or such as (for example including Tween-20) polysorbate include nonionic surfactants such as Porokishima or PEG.

  Albumin fusion proteins are typically formulated in such excipients at a pH of about 3-8, at a concentration of about 0.1 mg / ml to 100 mg / ml, preferably 1-10 mg / ml. . It is understood that the use of certain aforementioned excipients, carriers or stabilizers results in the formation of polypeptide salts.

  Any pharmacological substance used for therapeutic administration can be sterile. Sterilization is simply accomplished by filtration through a sterile filtration membrane (eg, a 0.2 micron membrane). Albumin fusion proteins and / or polynucleotides are typically placed in containers with sterile access holes, such as vials with stoppers that can be punctured by intravenous solution bags or hypodermic needles.

  Albumin fusion proteins and / or polynucleotides can usually be stored in unit or multidose containers, such as sealed ampoules or vials, as an aqueous solution or in lyophilized form for reconstitution. As an example of a lyophilized formulation, a 10-ml vial is filled with 5 ml of sterile filtered 1% (w / v) aqueous albumin fusion protein and / or polynucleotide solution and the resulting mixture is lyophilized. An infusion solution is prepared by reconstituting lyophilized albumin fusion protein and / or polynucleotide using bacteriostatic injection water.

  In certain and preferred embodiments, the albumin fusion protein formulation includes 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromolar sodium octanoate / fusion protein 1 milligram, 15 micrograms / polysorbate 80 1 Milliliter, pH 7.2 is included. In another specific and preferred embodiment, the albumin fusion protein formulation comprises 0.01 M sodium phosphate, 0.15 mM sodium chloride, 0.16 micromolar sodium octanoate / fusion protein 1 milligram, 15 micrograms / polysorbate 80 Contains 1 milligram, pH 7.2. The pH and buffer are selected to suit physiological conditions and salt is added as a tonicifier. Sodium octanoate has been selected for its reported ability to increase the temperature stability of proteins in solution. Finally, polysorbate is added as a genetic surfactant, which reduces the surface tension of the solution and reduces nonspecific adsorption of the albumin fusion protein to the container closure system.

  The invention also provides a pharmacological pack or kit comprising one or more containers filled with one or more components of an albumin fusion protein and / or polynucleotide of the invention. Notification in a form supported by a governmental organization that regulates the manufacture, use, or sale of a pharmaceutical or biological product is associated with such container (s), and the notification is manufactured, used for human administration Or reflects the permission of the sales organization. In addition, albumin fusion proteins and / or polynucleotides may be used in combination with other therapeutic compounds.

  The albumin fusion protein and / or polynucleotide of the present invention may be administered alone or in combination with an adjuvant. Adjuvants that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, alum, alum + deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21. (Genentech, Inc.), BCG (eg, THERCYS®), MPL and non-growth preparations of Corynebacterium parvum. In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with alum. In other specific embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with QS-21. Adjuvants that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, monosphoryl lipid immunomodulators, AdjuVax100a, QS-21, QS-18, CRL1005, aluminum salts, MF-59 And Virosomal adjuvant technology. Vaccines that may be administered with the albumin fusion protein and / or polynucleotide of the present invention include, but are not limited to, MMR (measles, mumps, rubella), polio, chickenpox, tetanus / diphtheria toxin, hepatitis A, Included are vaccines directed against protection against hepatitis B, H. influenzae, pertussis, pneumonia, influenza, Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, acute leukomyelitis, rabies, typhoid and pertussis. The combinations may be administered in combination, for example, either as a mixture or separately but simultaneously or simultaneously or sequentially. This includes a presentation in which a mixed drug is administered together with a therapeutic mixture, and a procedure in which the mixed drug is administered separately but simultaneously, for example to the same individual via separate intravenous lines. included. Administration “in combination” further includes one compound or agent given first, followed by another.

  The albumin fusion proteins and / or polynucleotides of the present invention may be administered alone or in combination with other therapeutic agents. Albumin fusion protein and / or polynucleotide agents that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, chemotherapeutic agents, antibiotics, steroids and non-steroidal anti-inflammatory agents. Agents, conventional immunotherapeutic agents, and / or therapeutic treatments described below. The combinations may be administered in combination, for example, either as a mixture or separately but simultaneously or simultaneously or sequentially. This includes a presentation in which a mixed drug is administered together with a therapeutic mixture, and a procedure in which the mixed drug is administered separately but simultaneously, for example to the same individual via separate intravenous lines. included. Administration “in combination” further includes one compound or agent given first, followed by another.

  In one embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with an anticoagulant. Anticoagulants that may be administered with the compositions of the present invention include, but are not limited to, heparin, low molecular weight heparin, warfarin sodium (eg, COUMADIN®), dicoumarol, 4-hydroxycoumarin, anisindione (eg, MIRADON). (Trademark)), acenocoumarol (for example nikumarone, SINTHROME (TM)), indan-1,3-dione, fenprocumone (for example MARCUMAL (TM)), ethyl biscouma acetic acid (for example TROMEXAN (TM)) and aspirin It is. In certain embodiments, the compositions of the invention are administered in combination with heparin and / or warfarin. In another specific embodiment, the compositions of the invention are administered in combination with warfarin. In other specific embodiments, the compositions of the invention are administered in combination with warfarin and aspirin. In other specific embodiments, the compositions of the invention are administered in combination with heparin. In another specific embodiment, the compositions of the invention are administered in combination with heparin and aspirin.

  In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with a thrombolytic drug. Thrombolytic drugs that may be administered with the compositions of the present invention include, but are not limited to, plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinase (eg, KABIKINASE ™), antires Place (eg, EMINASE ™), tissue plasminogen active (t-PA, Artebase, ACTIVASE ™), urokinase (eg, ABBOKINASE ™), saulplase (prourokinase, single chain urokinase) , And aminocaproic acid (eg, AMICAR ™). In certain embodiments, the compositions of the invention are administered in combination with tissue plasminogen active and aspirin.

  In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with antiplatelet drugs. Antiplatelet drugs that may be administered in combination with the compositions of the present invention include, but are not limited to, aspirin, dipyridamole (eg, PERSANTINE ™), and ticlopidine (eg, TICLID ™). .

  In certain embodiments, the use of an anticoagulant, thrombolytic and / or antiplatelet drug in combination with an albumin fusion protein and / or polynucleotide of the present invention comprises thrombosis, arterial thrombosis, venous thrombosis, Contemplated for the prevention, diagnosis and / or treatment of thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic stroke, unstable angina. In certain embodiments, the use of an anticoagulant, thrombolytic and / or antiplatelet drug in combination with an albumin fusion protein and / or polynucleotide of the present invention may be used to prevent occlusion of a saphenous graft. Mechanical valves and / or mitrals to reduce the risk of periprocedural thrombosis, such as may occur with plastic surgery, and to reduce the risk of stroke in patients with atrial cells, including non-rheumatic atrial fibrillation Intended to reduce the risk of embolism associated with valve disease. Other therapeutic uses of the present invention alone or in combination with antiplatelet, anticoagulant and / or antithrombotic drugs include, but are not limited to, extracorporeal devices (eg, intravascular cannulas, hemodialysis patients) Prevention of blockages in vascular access shunts, vascular dialysis devices, and cardiopulmonary bypass devices).

  In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are treated with antiretroviral agents, nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and / or It is administered in combination with protease inhibitors (PIs). NRTIs that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, RETROVIR ™ (Zidovudine / AZT), VIDEOX ™ (Didanocin / ddl), HIVID (Trademark) (zarcitabine / ddC), ZERIT (TM) (stavudine / d4T), EPIVIR (TM) (lamivudine / 3TC), and COMBIVIR (TM) (zidovudine / lamivudine). NNRTIs that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, VIRAMUNE ™ (Nevirapin), RESCRIPTOR ™ (Delavirudine), and SUSTIVA ™ (Efavi lens) is included. Protease inhibitors that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, CRIXIVAN ™ (Indinavir), NORVIR ™ (Ritonavir), INVIRASE ™ ) (Saquinavir), and VIRACEPT ™ (Nelfinavir). In certain embodiments, the antiretrovirus, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and / or protease inhibitor is used to treat AIDS and to treat HIV infection. Any combination with the albumin fusion proteins and / or polynucleotides of the invention may be used.

  Additional NRTIs are structurally related to LODENOSINE ™ (F-ddA, acid-stable adenosine NRTI, Triangle / Abbott), COVIRACIL ™ (amtricitabine / FTC, lamivudine, but in vitro 3-10 times more active, Triangle / Abbott), dOTC (BCH-10652, also structurally related to lamivudine, but active against an essential proportion of lamivudine-resistant isolates , Biochem Pharma, Adefovir (FDA refused permission for anti-HIV treatment, Gilead Sciences, PREVEO) a (Adefovir Dipivoxil, active prodrug of adefovir, the active form is PMEA-pp), TENOFOVIR ™ (Bis-POC PMPA, PMPA prodrug, Gilead), DAPD / DXG (DAPD activity Metabolites, Triangle / Abbott), D-D4FC (relevant to 3TC, activity against AZT / 3TC-resistant virus), GW420867X (Glaxo Wellcome), ZIAGEN ™ (Abacavir / 159U89) (Glaxo Wellcome Inc.), CS-87 (3 ′ azido-2 ′, 3′-dideoxyuridine, International Patent No. WO 99/6. 936), and b-L-FD4C and b-L-FddC the S- acyl-2-thioethyl (SATE) - containing prodrugs form (WO WO98 / seventeen thousand two hundred eighty-one) include.

  Additional NNRTIs include COACTINON ™ (Emvirine / MKC-442, powerful NNRTI in the HRPT class, Triangle / Abbott), CAPRAVILINE ™ (AG-1549 / S-1153, K103N mutation) New generation NNRTI with activity against viruses, Agouron, PNU-142721 (20-50 times greater activity than its predecessor delaviridine and active against K103N mutant, Pharmacia & Upjohn)), DPC-961 and DPC-963 (the second generation metabolite of efavirenz, designed to be active against viruses with the K103N mutation, DuPont), GW-420867X (25 times stronger than HBY097, active against K103N mutant, Glaxo Wellcome), CALANOLIDE A (naturally occurring from latex tree) Agents, activity against viruses containing either or both of the Y181C and K103N mutations), and Propolis (International Patent No. 99/49830).

  Additional protease inhibitors include LOPINAVIR ™ (ABT378 / r, Abbott Laboratories), BMS-232632 (Azapeptide, Bristol-Myres Squibbb), TIPRANVIR ™ (trademark) 140690, non-peptidic dihydropyrone, Pharmacia & Upjohn, PD-178390 (non-peptidic dihydropyrone, Park-Davis), BMS 232632 (Azapeptide, Bristol-Myers squib (Br -Myers Squibb)), L-756, 423 (Indinavir analog, Mel Merck), DMP-450 (cyclic urea compounds, Avid & DuPont), AG-1776 (peptidomimetics with in vitro activity against protease inhibitor resistant viruses, Agouron), VX -175 / GW-433908 (amprenavir phosphate prodrug, Vertex & Glaxo Welcom), CGP 61755 (Ciba), and AGENERASE ™ (Amprenavir, GlaxoWelcome ( Glaxo Wellcome Inc.)).

  Additional antiretroviral agents include fusion inhibitors / gp41 binding agents. Fusion inhibitors / gp41 binding agents include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain that binds to gp41 in its quiescent state and prevents deformation to the fusion state, Trimeris) and T-1249 (second generation fusion inhibitor, Trimeris).

  Additional antiretroviral agents include fusion inhibitors / chemokine receptor antagonists. Fusion inhibitors / chemokine receptor antagonists include AMD3100 (bicyclam), SDF-1 and its analogs, and ALX40-4C (cationic peptide), T22 (18 amino acid peptide, Trimeris), and T22 analogs CXCR4 antagonists such as T134 and T140, CXCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779, and CCR5 / CXCR4 antagonists such as NSC 651016 (distamycin analog) included. Also included are CCR3B, CCR3 and CCR6 antagonists. Chemokine receptor agonists such as RANTES, SDF-1, MIP-1a, MIP-1b, etc. can also inhibit fusion.

  Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acid, L-quinolinic acid (dicaffeoyltartar (DCTA) acid), quinalizarin (QLC) and related anthoraquinones, ZINTEVIR ™ (AR 177, in fact, Rather than as integrase inhibitors, oligonucleotides that probably work on the cell surface, Arondex, and naphthols such as those disclosed in International Patent No. WO 98/50347 are included.

  Additional antiretroviral agents include ribonucleoside reductase inhibitors such as BCX-34 (purine nucleoside phosphorylase inhibitor, Biocrist), DIDOX ™ (Molecules for Health, VX-497 Included are hydroxyurea-like compounds, such as inosine monophosphate dehydrogenase (IMPDH) inhibitors such as (Vertex), and mycophoric acid such as sercept (mycophenolate mofetil, Roche).

  Further antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear translocation such as arylene bis (methylketone) compounds, AOP-RANTES, NNY-RANTES, RANTES-IgG fusion proteins, RANTES and glycos. A soluble complex of saminoglycan (GAG), and inhibitors of HIV entry, such as AMD-3100, nucleocapsid zinc finger inhibitors such as dithione compounds, targets of HIV Tat and Rev, and ABT-378 A pharmaco enhancer is included.

  Other antiretroviral and adjunct treatments include MIP-1a, MIP-1b, SDF-1a, IL-2, PROLEUKIN ™ (Aldesleukin / L2-7001, Chiron), IL-4, Cytokines and lymphokines such as IL-10, IL-12, and IL-13, interferons such as IFN-alpha 2a, IFN-alpha 2b, or IFN-beta, TNFs, NFkB, GM-CSF, M-CSF, And IL-10 antagonists, agents that modulate immune activation such as cyclosporine and prednisone, Remune ™ (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (B / E )Recombinant Envelope glycoprotein, rgp120CM235, MNrgp120, SF-2rgp120, gp120 / soluble CD4 complex, Delta JR-FL protein, branched synthetic domain derived from discrete gp120C3 / C4 domain, fusion-complement immunogen, Gag, Pol, Nef, and Vaccines such as the Tat vaccine, genetic suppressor elements (GSEs, International Patent No. WO 98/54366), and Intrakin (targeted against the ER to block the surface expression of the newly synthesized CCR5 Genetically modified CC chemokines) (Yang et al., PNAS 94: 11567-72 (1997); Chen et al., Nat. Med. 3: 1110-16 (19 7) gene-based therapy, such as anti-CXCR4 antibody 12G5, anti-CCR5 antibodies 2D7, 5D7, PA8, PA9, PA10, PA11, PA12 and PA14, anti-CD4 antibody Q4 120 and RPA-T4, anti-CCR3 antibody 7B11 Anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-a antibodies, and antibodies such as monoclonal antibody 33A, TCDD, 3, International patent aryl hydrocarbons such as 3 ′, 4,4 ′, 5-pentachlorobiphenyl, 3,3 ′, 4,4′-tetrachlorobiphenyl, and a-naphthoflavone (WO 98/30213) ( AH) receptor agonists and antagonists, and gL-glutamyl-L-cysteine Chiruesuteru (g-GCE, WO WO 99/56764) include antioxidants such as.

  In a further embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with one or more antiviral agents. Antiviral agents that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, acyclovir, ribavirin, ribavirin analogs, amantadine, remantidine, maxiamine, or timalfacin. In particular, the interferon albumin fusion protein can be administered in combination with any of these agents. Furthermore, an interferon alpha albumin fusion protein can also be administered with any of these agents, and preferably an interferon alpha 2a or 2b albumin fusion protein can be administered with any of these agents. In addition, interferon beta albumin fusion proteins can also be administered with any of these agents. Furthermore, any IFN hybrid albumin fusion protein can be administered in combination with any of these agents.

  In the most preferred embodiment, the interferon albumin fusion protein of the invention is administered in combination with ribavirin or a ribavirin analog. In a preferred embodiment, ribavirin or ribavirin derivatives that may be administered in combination with an interferon albumin fusion protein include, but are not limited to, COPEGUS® (Hoffman-La Roche, Nutley, N .; J.), REBETOL (R) (Schering Corp., Kenilworth, NJ), VIRAZOLE (R) (Valeant, Costa Mesa, CA), RIBAVIN (R) (Lupin ( Lupin), Baltimore, MD), RIBAZID ™ (Epla, Kirachi, Pakistan), Tribavirin, VIRAMIDINE ™ (Valeant, Costa Mesa, CA), and RIBASPHERE ™ (Three Rivers Pharmaceuticals, Cranbury Township, PA). In a further preferred embodiment, the interferon alpha albumin fusion protein is administered in combination with ribavirin or a ribavirin derivative. In a further preferred embodiment, the interferon 2a albumin fusion protein is administered in combination with ribavirin or a ribavirin derivative. In a further preferred embodiment, the interferon alpha 2b albumin fusion protein is administered in combination with ribavirin or ribavirin derivatives. In a further preferred embodiment, the interferon beta albumin fusion protein is administered in combination with ribavirin or a ribavirin derivative. In a further preferred embodiment, the hybrid interferon albumin fusion protein is administered in combination with ribavirin or a ribavirin derivative.

  In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention may be administered alone or in combination with one or more antiviral agents for the treatment of viral infections. In a preferred embodiment, the interferon-albumin fusion protein of the invention may be administered in combination with one or more antiviral agents. In a further preferred embodiment, the viral infection is the result of an infection with hepatitis virus. In the most preferred embodiment, the hepatitis virus is hepatitis C virus (HCV). Antiviral agents that may be administered with the albumin fusion protein and / or polynucleotide of the present invention include, but are not limited to, small molecule inhibitors of viral enzymes, small molecule inhibitors of RNA polymerase, nucleic acid based antiviral agents , Antisense oligonucleotide inhibitors, thiazolides, novel immunomodulatory agents, and interferon enhancers. Antiviral enzyme inhibitors that may be administered in combination with albumin fusion proteins and / or polynucleotides of the invention include, but are not limited to, VX-950 (protease inhibitor, Vertex, Cambridge, MA). VX-497 (merimeposib, oral, IMPDH inhibitor, Vertex, Cambridge, MA), BILB 1941 (protease inhibitor, Boehringer Ingelheim, Germany, SCH7 (protease inhibitor, Schering) (Schering Corp.), Kenilworth, NJ), MX-3253 (glucosidase inhibitor, Migenix, Vanco) ver, BC, IDN-6556 (caspase inhibitor, Pfizer, New York, NY), UT231B ((glucosidase inhibitor), United Therapeutics, Silver Spring, MD protease, R26 inhibitor) F. Hoffman-La Roche, Switzerland, ITMN-B (ITMN-191, protease inhibitor, InterMune, Brisbane, Calif.), Celgosvir (MBI-3253, α-glucosidase) Inhibitor, Migenix, Inc., Vancouver, BC), SCH 503034 Protease inhibitors, including Schering Corp., Kenilworth, NJ, ACH 806 (GS9132, oral protease inhibitor, Achillion, New Haven, CT / Gilead Sciences, Foster City, CA). Antiviral polymerase inhibitors that may be administered in combination with the albumin fusion proteins and / or polynucleotides of the present invention may be nucleoside analogs or non-nucleoside inhibitors (NNIs). Viral polymerase inhibitors inhibit HCV RNA polymerase. In one embodiment, the antiviral polymerase inhibitor is a nucleoside analog including, but not limited to, NM283 (23'-C-methyl-cytidine, Idenix, Cambridge, MA) and 2'-C-methyl nucleoside. It may be a body. In other embodiments, the antiviral polymerase inhibitors include, but are not limited to, JTK-103, JTK-003, and JTK-109 (Japan Tobacco, Tokyo, Japan), R803 (Rigel, South San Francisco, CA), HCV-371, HCV-086, and HCV-796 (ViroPharm, Exton, PA / Wyeth, Madison, NJ), and XTL-2125 (BC2125, XTL Bio (XTLbio), New Yor, It may be a non-nucleoside inhibitor comprising Agents based on antiviral nucleic acids that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, antisense oligonucleotides, ribozymes, and siRNAs or short hairpin RNAs (shRNAs). Is included. Antiviral antisense oligonucleotide inhibitor agents that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, NEUGENE® AVI-4065 (AVI biopharmaceutical ( Biopharma), Portland, OR). In other embodiments, thiazolide may be administered in combination with an albumin fusion protein and / or polynucleotide of the present invention. In a preferred embodiment, the thiazolide that may be administered in combination with an albumin fusion protein and / or polynucleotide of the present invention includes, but is not limited to, ALINIA® (Nitazoxanide, Romark Laboratories LC (Romark). Laboratories, L.C.,), Tampa, FL). Antiviral immunomodulatory agents that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, ZADXIN® (thymosin alpha 1, timalfacin, Cyclone Pharmaceuticals, Inc. (SciClone Pharmaceuticals Int'l), Hong Kong) and, but not limited to, ANA245 (TLR-7 agonist, Anadys Pharmaceuticals, San Diego, CA), ANA 945A Pro Shootys (Anady's Pharmaceuticals, San Diego, CA) and CPG-1 101 (ACTILON (TM), TLR-9 agonist, Corey Pharmaceutical Group (Coley Pharmaceutical Group), Wellesley, MA) toll-like receptor comprising (TLR) include agonist. Interferon enhancers that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, EMZ702 (Transition Therapeutics, Toronto, Ontario). Furthermore, antiviral antibodies that may be used in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, Tarvacin (a human that targets phosphatidylserine on the surface of tumor epithelial cells). Monoclonal antibody, Peregrine Pharmaceuticals, Inc., Tustin, Calif.).

  In a preferred embodiment, the albumin fusion protein that may be administered alone or in combination with one or more antiviral agents comprised by the present invention is an interferon-albumin fusion protein. In a further embodiment, the interferon portion of the interferon-albumin fusion protein is interferon alpha. Non-limiting examples of interferon alpha encompassed by the present invention include, but are not limited to, the interferon faprotein disclosed in the therapeutic protein column of Table 1. In certain embodiments, the interferon alpha moiety is interferon alpha-2a, interferon alpha-2b, interferon alpha-2c, consensus interferon, interferon alphacon-1, interferon alpha-n1, interferon alpha-n3, such as INTRON®. ) A (Schering Corp., Kenilworth, NJ), ROFERON® A (Hoffman-La Roche, Nutley, NJ), Berofor alpha interferon (Behringer) Ingelheim Pharmaceutical Company (Boehringer Ingelheim Pharmac) (eutical, Inc.), Ridgefied, Conn.), OMNIFERON ™ (Viragen, Inc., Plantation, FL), MULTIFERON ™ (Viragen, Inc.), Plantation, FL WELLFERON (registered trademark) (GlaxoSmithKline, London, Great Britian), INFGENGEN (registered trademark) (Amgen, Inc.), Thousands Oaks (registered trademark), SUMIFERON (registered trademark) , Japan, BELEROFON® (Nautilus Biotech) Biotech, France), MAXY-ALPHA (TM) (Maxigen, Redwood City, CA / Hoffman-La Roche, Nutley, NJ) and any commercially available interferon alpha. Or a purified interferon product or fragment thereof, or comprises them. In a further embodiment, the interferon alpha site of the IFN-alpha-HSA fusion protein consists of or comprises an interferon that has been modified or formulated for extended or controlled release. For example, the interferon alpha moiety includes, but is not limited to, interferon-alpha-XL (Flamel Technologies, France) and LOCTERON ™ (BioLex Therapeutics / OctoPlus, Pittsbor, , NC) or consisting of or containing commercially available extended release or controlled release interferon alpha. In a further embodiment, the interferon alpha site of the IFN-alpha-HSA fusion protein may be modified by attachment of a chemical site. For example, the interferon alpha site may be modified by pegylation. Thus, in a further embodiment, the interferon alpha site of the IFN-alpha-HSA fusion protein consists of or comprises a PEGylated form of interferon alpha-2a, 2b or consensus interferon, and includes PEG-INTRON® (Schering) (Schering Corp.), Kenilworth, NJ), PEGASYS® (Hoffman-La Roche, Nutley, NJ), PEG-OMNIFERON ™ (Viragen, Inc.) Inc.), Plantation, FL), commercially available pegylated interferon alpha, or fragments thereof. In a further preferred embodiment, the interferon portion of the albumin fusion protein is interferon alpha 2a or 2b interferon, and the interferon albumin fusion protein can be administered in combination with any of these agents. Furthermore, in other embodiments, the interferon portion of the interferon-albumin protein is interferon beta or is an interferon hybrid. In further embodiments, the non-fused interferon portion of the interferon-albumin fusion protein may be used alone or in combination with one or more antiviral agents encompassed by the present invention.

  In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention may be administered in combination with an anti-opportunistic infection agent. Anti-opportunistic drugs that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, TRIMETHOPRIM-SULFAMETOXAZOLE ™, DAPSONE ™, PENTAMIDINE ™, ATOVAQUEONE ( Trademarks), ISONIAZID (TM), RIFAMPIN (TM), PYRAZINAMIDE (TM), ETHAMBUTOL (TM), RIFABUTIN (TM), CLARITHROMYCIN (TM), AZITROMYCIN (TM), GANICLOVIR (C) Trademark), FLUCONAZOLE (trademark), ITRACONAZOLE (quotient) ), KETOCONAZOLE (TM), ACYCLOVIR (TM), FAMCICOLVIR (TM), PYRIMETAMAMINE (TM), LEUCOVORIN (TM), NEUPOGEN (TM) (Filgrastim / G-CSF), and LEUKINE (TM) -CSF). In certain embodiments, the albumin fusion proteins and / or polynucleotides of the present invention may contain TRIMETHOPRIM-SULFAMETOXAZOLE ™, DAPSONE (in order to prevent or treat an opportunistic Pneumocystis carinii infection prophylactically. ™, PENTAMIDINE ™, and / or any combination with ATOVAQUINE ™. In other specific embodiments, the albumin fusion proteins and / or polynucleotides of the present invention may be used to prevent or treat ISONIAZID ™, RIFAMPIN to prevent or prevent opportunistic Mycobacterium avium complex infections. (TM), PYRAZINAMIDE (TM), and / or administered in combination with ETHAMBUTOL (TM). In other specific embodiments, the albumin fusion proteins and / or polynucleotides of the present invention may be used to prevent or treat opportunistic Mycobacterium tuberculosis infections. Used in combination with CLARITHROMYCIN ™ and / or AZITHROMYCIN ™. In other specific embodiments, the albumin fusion proteins and / or polynucleotides of the present invention may be used to prevent or treat opportunistic cytomegalovirus infections, such as GANICLOVIR ™, FOSCARNET ™, and / or Used in combination with CIDOFOVIR ™. In other specific embodiments, the albumin fusion proteins and / or polynucleotides of the present invention may be used to prevent or treat opportunistic fungal infections in order to prevent or treat FLUCONAZOLE ™, ITRACONAZOLE ™, and / or KETOCONAZOOLE ( Used in combination with a trademark. In other specific embodiments, the albumin fusion proteins and / or polynucleotides of the present invention provide ACYCLOVIR ™ and / or for the prophylactic treatment or prevention of opportunistic type I and / or type II herpes simplex infections. Used in combination with FAMCICOLVIR ™. In other specific embodiments, the albumin fusion proteins and / or polynucleotides of the present invention may be used to prevent or treat opportunistic Toxoplasma gondii infections with PYRIMETHAMINE ™ and / or LEUCOVORIN ™ ). In other specific embodiments, the albumin fusion proteins and / or polynucleotides of the invention are combined with LEUCOVORIN ™ and / or NEUPOGEN ™ to prevent or treat opportunistic bacterial infections prophylactically. use.

  In a further embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, amoxicillin, beta-lactamas, aminoglycosides, beta-lactams (glycopeptides), beta-lactamas, clinda Mycin, chloramphenicol, cephalosporin, ciprofloxacin, erythromycin, fluoroquinolone, macrolide, metronidazole, penicillin, quinolone, rapamycin, rifampin, streptomycin, sulfonamide, tetracycline, trimethoprim, trimethoprim-sulfamethoxazole And vancomycin.

  In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with an immunostimulatory agent. Immunostimulatory agents that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, levamisole (eg, ERGAMISOL ™), isoprinosine (eg, INOSIPLEX ™) , Interferons (eg, interferon alpha), and interleukins (eg, IL-2).

  In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with an immunosuppressive agent. Immunosuppressive agents that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxysergualin, and other immunosuppressive agents that work by inhibiting the functioning of responding T cells are included. Other immunosuppressive agents that may be administered in combination with the albumin fusion proteins and / or polynucleotides of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxalene, rapamycin, leflunomide, mizoribine (BREDININ ™ )), Brequinal, deoxyspergualin and azaspirane (SKF 105685), ORTHOCLONE OKT® 3 (Muromonab-CD3), SANDIMMUNE ™, NEORAL ™, SANGDYA ™ (cyclosporine), PROGRAF® (Trademark) (FK506, tacrolimus), CELLCEPT (registered trademark) (mycophenolate motefil, active metabolite of mycophenolic acid), IMURAN (trademark) Azathiopyrine), glucocorticosteroids, DELTATASONE ™ (prednisone) and HYDELTRASOL ™ (prednisone), FOLEX ™ and MEXATE ™ methotrexate), OXSOLALEN-ULTRA ™ (methoxalene) and RAPAMUNE Adrenocorticol steroids such as (sirolimus) are included. In certain embodiments, immunosuppressive agents may be used to prevent rejection of organ or bone marrow transplants.

  In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered alone or in combination with one or more intravenous immunoglobulin modulators. Intravenous immunoglobulin modulators that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, GAMMAR ™, IVEEGAM ™, SANDOGLOBULIN ™, GAMMAGARD S / D. (Trademark), ATGAM (TM) antithymocyte globulin), and GAMIMUNE (TM). In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with intravenous immunoglobulin modulators in transplantation therapy (eg, bone marrow transplantation).

  In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention are either alone or in vivo for a patient or for cells for treatment of cancer. And administered alone or as part of a combination therapy. In certain embodiments, albumin fusion proteins, particularly IL-2-albumin fusions, are prepared according to Dudley et al. (As described in (Science Express, 19 September 2002, at www.scienceexpress.org, all of which are incorporated by reference) for cancer, such as adoptive cell transport therapy for metastatic melanoma. Dosing repeatedly during passive immunotherapy.

  In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered alone or in combination with anti-inflammatory agents. Anti-inflammatory agents that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, corticosteroids (eg betamethasone, prednisolone, prednisone and triamcinolone), non-steroidal anti-inflammatory drugs (eg , Diclofenac, diflunisal, etodolac, fenoprofen, fluoctaphenine, flubiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, Tenoxicam, thiaprofenic acid and tolmethine), and antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, aryl Tyric acid derivative, arylcarboxylic acid, arylpropionic acid derivative, pyrazole, pyrazolone, salicylic acid derivative, thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amiditolin, Vendazac, benzydamine, bucolome, difenpyramide, ditazole, emorphazone, guaiazulene, nabumetone, nimesulide, orgothein, oxaceptol, paraniline, perisoxal, pifoxime, proguazone, proxazole and tenidap.

  In a further embodiment, the composition of the invention is administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the present invention include, but are not limited to, Angiostatin (Entremed, Rockville, MD), Troponin-1 (Boston Life) Sciences (Boston Life Sciences), Boston, MA), anti-invasive factor (Invasive Factor), retinoic acid and its derivatives, paclitaxel (Taxol), suramin (Suramin), tissue inhibitor of metalloproteinase-1 1) Tissue Inhibitor of Meta, a metalloproteinase-2 tissue inhibitor lloproteinase-2), VEGI, plasminogen activator inhibitor-1 (Plasminogen Activator Inhibitor-1), plasminogen activator inhibitor-2 (Plasminogen Activator Inhibitor-2) and lighter “d group” transition metals Various forms are included.

  Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species can form transition metal complexes. Suitable complexes of the transition metal species include oxo transition metal complexes.

  Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as ammonia metavanadate, sodium metavanadate and sodium orthovanadate. Suitable vanadyl complexes include vanadyl acetylacetonate and vanadyl sulfate, including, for example, vanadyl sulfate hydrides such as vanadyl sulfate mono- and trihydrate.

  Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonia tungstate, calcium tungstate, sodium tungstate dihydrolate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxomolybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonia molybdate and its hydrate, sodium molybdate and its hydrate, and potassium molybdate and its hydrate. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid and sugars.

  A wide variety of other anti-angiogenic factors may also be used in the context of the present invention. Representative examples include, but are not limited to, platelet factor 4, protamine sulfate, sulfonated chitin derivatives (prepared from Queen Crab Shell), (Murata et al., Cancer Res. 51: 22-26 (1991). )), Sulfonated polysaccharides, peptidoglycan conjugates (SP-PG) (the function of the compound is enhanced by the presence of steroids such as estrogen, and tamoxifen citrate), staurosporine, for example, Regulators of matrix metabolism including proline analogs, cishydroxyproline, d, L-3,4-dehydroproline, thiaproline, alpha, alpha-dipyridyl, aminopropionitrile fumarate, 4-propyl-5- (4- Pyridinyl) 2 (3H) -oxazolone, methotrexate, mitoxatron, heparin, interferons, 2 globulin-serum, ChIMP-3 (Pavloff et al., J. Bio. Chem. 267: 17321-17326, (1992)), thymostatin ( (Chymostatin) (Tomkinson et al., Biochem J. 286: 475-480, (1992)), cyclodextrin tetradecasulfate, eponemycin, camptothecin, fumagillin (Ingber et al., Nature 348: 555-557, 90). ), Gold sodium thiomalate (“GST”, Matsubara and Ziff, J. Clin. Invest. 79: 1440). 1446, (1987)), anti-collagenase-serum, alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262 (4): 1659-1664 (1987)), bisantren (National Cancer Institute), Lobenzalite disodium (N- (2) -carboxyphenyl-4-chloroanthronylate disodium or “CCA” (Takeuchi et al., Agents Actions 36: 312-316, (1992)), and BB94 Metalloproteinase inhibitors are included.

  Additional anti-angiogenic factors that may be used within the conditions of the present invention include thalidomide, (Celgene, Warren, NJ), vascular steroids, AGM-1470 (H. Brem and J. Folkman J Pediatr. Surg. 28: 445-51 (1993)), integrin alpha vbeta3 antagonist (C. Storgard et al., J Clin. Invest. 103: 47-54 (1999)), carboxinaminol midazole, carboxamidotriazole ( CAI) (National Cancer Institute, Bethesda, MD), Combretastatin A-4 (CA4P) (OXiGENE, Boston, MA) , Squalamine (Magainin Pharmaceuticals), Plymouth Meeting, PA, TNP-470 (Tap Pharmaceuticals, Deerfield, IL), ZD-01Astra APRA (CT2584), Benefin, Virosatin-1 (SC339555), CGP-41251 (PKC412), CM101, Dexrazoxane (ICRF187), DMXAA, Endostatin, Flavopyridiol, Genestein, GTE, ImmTher, Iressa (Iressa) ZD1839), octreotide (Somatos) Tachin), Panretin, Penacillamine, Photopoint, PI-88, Pyriminostat (AG-3340) Pulllittin, Slistasta (FCE26644), Tamoxifen (Norbadex), Tazarotene, Tetrathiomolybdate, Xeroda (Capecitabine), and 5-Fluorouracil Is included.

  Anti-angiogenic agents that may be administered in combination with the compounds of the present invention include, but are not limited to, inhibiting extracellular matrix proteolysis, blocking endothelial cell-extracellular matrix adhesion molecules, growth factors Can work through various mechanisms including neutralizing the function of angiogenesis inducers such as and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenesis inhibitors that interfere with extracellular matrix proteolysis and may be administered in combination with the compositions of the present invention include, but are not limited to, AG-3340 (Agouron, La Jolla, CA), BAY-12-9566 (Bayer, West Haven, CT), BMS-275291 (Bristol Myers Squibb), Princeton, NJ, CGS-27032A (Novartis, Novartis, Novartis, Novartis) NJ), Marimastat (British Biotech, Oxford, UK), and Metastat. (Aeterna, St-Foy, Quebec). Examples of anti-angiogenesis inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and may be administered in combination with the compositions of the present invention include, but are not limited to, EMD-121974 ( Merck KcgaA Darmstadt, Germany and Vitaxin (Ixsys, La Jolla, CA / Medimune, Gaithersburg, MD). Examples of anti-angiogenic agents that work by directly neutralizing or inhibiting angiogenesis inducers and may be administered in combination with the compositions of the present invention include, but are not limited to, angiozyme. ) (Ribozyme, Boulder, CO), anti-VEGF antibodies (Genentech, S. San Francisco, CA), PTK-787 / ZK-225846 (Novatis, Basel, Switzerland) 101 (Sugen, S. San Francisco, Calif.), SU-5416 (Sugen / Pharmacia Upjohn, Bridgewater, NJ), and SU-6668 (Sugen (Sugen)) are included. Other anti-angiogenic agents work by indirectly inhibiting angiogenesis. Examples of indirect inhibitors of angiogenesis that may be used in combination with the compositions of the present invention include, but are not limited to, IM-862 (Cytran, Kirkland, WA), interferon-alpha, IL-12 (Roche, Nutley, NJ), and pentosan polysulfate (George University University, Washington, DC).

  In certain embodiments, the use of the compositions of the present invention in combination with an anti-angiogenic agent can be used to treat, prevent and / or treat autoimmune diseases, such as, for example, autoimmune diseases as described herein. Or intended for mitigation.

  In certain embodiments, the use of the composition of the present invention in combination with an anti-angiogenic agent is contemplated for the treatment, prevention and / or alleviation of arthritis. In a more particular embodiment, the use of the composition of the invention in combination with an anti-angiogenic agent is contemplated for the treatment, prevention and / or alleviation of rheumatoid arthritis.

  In other embodiments, a polynucleotide encoding a polypeptide of the invention is administered in combination with an anti-angiogenic protein or a polynucleotide encoding an angiogenic protein. Examples of angiogenic proteins that may be administered with the compositions of the present invention include, but are not limited to, acidic and basic fibroblast growth factor, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha And beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte / macrophage colony stimulating factor, and oxidation Nitrogen synthase is included.

  In a further embodiment, the composition of the invention is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, nitrogen mustards (eg, mechlorethamine, cycloformamide, cyclophosphamide ifosfamide). Alkylating agents such as melphalan (L-sarcolicine), and chlorambucil), ethyleneimines and methylmelamines (eg hexamethylmelamine and thiotepa), alkylsulfonates (eg busulfan), nitrosourea (eg carmustine (BCNU)) ), Lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin)), triazenes (eg, dacarbazine (DTIC, dimethyltriazenoy) Midazolecarboxamide)), folic acid analogues (eg methotrexate (amesopterin)), pyrimidine analogues (eg fluorouracil (5-fluorouracil, 5-FU), furoxyuridine (fluorodeoxyuridine, FudR), and cytarabine (cytosine arabinoside) )), Purine derivatives and related inhibitors (eg, mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG), and pentostatin (2′-deoxycoformycin)), vinca alkaloids (Eg, vinblastine (VLB, vinblastine sulfate)) and vincristine (vincristin sulfate)), epipodophyllotoxins (eg, etoposide and teniposi) ), Antibiotics (eg, dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, primycin (misalamycin) and mitomycin (mitomycin C), enzymes (eg, L-asparaginase), biology Response modifiers (eg, interferon-alpha and interferon-alpha-2b), platinum-modulating compounds (eg, cisplatin (cis-DDP) and carboplatin), ansolacenesion (mitoxatron), substituted ureas (eg, hydroxyurea) Methylhydrazine derivatives (for example, procarbazine (N-methylhydrazine, MIH), adrenocorticosteroids (for example, prednisone), progesti (Eg, hydroxyprogesterone caproic acid, medroxyprogesterone, medroxyprogesterone acetate, and megestrol acetate), estrogens (eg, diethylsitylbestrol (DES), diethylsitylbestrol diphosphate, estradiol) , And ethinylestradiol), antiestrogens (eg tamoxifen), androgens (testosterone propionic acid and fluoxymesterone), antiandrogens (eg flutamide), gonadotropin-releasing hormone analogues (eg leuprolide), other Hormones and hormone analogs such as methyltestosterone, estramustine, estramustine sodium phosphate, chlorotrianicene, and test Kuton), and others (eg, dicarbazine, glutamic acid and mitotane).

  In one embodiment, the composition of the invention is administered in combination with one or more of the following drugs. Ifliximab (also known as Remicade ™ (also known as Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (Hoechst Marion Lucel) (Also known as Arava ™ from Marion Roussel), Kineret ™ (Anakinra (IL-1 receptor antagonist, also known as Amgen, Inc.).

  In certain embodiments, the compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), or in combination with one or more components of CHOP. In one embodiment, the composition of the invention is administered in combination with an anti-CD20 antibody, a human monoclonal anti-CD20 antibody. In other embodiments, the composition of the invention is combined with anti-CD20 antibody and CHOP, or a combination of anti-CD20 antibody and any one or more components of CHOP, especially cyclophosphamide and / or prednisone. In combination. In certain embodiments, the compositions of the invention are administered in combination with rituximab. In a further embodiment, the composition of the invention is administered with rituximab and CHOP, or any combination of rituximab and one or more components of CHOP, especially cyclophosphamide and / or prednisone. In certain embodiments, the compositions of the invention are administered in combination with tositumomab. In a further embodiment, the composition of the invention is administered with tositumomab and CHOP, or any combination of tositumomab and one or more components of CHOP, particularly cyclophosphamide and / or prednisone. The anti-CD20 antibody may optionally be conjugated to a radioisotope, toxin or cytotoxic prodrug.

In other specific embodiments, the compositions of the invention are administered in combination with Zevalin ™. In a further embodiment, the composition of the present invention is combined with Zevalin ™ and CHOP, or Zevalin ™ with any combination of one or more components of CHOP, especially cyclophosphamide and / or prednisone. Administer. Zevalin ™ may be combined with one or more radioisotopes. Particularly preferred isotopes are ⁹⁰ Y and ¹¹¹ In.

  In a further embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with cytokines. Cytokines that may be administered with the albumin fusion protein and / or polynucleotide of the present invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40. CD40L, IFN-gamma and TNF-alpha. In other embodiments, the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL- 18, IL-19, IL-20, and IL-21.

  In one embodiment, the albumin fusion protein and / or polynucleotide of the present invention is administered in combination with a TNF family member. TNF, TNF-related, or TNF-like molecules that may be administered with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, a soluble form of TNF-alpha, lymphotoxin-alpha (LT-alpha , Also known as TNF-beta), LT-beta (found in the complex heterotrimer LT-alpha 2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L , TNF-gamma (International Patent Specification No. WO 96/14328), AIM-1 (International Patent Specification No. WO 97/33899), Endokine-Alpha (International Patent Specification No. WO 98/07880). No.), OPG, and Neurokine-Alpha (International Special No. WO 98/18921), OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Patent Specification No. WO 96/34095) ), DR3 (International Patent Specification No. WO 97/33904), DR4 (International Patent Specification No. WO 98/32856), TR5 (International Patent Specification No. WO 98/30693), TRANSK, TR9 (International Patent Specification No. WO 98/56892), TR10 (International Patent Specification No. WO 98/54202), 312C2 (International Patent Specification No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153 are included.

  In a further embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with an angiogenic protein. Angiogenic proteins that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, glioma derived growth factors, such as those disclosed in European Patent No. EP-399816. Factor (GDGF)), as disclosed in European Patent No. EP-682110, as disclosed in Platelet Derived Growth Factor-A (PDGF-A), European Patent No. EP-282317. Platelet-derived growth factor-B (Plalet Derived Growth Factor-B (PDGF-B)), such as disclosed in International Patent Specification WO 92/06194. ntal Growth Factor (PlGF)), Hauser et al. , Growth Factors, 4: 259-268 (1993), Placental Growth Factor-2 (PlGF-2), International Patent Specification No. WO 90/13649. Vascular Endothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor-A (as disclosed in European Patent No. EP-506477). VEGF-A)), Vascular Endothelial Growth Factor-2 (as disclosed in International Patent Specification No. WO 96/39515). EGF-2)), Vascular Endothelial Growth Factor B (VEGF-3)), Vascular Endothelial Growth Factor B-186, as disclosed in International Patent Specification No. WO 96/26736. (Vascular Endothelial Growth Factor B-186 (VEGF-B186)), Vascular Endothelial Growth Factor-D (VEGF-D) as disclosed in International Patent Specification No. WO 98/02543. D)), Vascular Endothelial Growth Factor-D (V) as disclosed in International Patent Specification No. WO 98/07832. GF-D)) and DE DE19639601 degree in the disclosed vascular endothelial growth factor -E (Vascular Endothelial Growth Factor-E (VEGF-E)) are included. All of the above references are incorporated herein by reference.

  In a further embodiment, the albumin fusion protein and / or polynucleotide of the present invention is administered in combination with Fibroblast Growth Factors. Fibroblast growth factors that may be administered with the albumin fusion protein and / or polynucleotide of the present invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15 are included.

  In a further embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with a hematopoietic growth factor. Hematopoietic growth factors that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim, LEUKINE ™, PROKINE ™, granulocyte colony) Stimulating factor (G-CSF) (filgrastim, NEPOGEN ™), macrophage colony stimulating factor (M-CSF, CSF-1), erythropoietin (epoetin alfa, EPOGEN ™, PROCRIT ™), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony-stimulating factor, PIXY321 (GMCSF / IL-3 fusion protein), interleukins, especially one or more IL-1 to IL-12, interphase Elon-gamma or thrombopoietin is included.

  In certain embodiments, the albumin fusion proteins and / or polynucleotides of the present invention are, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxyprenolol, penbutolol, pindolol, propranolol, Administered in combination with adrenergic blockers such as sotalol and timolol.

  In other embodiments, the albumin fusion protein and / or polynucleotide of the present invention is treated with an arrhythmia therapeutic agent (eg, adenosine, amidarone, bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine, moricidin. Phenytoin, procainamide, N-acetylprocainamide, propaphenone, propanolol, quinidine, sotalol, tocainide and verpamil).

In other embodiments, the albumin fusion proteins and / or polynucleotides of the present invention are combined with carbon anhydrase-inhibitors (eg, acetazolamide, dichlorophenamide and methazoleamide), osmotic diuretics (eg, glycerin, isosorbide, mannitol and ^{urea), Na} + -K + -2Cl ^- diuretics that inhibit symport (e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrylic acid, muzolimine, and torsemide), thiazide and thiazide - like diuretics ( For example, bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methylcrothiazide, polythiazide, triclomethiazide, chlorsalidon, indapamide Metolazone and Kinesazon), potassium sparing diuretics (e.g., amiloride and triamterene), and mineralocorticoid receptor antagonists (e.g., spironolactone, canrenoate, and potassium canrenoate), such as, administered in combination with a diuretic.

In one embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with treatment of endocrine and / or hormonal imbalance disorders. Treatment of endocrine and / or hormonal imbalance diseases includes, but is not limited to, radioactive isotopes of elements such as ¹²⁷ I, ¹³¹ I and ¹²³ I, groups such as HUMATROPE ™ (recombinant somatropin) Replacement growth hormone, growth hormone analogs such as PROTROPIN ™ (Somatrem), dopamine agonists such as PARLODEL ™ (bromocriptine), somatostatin analogues such as SANDOSTATIN ™ (octreotide), PREGNYL ™ A. P. L. Gonadotropin modulators, FACTREL ™ and LUTREPULSE, such as TM ™ and PROFASI ™ (cholinergic gonadotropin (CG)), PERGONAL ™ (menotropin), and METRODIN ™ (urophorotropin (uFSH)) Synthetic human gonadotropin-releasing hormone regulators such as (TM) (gonadorelin hydrochloride), LUPRON (TM) (Luprolide Acetate), SUPPRELIN (TM) (histrelin acetate), SYNAREL (TM) (Nafarelin acetate), and ZOLADEX (TM) Synthetic gonadotropin agonists such as (Goserelin acetate), thyrotropin-releasing hormones such as RELEFACT TRH ™ and THYPINE ™™ (Protillin) Synthetic preparations of recombinant human TSH, such as THYROGEN ™, LT- ₄ ™, SYNTHROID ™ and LEVOTHROID ™ (levothroxin sodium), LT- ₃ ™, Modulators of sodium salts of natural isomers of thyroid hormones such as CYTOMEL ™ and TRIOSTAT ™ (liothyloin sodium), and THYROLAR ™ (Riotrix), 6-n-propylthiouracil (propylthio) Anti-thyroid compounds such as uracil), 1-methyl-2-mercaptoimidazole and TAPAZOLE ™ (methimazole), NEO-MERCAZOLE ™ (carbimazole), beta-adrenergic agonists such as propranolol and esmolol Dexamethasone and iodinated radiocontrast agents, such as sex receptor antagonists, Ca2 ⁺ channel blockers, TELEPAQUE ™ (iopanonic acid) and ORGRAFIN ™ (sodium ipodoate).

  Additional treatments for endocrine and / or hormonal imbalance diseases include, but are not limited to, ESTRACE ™ (estradiol), ESTINYL ™ (ethinylestradiol), PREMARIN ™, ESTRATATAB ™, ORTHO -EST (TM), OGEN (TM) and estropipete (estrone), ESTROVIS (TM) (quinestrol), ESTRADERM (TM) (estradiol), DELASTROGEN (TM) and VALERGEN (TM) (estradiol valerate), DEPO- Estrogens or conjugated estrogens such as ESTRADIOL CYPIONATE ™ and ESTROJECT LA ™ (estradiol cypionate), N Antiestrogens such as OLVADEX ™ (tamoxifen), SEROPHENE ™ and CLOMID ™ (clomiphene), DURALUTIN ™ (hydroxyprogesterone caproate, MPA ™ and DEPO-PROVERA ™ (medoacetate) Roxyprogesterone), PROVERA (TM) and CYCRIN (TM) (MPA), MEGACE (TM) (megestrol acetate), NORLUTIN (TM) (norethindrone) and NORLUTATE (TM) and AYGETIN (TM) (norethindrone acetate) Progestins, such as RU486 ™, progesterone implants, such as NORPLANT SYSTEM ™ (norgestrel subcutaneous implant) Antiprogestins such as Mifepristone), ENOVID ™ (norethinoderel + mestranol), PROGESTER ™™ (intrauterine device that releases progesterone), LOESTRIN ™, BREVICON ™, MODICON ™, GENORA (TM), NELONA (TM), NORINYL (TM), OVACON-35 (TM) and OVACON-50 (TM) (ethynylestradiol / norethindrone), LEVLEN (TM), NORDETTE (TM), TRI-LEVLEN (TM) ) And TRIPHASIL-21 ™ (ethynylestradiol / levonorgestrel), LO / OVRAL ™ and OVRAL ™ (ethynylestradiol / no Lugesterel), DEMULEN ™ (ethynylestradiol / ethinodioldiacetic acid), NORNYL ™, ORTHO-NOVUM ™, NORETIN ™, GENORA ™, and NELOVA ™ (Noretindrone / Mestranol), DESOGEN (Trademark) and ORTHO-CEPT (trademark) (ethynylestradiol / desogestrel), hortho-cyclen (trademark) and hortho-trickylen (trademark) (ethynylestradiol / norgestimate), micronor (trademark) and NOR-QD (trademark) And hormonal contraceptives such as OVRETTE ™ (norgestrel).

  Additional treatments for endocrine and / or hormonal imbalance disorders include, but are not limited to, testosterone esters such as methenolone acetate and testosterone undecanoate, TESTOJECT-50 ™ (testosterone), TESTEX ™. (Testosterone propionate), DELATESTRYL ™ (testosterone enanthate), DEPO-TESTOSTONEONE ™ (testosterone cypionate), DANOCRINE ™ (danazol), HALOTESTIN ™ (fluoxymesterone), ORETON METHYL (Trademark), TESTRED (trademark) and VIRILON (trademark) (methyltestosterone), and OXANDRIN (trademark) (oxandrolone) Such as parenteral and oral androgens such as, testosterone transdermal system such as TESTODER ™, ANDROCUR ™ (cyproterone acetate), EULEXIN ™ (flutamide), and PROSCAR ™ (finasteride) Androgen receptor antagonists and 5-alpha-reductase inhibitors, adrenocorticotropin hormone preparations such as CORTROSYN ™ (cosyntropin), ACLOVATE ™ (alcomethasone dipropionate), CYCLOCORT ™ (amicinonide), BECLOVENT (Trademark) and VANCERIL (trademark) (beclomethasone dipropionate), CELESTONE (trademark) (betamethasone), BENISONe (trademark) And UTICORT ™ (betamethasone benzoate), DIPROSON ™ (betamethasone dipropionate), CELESTON PHOSPHATE ™ (betamethasone sodium phosphate), CELESTONE SOLUSPAN ™ (phosphate and betamethasone sodium acetate), BETA- VAL (TM) and VALISEONE (TM) (betamethasone valerate), TEMOVEATE (TM) (clobetasol propionate), CLODERM (TM) (crocortron pivalate), CORTE (TM) and HYDROCORTONE (TM) (cortisol (hydrocortisone)) , HYDROCORTONE ACETATE ™ (cortisol acetate (hydrocortisone)), LOCOID ™ Cortisol butyrate (hydrocortisone)), HYDROCORTONE PHOSPHATE ™ (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORE ™ and SOLU CORTEF ™ (cortisol (hydrocortisone) sodium succinate), WESTCORT ™ Cortisol (hydrocortisone) valeric acid), CORTISON ACEATE ™ (cortisone acetate), DESOWEN ™ and TRIDESILON ™ (desonide), TOPICORT ™ (desoxymethasone), DECADRON ™ (dexamethasone), DECADRON LA ( (Trademark) (dexamethasone acetate), DECADRON PHOSPHATE (trademark) and HEXA ROL PHOSPHATE (TM) (dexamethasone sodium phosphate), FLORONE (TM) and MAXIFLOR (TM) (diflorazone diacetate), FLORINEF ACETATE (TM) (fludrocortisone acetate), AEROBID (TM) and NASALIDE (TM) (flunisolide) ), FLUONID ™ and SYNALAR ™ (fluocinolone acetonide), LIDEX ™ (fluocinonide), FLUOR-OP ™ and FML ™ (fluoromesolone), CORDRAN ™ (full) Landrenolide), HALOG ™ (halcinonide), HMS LIZUIFILM ™ (Medrison), MEDROL ™ (methylprednisolone), DEPO-MEDRO (Trademark) and MEDROL ACEDATE (TM) (methylprednisone acetate), A-METHAPPRED (TM) and SOLUMEDROL (TM) (methylprednisolone sodium succinate), ELOCON (TM) (mometasone furoate), HALDRONE (TM) (acetic acid) Parameterzone), DELTA-CORTEF ™ (prednisolone), ECONOPRED ™ (prednisolone acetate), HYDELTRASOL ™ (prednisolone sodium phosphate), HYDELTRA-T. B. A (TM) (prednisolone tebutate), DELTATASONE (TM) (prednisone), ARISCOCORT (TM) and KENACORT (TM) (triamcinolone), KENALOG (TM) (triamcinolone acetonide), ARISCOCORT (TM) and KENACORT DIACATE ™ (triamcinolone diacetate), and adrenocortical hormones such as ARISTOSPAN ™ (triamcinolone hexaacetonide) and their synthetic analogues, CYTADREN ™ (aminoglutesamide), NIZORAL ™ (ketoconazole) Biosynthesis of corticosteroids and steroids such as MODASTANE ™ (trilostane) and METOPIRONE ™ (methyrapone) Inhibitors of activity, bovine, porcine or human insulin or mixtures thereof, insulin analogues, recombinant human insulins such as HUMULIN ™ and NOVOLIN ™, ORAMIDE ™ and ORINASE ™ (tolbutamide), DIABINESE (TM) (chlorpropamide), TOLAMIDE (TM) and TOLINASE (TM) (toluazamid), DYMELOR (TM) (acetohexamide), glibenclamide, MICRONASE (TM), DIBETATA (TM) and GLYNASE (TM) ( Glyburide), GLUCOTROL ™ (glipizide), and DIAMICRON ™ (gliclazide), GLUCOPHAGE ™ (metformin), ciglitazone, pioglita Oral hypoglycemic drugs such as zon and alpha-glucosidase inhibitors, somatostatins such as bovine and porcine glucagon, SANDOSTATIN ™ (octreotide), and diazoxides such as PROGLYCEM ™ (diazoxide).

  In one embodiment, the albumin fusion protein and / or polynucleotide of the invention is administered in combination with a treatment for uterine motility disease. Treatments for uterine motility diseases include, but are not limited to, conjugated estrogens (eg, PREMARIN® and ESTRATAB®), estradiols such as CLIMARA® and ALORA®) , Estrogenic drugs such as estoppiate, chlorotrianine, progestin drugs (eg, AMEN® (medroxyprogesterone), MICRONOR® (noretidolone acetate), PROMETRIUM® progesterone, and megestrol acetate ) And, for example, conjugated estrogens / medroxyprogesterone (eg, PREMPRO ™ and PREMPPHASE®) and noresindolone acetate / ethynylestradi Lumpur (e.g., FEMHRT (TM)), such as, include estrogen / progesterone combination therapy.

In a further embodiment, the albumin fusion protein and / or polynucleotide of the present invention comprises iron sulfate (iron sulfate, FEOSOL ™), iron fumarate (eg FEOSTAT ™), iron gluconate (eg FERGON ( Trademark)), polysaccharide-iron complexes (eg, NIFERREX ™), iron dextran injection (eg, INFED ™), copper sulfate, proxydin, riboflavin, vitamin B ₁₂ , cyancobalamin injection (eg, REDISOL (TM), RUBRAMIN PC (TM), hydroxocobalamin, formic acid (e.g. FOLVITE (TM)), leucovorin (folinic acid, 5-CHOH4PteGlu, citrobolum factor) or WELLCOVORIN (locovorin) Calcium salts), including but not limited to transferrin or ferritin, administered in combination with effective drug in the treatment of iron deficiency and hypochromic anemia.

  In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with agents used to treat psychiatric illnesses. Psychiatric drugs that may be administered with the albumin fusion protein and / or polynucleotide of the present invention include, but are not limited to, antipsychotic drugs (eg, chloropromazine, chloroprosixin, clozapine, fluphenazine, haloperidol, loxapine) , Mesoridazine, morindon, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoroperiazine and triflupromazine), antidepressants (eg, carbamazepine, divalproex sodium, lithium carbonate, and lithium citrate) ), Antidepressants (eg, amitriptyline, amoxapine, bupropion, titalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, i Pramine, isocarboxazide, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertalarin, tranylcypromine, trazodone, trimipramine and venlafaxine), anxiolytics (eg, alprazolam, buspirone, chlordiazepoxide, Chlorazepate, diazepam, halazepam, lorazepam, oxazepam and parazepam), and stimulants (eg, d-amphetamine, methylphenidate and pemoline).

  In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with drugs used for the treatment of nervous system diseases. Nervous agents that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, antiepileptic drugs (eg, carbamazepine, clonazepam, ethoximid, phenobarbital, phenytoin, primidone, valproic acid, Divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian drugs (eg, levodopa / carbidopa, selegiline, amantidine, bromocriptine, Ropinirole, pramipexole, benztropine, biperidene, etopropazine, procyclidine, trihexyphenidyl, tolcapo ) And ALS treatment (e.g. riluzole) is included.

  In other embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with vasodilators and / or calcium channel inhibitors. Vasodilators that may be administered with the albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, angiotensin converting enzyme (ACE) inhibitors (eg, papaverine, isoxspirin, benazepril, Captopril, cilazapril, enalapril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril and nilidrin) and nitrates (eg, isosorbide dinitrate, isosorbide mononitrate and nitroglycerin). Examples of calcium channel inhibitors that may be administered in combination with albumin fusion proteins and / or polynucleotides of the present invention include, but are not limited to, amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, Nimodipine and verapamil are included.

In certain embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with a treatment for gastrointestinal disease. Treatments for gastrointestinal diseases that may be administered with albumin fusion proteins and / or polynucleotides of the invention include, but are not limited to, H ₂ histamine receptor antagonists (eg, TAGAMET ™ (cimetidine), ZANTAC ™ ) (Ranitidine), PEPCID ™ (famotidine), and AXID ™ (nizatidine)), inhibitors of H ⁺ , K ⁺ ATPase (eg, PREVACID ™ (lansoprazole) and PRILOSEC ™ (omeprazole) ), Bismuth compounds (eg, PEPTO-BISMOL ™ (bismuth subsalicylate) and DE-NOL ™ (bismuth subcitrate)), various antacids, sucralfate, prostaglandin analogs ( For example, CYTOTEC ™ (misoprostol)), a murucalin cholinergic antagonist, constipation drugs (eg, surfactant constipation drugs, irritant laxatives, saline and isotonic laxatives), anti-diarrheal drugs ( For example, synthetic analogues of somatostatin, such as LOMOTIL ™ (diphenoxylate), MOTOFEN ™ (diphenoxine), and IMODIUM ™ (loperamide hydrochloride)), SANDOSTATIN ™ (octreotide), antiemetics ( For example, ZOFRAN ™ (ondansetron), KYTRIL ™ (granisetron hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, prochlorperazine, promethazine, thiethylperazine, trifluproma Gin, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol and nabilone), D2 antagonists (eg, metoclopramide, trimethobenzamide and chlorpromazine), bile acid, chenodeoxycholic acid, ursodeoxycholine acid, and pan Pancreatic enzyme regulators such as creatine and pancrelipase are included.

  In further embodiments, the albumin fusion proteins and / or polynucleotides of the invention are administered in combination with other therapeutic or prophylactic regimes such as, for example, radiation therapy.

The invention also provides a pharmacological pack or kit comprising one or more containers filled with one or more components of a pharmacological composition comprising an albumin fusion protein of the invention. A notice in the form prescribed by the government that regulates the manufacture, use or sale of a medicinal product or biological product is optionally associated with such a container, and such notice may be produced for human administration. Reflects permission from the authorities of use or sales.

Gene therapy

  A construct encoding an albumin fusion protein of the present invention can be used as part of a gene therapy protocol to deliver a therapeutically effective amount of an albumin fusion protein. A preferred approach for in vivo introduction of nucleic acids into cells is by using a viral vector encoding an albumin fusion protein of the invention that contains the nucleic acid. Infection of cells with a viral vector has the advantage that a large proportion of target cells can receive the nucleic acid. Furthermore, for example, a molecule encoded in a viral vector is effectively expressed in a cell that has taken up the viral vector nucleic acid due to the cDNA contained in the viral vector.

  Retroviral and adeno-associated viral vectors can be used as recombinant gene delivery systems for the delivery of foreign nucleic acids encoding albumin fusion proteins in vivo. These vectors provide efficient transport of the nucleic acid into the cell so that the transported nucleic acid is stably integrated into the host chromosomal DNA. Development of specialized cell lines that produce only non-replicatable retroviruses (called “packaging cells”) has increased the use of retroviruses for gene therapy, and defective retroviruses are Characterized for use in gene delivery for therapeutic purposes (for review see Miller, AD (1990) Blood 76: 271)). Non-replicating retroviruses can be packaged into virions that can be used to infect target cells through the use of helper viruses by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses are described in Current Protocols in Molecular Biology, Ausubel, F., et al. M.M. et al. (Eds.) Green Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.

  Another viral gene delivery system useful in the present invention uses adenovirus-derived vectors. The gene of the adenovirus genome encodes and expresses the gene product of interest, but is operable to inactivate with respect to its ability to replicate during the normal lytic virus life cycle. For example, Berkner et al. BioTechniques 6: 616 (1988); Rosenfeld et al. , Science 252: 431-434 (1991), and Rosenfeld et al. , Cell 68: 143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (eg, Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Recombinant adenoviruses are advantageous in certain situations where infection of non-dividing cells is not possible and can be used to infect a wide variety of cell types, including endothelial cells (Rosenfeld et al. (Cited above (1992)). In addition, virus particles are relatively stable and susceptible to purification and concentration, and as described above, can be modified to affect the spectrum of infection. Furthermore, in the situation where the introduced adenoviral DNA (and the foreign DNA contained therein) is not integrated into the host cell genome, but remains episomal, thereby integrating the introduced DNA into the host cell genome, Essential problems that can arise as a result of insertional mutagenesis are avoided (eg retroviral DNA). In addition, the adenoviral genome transport capacity for foreign DNA is large (˜8 kilobases) compared to other gene delivery vectors (Berkner et al., Cited supra; Haj-Ahmand et al., J. Virol. 57). : 267 (1986)).

  In other embodiments, the non-viral gene delivery system of the invention relies on an endocytotic pathway for uptake of the nucleotide molecule of interest by the targeted cell. Exemplary gene delivery systems of this type include liposome-derived systems, poly-lysine conjugates, and artificial viral envelopes. In an exemplary embodiment, the nucleic acid molecules encoding the albumin fusion proteins of the present invention are (optionally) targeted to liposomes with positive charges on their surface that are targeted with antibodies to cell surface antigens of the target tissue. It can be trapped (eg lipofection). (Mizuno et al. (1992) No Shinkei Geka 20: 547-5 51, PCT specification No. W09 / 06309, Japanese patent No. 1047381 and European patent specification No. EP-A-43075)

A gene delivery system for a gene encoding an albumin fusion protein of the invention can be introduced into a patient by any number of methods. For example, a pharmacological composition of a gene delivery system can be introduced systemically, for example, by intravenous injection, and specific transduction of proteins in target cells primarily results in gene delivery vehicle, receptor gene expression. It arises from the specificity of transfection provided by cell-type or tissue-type expression by a regulatory transcriptional regulatory sequence, or a combination thereof. In other embodiments, the initial delivery of the recombinant gene is more limited by introduction into a highly localized animal. For example, gene delivery excipients can be introduced by catheter (US Pat. No. 5,328,470) or by stereotaxic injection (Chen et al. (1994) PNAS 91: 3 054-3 05 7). The pharmacological preparation of the gene therapy structure may comprise a sustained release matrix consisting essentially of the gene delivery system in an acceptable diluent or embedded with gene delivery excipients. If the albumin fusion protein can be produced intact from a recombinant cell, eg, a retroviral vector, the pharmacological preparation can include one or more cells that produce the albumin fusion protein.

Further gene therapy methods

  Also encompassed by the present invention are gene therapy methods for treating or preventing diseases, illnesses and conditions. The gene therapy method relates to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into animals to achieve expression of the albumin fusion protein of the invention. The method requires a nucleotide encoding an albumin fusion protein of the invention operably linked to a promoter and other genetic elements required for expression of the fusion protein by the target tissue. Such gene therapy and delivery techniques are known in the art, see, eg, International Patent No. WO 90/11092, which is incorporated herein by reference.

  Thus, for example, ex vivo, a polynucleotide comprising a promoter operably linked to a polynucleotide encoding an albumin fusion protein of the invention, and then modified cells are treated with the fusion protein of the invention. By providing the patient, the cells from the patient can be modified. Such methods are known in the art. See, eg, Belldegrun, A., which is incorporated herein by reference. , Et al. , J. et al. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M .; et al. , Cancer Research 53: 1107-1112 (1993); Ferrantini, M .; et al. , J. et al. Immunology 153: 4604-4615 (1994); Kaido, T .; , Et al. , Int. J. et al. Cancer 60: 221-229 (1995); Ogura, H .; , Et al. , Cancer Research 50: 5102-5106 (1990); Santodonato, L .; , Et al. , Human Gene Therapy 7: 1-10 (1996); Santodonato, L .; , Et al. , Gene Therapy 4: 1246-1255 (1997); and Zhang, J. et al. -F. et al. , Cancer Gene Therapy 3: 31-38 (1996)). In one embodiment, the cell to be modified is an arterial cell. Arterial cells may be reintroduced to the patient via direct injection into the artery, tissue surrounding the artery, or via catheter injection.

  As discussed in more detail below, the polynucleotide construct delivers an injectable substance into an animal cell, such as an injection into a tissue interstitial space (heart, muscle, skin, lung, liver, etc.). It can be delivered by any method. The polynucleotide construct may be delivered in a pharmacologically acceptable liquid or aqueous carrier.

  In one embodiment, the polynucleotide encoding the albumin fusion protein of the invention is delivered as a naked polynucleotide. The phrase “naked” polynucleotide, DNA or RNA is any that serves to assist, drive or facilitate entry into cells, including viral sequences, viral particles, liposomal formulations, lipofectin or precipitants, etc. By sequence without delivery excipients is meant. However, polynucleotides encoding albumin fusion proteins of the invention can also be delivered in liposome and lipofectin formulations and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference. ing.

  Polynucleotide vector constructs for use in gene therapy methods are preferably constructs that are not integrated into the host genome or that do not contain sequences that allow replication. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene, pSVK3P available from Pharmacia, BPV, pMSG and pSVL, pE available from Invitrogen1 / V5, pcDNA3.1, and pRc / CMV2. Other suitable vectors will be readily apparent to those skilled in the art.

  Any strong promoter known to those skilled in the art can be used to drive expression of the polynucleotide sequence. Suitable promoters include adenovirus promoters such as the adenovirus major late promoter, or heterologous promoters such as the cytomegalovirus (CMV) promoter, respiratory syncytial virus (RSV) promoter, MMT promoter, metallothionein promoter and the like. Inducible promoters, heat shock promoters, albumin promoters, ApoAI promoters, human globulin promoters, viral thymidine kinase promoters such as herpes simplex thymidine kinase promoters, retroviral LTRs, b-actin promoters, and human growth hormone promoters. The promoter may also be a natural promoter for the gene corresponding to the therapeutic protein portion of the albumin fusion protein of the invention.

  Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the temporary nature of intracellular polynucleotide synthesis. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods up to 6 months.

  Polynucleotide construct, muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, joint, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nerve It can be delivered to the interstitial space of tissues in animals, including systems, eyes, glands, and connective tissues. In the interstitial space of tissue, intercellular, fluid, muropolysaccharide matrix between reticulated fibers of organ tissue, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrous tissue, or sheathed connective tissue of muscle cells Include the same matrix within or within bone loss. Similarly, the space occupied by circulating plasma and lymph channel lymph. Delivery to the interstitial space of muscle tissue is preferred for reasons discussed below. It can be conveniently delivered by injection into a tissue containing these cells. For example, delivery and expression can be achieved in non-differentiated or not fully differentiated cells, such as blood stem cells or dermal fibroblasts, but are preferably delivered to differentiated persistent non-dividing cells, Expressed. In vivo cells are competent in particular in their ability to take up and express polynucleotides.

  For naked nucleic acid sequence injections, an effective amount of DNA or RNA is in the range of about 0.05 mg / kg body weight to about 50 mg / kg body weight. Preferably, per dose is from about 0.005 mg / kg to about 20 mg / kg, more preferably from about 0.05 mg / kg to about 5 mg / kg. Of course, those skilled in the art will appreciate that this dose may vary according to the tissue site of the injection. Appropriate and effective doses of nucleic acid sequences are readily determined by those skilled in the art and may depend on the condition being treated and the route of administration.

  The preferred route of administration is by the parenteral route of injection into the interstitial space of the tissue. However, other parenteral routes may also be used, such as inhalation of aerosol formulations to lung or bronchial tissue, throat or nasal mucosa, among others. Furthermore, naked DNA constructs can be delivered to the artery during angioplasty by the catheter used in the procedure.

  Naked polynucleotides can be obtained by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter injection, and what are termed “gene guns”. Deliver. These delivery methods are known in the art.

  The construct may also be delivered in delivery vehicles such as viral sequences, viral particles, lysosomal formulations, lipofectin, precipitants, and the like. Such delivery methods are known in the art.

  In certain embodiments, the polynucleotide construct is complexed in a liposome preparation. Liposome preparations for use in the present invention include cation (positive charge), anion (negative charge) and neutral preparations. However, cationic liposomes are particularly preferred because close charge complexes can be formed between cationic liposomes and polyanionic nucleic acids. Cationic liposomes are in functional form in plasmid DNA (see, eg, Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84: 7413-7416, incorporated herein by reference). ), MRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86: 6077-6081), and purified transcription factor (herein incorporated by reference). It has been shown to mediate intracellular delivery of Debs et al., J. Biol. Chem. (1990) 265: 10189-10192), incorporated into the literature.

  Cationic liposomes are readily available. For example, N (1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTMA) liposomes are particularly useful, under the trademark Lipofectin, Gibco BRL (GIBCO BRL), Grand Island, N. Y (also see Felgner et al., Proc. Natl Acad. Sci. USA (1987) 84: 7413-7416, which is incorporated herein by reference). Other commercially available liposomes include transfections (DDAB / DOPE) and DOTAP / DOPE (Boehringer).

  Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. For example, with respect to the description of the synthesis of DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonio) propane) liposomes, PCT specification WO 90 (incorporated herein by reference). See / 11092. The preparation of DOTMA liposomes has been described in the literature, e.g. P.A., incorporated herein by reference. Felgner et al. , Proc. Natl. Acad. Sci. USA 84: 7413-7417. Similar methods can be used to prepare liposomes from other cationic lipid materials.

  Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), Or easily prepared using readily available materials. Is possible. Such materials include phosphatidyl, choline, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), and the like. These materials can also be mixed with DOTMA and DOTAP starting materials in appropriate ratios. Methods for producing liposomes using these substances are well known in the art.

  For example, commercially available dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) with the addition of cholesterol, or to make conventional liposomes, or None, and can be used in various combinations. Thus, for example, DOPG / DOPC excipients can be prepared by drying each 50 mg of DOPG and DOPC into a sonication vial under a stream of nitrogen gas. The sample is placed under a suction pump overnight and hydrated with undressing warm water the next day. The sample is then sonicated in a capped vial at maximum setting using a Heat Systems model 350 sonicator equipped with a reverse cap (bath type) probe for 2 hours while circulating the bath at 15 ° C. Alternatively, negatively charged vesicles can be prepared by extruding nucleopore membranes to produce multilamellar vesicles, without sonication, or to produce multi-layered vesicles of separated size. Other methods are known and available to those skilled in the art.

Liposomes include multilamellar vesicles (MLVs), small multilamellar vesicles (SUVs), or large multilamellar vesicles (LUVs), with SUVs being preferred. Various liposome-nucleic acid complexes are prepared using methods well known in the art. See, for example, Straubinger et al., Which is incorporated herein by reference. , Methods of Immunology (1983), 101: 512-527. For example, MLVs containing nucleic acids can be prepared by depositing a thin film of phospholipids on the wall of a glass tube, followed by hydration of a solution of the material to be encapsulated, and SUVs are Prepared by extended sonication of MLVs to produce a homogeneous membrane. The substance to be encapsulated is added to the resulting suspension of MLVs and then sonicated. When using liposomes containing cationic lipids, dry lipid films are resuspended in a suitable solution such as sterile water or an isotonic buffer solution such as 10 mM Tris / NaCl, sonicated and then made. The prepared liposome is directly mixed with DNA. Liposomes and DNA form very stable complexes due to the binding of positively charged liposomes to cationic DNA. SUVs are utilized with small nucleic acid fragments. LUVs are prepared by a number of methods well known in the art. Commonly used methods include Ca ²⁺ -EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394: 483; Wilson et al., Cell, incorporated herein by reference. 17:77 (1979)), ether injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta 443: 629 (1976); Ostro et al., Biochem. Biophys. Res. Commun. 76:36. Fraley et al., Proc. Natl. Acad. Sci. USA 76: 3348 (1979)), detergent dialysis (Enoch, H. and St.). Rittmatter, P., Proc. Natl. Acad. Sci., USA 76: 145 (1979)) and reverse phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255: 10431 (1980); F. and Papahadjopoulos, D., Proc.Natl.Acad.Sci.USA 75: 145 (1978); Schaefer-Ridder et al., Science 215: 166 (1982)).

  In general, the ratio of DNA to liposome is from about 10: 1 to about 1:10. Preferably, the ratio is from about 5: 1 to about 1: 5. More preferably, the ratio is from about 3: 1 to about 1: 3. Even more preferably, the ratio is about 1: 1.

  US Pat. No. 5,676,954 (incorporated herein by reference) reports on the injection of genetic material complexed with a cationic liposome carrier into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5, (incorporated herein by reference) 589,466, 5,693,622, 5,580,859, 5,703,055 and International Patent Specification WO 94/9469 are used to transfect DNA into cells and animals. Cationic lipids are provided for use in. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and International Patent Specification WO 94/9469 are directed to mammals. A method for the delivery of a DNA-cationic lipid complex is provided.

  In certain embodiments, the cells are modified ex vivo or in vivo with retroviral particles comprising RNA comprising a sequence encoding an albumin fusion protein of the invention. The retrovirus from which the retroviral plasmid vector is derived includes, but is not limited to, sarcoma leukemia virus (Moloney Murine Leukemia Virus), spleen necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, Harvey Sarcoma virus, Includes leukemia virus, gibbon leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus and breast cancer virus.

Retroviral plasmid vectors are used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that may be transfected include, but are not limited to, PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP + E. -86, GP + envAm12, and DNA cell lines as described in Miller, Human Gene Therapy 1: 5-14 (1990), all of which are incorporated herein by reference. The vector may be transduced into the packaging cells via any method known in the art. Such methods include, but are not limited to, electroporation, the use of liposomes, and CaPO ₄ precipitation. In other methods, retroviral plus vectors are encapsulated in liposomes or conjugated to lipids and then administered to the host.

  The producer cell line produces infectious retroviral vector particles that contain a polynucleotide encoding an albumin fusion protein of the invention. Such retroviral vector particles may then be used to transduce prokaryotic cells either in vitro or in vivo. Transduced eukaryotic cells express the fusion protein of the invention.

  In certain other embodiments, the cells are modified ex vivo or in vivo with a polynucleotide contained in an adenoviral vector. The adenovirus can be engineered to inactivate with respect to its ability to replicate and replicate in the normal lytic virus life cycle to encode and express the fusion protein of the invention. Adenovirus expression is achieved without integration of viral DNA into the host cell chromosome, thereby reducing concerns about insertional mutagenesis. In addition, adenovirus has been used as a multi-year live intestinal vaccine with a great safety profile (Schwartz et al. Am. Rev. Respir. Dis. 109: 233-238 (1974)). Finally, adenovirus-mediated gene delivery is the delivery of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, MA et al. (1991) Science 252: 431-434; Rosenfeld et al. (1992) Cell 68: 143-155). Furthermore, extensive research to attempt to establish adenovirus as a causative agent in human cancer is uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci.USA 76: 6606).

  Suitable adenoviral vectors useful in the present invention are described, for example, by Kozarsky and Wilson, Curr. Opin. Genet. Dev. 3: 499-503 (1993); Rosenfeld et al. Cell 68: 143-155 (1992); Engelhardt et al. , Human Genet. Ther. 4: 759-769 (1993); Yang et al. , Nature Genet. 7: 362-369 (1994); Wilson et al. , Nature 365: 691-692 (1993); and US Pat. No. 5,652,224. For example, the adenoviral vector Ad2 is useful and can be propagated in human 293 cells. These cells contain the E1 region of the adenovirus, structurally express Ela and Elb, and complement the defective adenovirus by providing the product of the gene that is missing from the vector. In addition to Ad2, various other adenoviruses (eg, Ad3, Ad5 and Ad7) are also useful in the present invention.

  Preferably, the adenovirus used in the present invention is replication defective. Replication-deficient adenoviruses require the help of helper virus and / or packaging cells to form infectious particles. The resulting virus can infect cells and can express the polynucleotide of interest that is operably linked to a promoter, but is not replicable in most cells. Non-replicatable adenovirus can be detected in one or more of the following genes E1a, E1b, E3, E4, E2a, or all or part of L1 to L5.

  In certain other embodiments, the cells are modified ex vivo or in vivo using an adeno-associated virus (AAV). AAV is a naturally occurring defective virus that requires a helper virus to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158: 97 (1992)). It is also one of several viruses that can integrate its DNA into non-dividing cells. Vectors containing as much as 300 base pairs of AAV can be packaged and integrated, but the space for foreign DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. For example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745 and See 5,589,377.

  For example, suitable AAV vectors for use in the present invention include all sequences necessary for DNA replication, capsid inclusion, and host-cell integration. Polynucleotide constructs are described in Sambrook et al. , Molecular Cloning: Inserted into an AAV vector using standard cloning methods, such as found in A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells infected with helper virus using any standard technique, such as lipofection, electrophoresis, calcium phosphate precipitation. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus, or herpes virus. Once the packaging cells have been transfected and infected, infectious AAV virions containing the polynucleotide construct are produced. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cell contains the polynucleotide construct integrated within its genome and expresses the fusion protein of the invention.

  Other methods of gene therapy include a heterologous regulatory region operatively linked via homologous recombination and an endogenous polynucleotide sequence (eg, encoding a polypeptide of the invention) (eg, U.S. Pat. No. 5,641,670, issued Jun. 24, 1997, International Patent Specification No. WO 96, issued September 26, 1996, incorporated herein by reference. No./29411, International Patent Specification No. WO 94/12650, issued Aug. 4, 1994, Koller et al., Proc. Natl. Acad. Sci. et al., Nature 342: 435-438 (1989)). The method includes activation of a gene that is present in the target cell but is not normally expressed in the cell, or is expressed at a lower level than desired.

  A polynucleotide construct is made using a standard technique known in the art, including a promoter having a target sequence adjacent to the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently homologous to the endogenous sequence to allow homologous recombination of the promoter-target sequence with the endogenous sequence. The target sequence is close enough to the 5 'end of the desired endogenous polynucleotide sequence so that the promoter is operably linked to the endogenous sequence in homologous recombination.

  The promoter and target sequence can be amplified using PCR. Preferably, the amplified promoter contains different restriction enzyme sites on the 5 'and 3' ends. Preferably, the 3 ′ end of the first target sequence comprises the same restriction enzyme site as the 5 ′ end of the amplified promoter, alternatively the 3 ′ end of the target sequence is identical to the 3 ′ end of the amplified promoter Of restriction sites. Amplified promoter and target sequences are designed and ligated together.

  Transfection-promoting agents such as liposomes, viral sequences, viral particles, whole viruses, lipofections, precipitation drugs, etc., where the promoter-target sequence structure is described as a raw polynucleotide or as described in more detail above. Delivered to the cells either in combination. The P promoter-target sequence can be delivered by any method including direct needle injection, intravenous injection, local administration, catheter injection, particle accelerator, and the like. The method is described in more detail below.

  The promoter target sequence structure is taken up by the cell. Homologous recombination occurs between the structure and the endogenous sequence, and the endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.

  A polynucleotide encoding an albumin fusion protein of the invention may include a secretory signal sequence that facilitates secretion of the protein. Typically, a signal sequence is located in the coding region of the polynucleotide to be expressed toward or at the 5 'end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest, and may be homologous or heterologous to the transfected cell. Furthermore, the signal sequence may be synthesized using methods known in the art.

  Any mode of administration of any of the above polynucleotide constructs can be used as long as that mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter injection, biolistic injectors, particle accelerators (ie "gene guns"), gelfoam sponge depots, other commercially available depot molecules, isotonic pumps (eg Alza minipumps) ), Oral or suppository solids (tablets or pills), pharmacological formulations, and decanting or topical application during surgery. For example, direct injection of a naked calcium phosphate precipitation plasmid into the rat liver and rat spleen or into the portal vein of the protein-encoding plasmid resulted in gene expression of foreign genes in the rat liver (Kaneda et al., Science 243: 375 (1989)).

  The preferred method of topical administration is by direct injection. Preferably, the albumin fusion protein of the invention complexed with a delivery vehicle is administered directly into the region of the artery or within a local area. Administration of the composition within a local area of the venous region means injection of the composition in intravenous centimeters, preferably millimeters.

  Another method of topical administration is to include the polynucleotide construct of the present invention within or around a surgical wound. For example, a patient can undergo surgery and a polynucleotide construct can be coated on the surface of the tissue inside the wound, or the construct can be injected into a region of tissue within the wound.

  A therapeutic composition useful for systemic administration includes a fusion protein of the invention complexed to a targeted delivery vehicle of the invention. Suitable delivery excipients for use with systemic administration include liposomes containing a ligand for targeting the excipient to a specific site. In certain embodiments, suitable delivery vehicles for use in systemic administration include liposomes comprising an albumin fusion protein of the invention for targeting the vehicle to a specific site.

  Preferred methods of systemic administration include intravenous administration, aerosol, oral and transdermal (topical) delivery. Intravenous injection can be performed using standard methods in the art. Aerosol delivery can also be performed using standard methods in the art (eg, Stribling et al., Proc. Natl. Acad. Sci. USA 189, which is incorporated herein by reference). : 11277-11281, 1992). Oral delivery can be performed by conjugating the polynucleotide construct of the invention to a carrier that is resistant to degradation by digestive enzymes in the stomach of the animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing the polynucleotide construct of the present invention with a fat-soluble reagent (eg, DMSO) that can pass into the skin.

  Determining the effective amount of substrate to be delivered includes, for example, the chemical structure and biological activity of the substrate, the age and weight of the animal, the actual condition requiring treatment and its severity, and the route of administration. It can depend on a number of factors including. The frequency of treatment depends on a number of factors, such as the amount of polynucleotide construct administered per time and the health and history of the subject. The actual amount, number of doses, and timing of administration can be determined by the attending physician or veterinarian.

The albumin fusion protein of the present invention can be administered to any animal, preferably mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cows, horses and pigs, with humans being particularly preferred.

Biological activity

  Albumin fusion proteins and / or polynucleotides encoding albumin fusion proteins of the invention can be used in assays to test for one or more biological activities. If an albumin fusion protein and / or polynucleotide shows activity in a particular assay, it is possible that a therapeutic protein corresponding to the fusion protein may be involved in diseases associated with biological activity. Thus, fusion proteins can be used to treat related diseases.

  In a preferred embodiment, the present invention provides a “therapeutic protein X” column of Table 1 in an amount effective to treat, prevent or alleviate a disease or condition in a patient where such treatment, prevention or alleviation is desired. The invention comprising a therapeutic protein portion corresponding to the disclosed therapeutic protein in (in the same column as the disease or condition to be treated, listed in the “preferred indication Y” column of Table 1) A method of treating a disease or condition listed in “Preferred indication Y” of Table 1, comprising administering an albumin fusion protein of

  In a further preferred embodiment, the invention relates to the indications in the examples in an amount effective to treat, prevent or alleviate the disease or condition in a patient where such treatment, prevention or alleviation is desired. A disease listed for a particular therapeutic protein in Table 1 “Preferred indication Y” comprising administering an albumin fusion protein of the invention comprising a therapeutic protein portion corresponding to the therapeutic protein, or Including a method of treating a disease.

  Albumin fusion proteins produced by cells when specifically encoded by a polynucleotide encoding SEQ ID NO: Y are specifically contemplated by the present invention. When these polypeptides are used to express an encoded protein from a cell, the signal sequence where the natural secretion and processing steps of the cell are clearly listed in columns 4 and / or 11 of Table 2. Produces proteins lacking The specific amino acid sequences of the listed signal sequences are set forth herein or are well known in the art. Thus, the most preferred embodiment of the present invention includes an albumin fusion protein produced by a cell (lacking the leader sequence shown in columns 4 and / or 11 of Table 2). Also most preferred is a polypeptide comprising SEQ ID NO: Y without the specific leader sequence listed in columns 4 and / or 11 of Table 2. Also preferred are compositions comprising these two preferred embodiments, including pharmacological compositions. These albumin fusion proteins are specifically contemplated for treating, preventing, or alleviating the diseases or conditions listed for the particular therapeutic protein in the “Preferred indication Y” column of Table 1.

  In a preferred embodiment, the fusion protein of the present invention comprises an endocrine system (see, for example, the “endocrine disease” item below), nervous system (eg, see “neurological disease” item below), immune system (for example, See, for example, “Immune System Disease” below, respiratory system (eg, see “Respiratory Disease” below), cardiovascular system (eg, see “Cardiovascular Disease” below) ), Reproductive system (eg, see “Reproductive System Disease” below), digestive system (eg, see “Gastrointestinal Disease” below), diseases related to cell proliferation and / or Or diseases (eg, see “hyperproliferative diseases” below) and / or blood related diseases and / or diseases (eg, see “blood related diseases” below). ) In the diagnosis of diseases associated with and / or disease, prognosis, may be used in the prevention and / or treatment.

  In certain embodiments, the albumin fusion protein of the present invention is used to diagnose and / or diagnose diseases and / or diseases associated with the tissue (s) in which the gene corresponding to the therapeutic protein portion of the albumin fusion protein of the present invention is expressed. Or it may be used for prognosis.

  Accordingly, the fusion protein of the present invention and the polynucleotide encoding the albumin fusion protein of the present invention include, but are not limited to, prohormone activation, neurotransmitter activity, cell signaling, cell proliferation, cell differentiation and cell migration. It is useful in the diagnosis, detection and / or treatment of diseases and / or diseases associated with activities including

More generally, the fusion proteins of the invention and polynucleotides encoding the albumin fusion proteins of the invention are useful in the diagnosis, detection and / or treatment of diseases and / or diseases associated with the following systems: is there.

Immune activity

  The albumin fusion protein of the present invention and the polynucleotide encoding the albumin fusion protein of the present invention can be used, for example, by activating or inhibiting immune cell proliferation, differentiation or mobilization (chemotaxis) to It may be useful in treating, preventing, diagnosing and / or prognosing a disease, illness and / or condition of the system. Immune cells develop through a process called hematopoiesis and produce bone marrow (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T cells) cells from pluripotent stem cells. The etiology of these immune diseases, diseases and / or conditions may be somatic, acquired (eg, by chemotherapy or toxins), or infectious, such as genetic, cancer and autoimmune diseases. Furthermore, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can be used as markers or detectors for specific immune system diseases or conditions.

  In other embodiments, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to treat diseases and disorders of the immune system and / or the polypeptide of the invention is expressed. May be used to inhibit or enhance the immune response generated by cells associated with the selected tissue (s).

  Treating, preventing, diagnosing immunodeficiency, including both congenital and acquired immunodeficiencies, the fusion protein of the invention and / or a polynucleotide encoding the albumin fusion protein of the invention; And / or may be useful in prognosing. Examples of B cell immunodeficiency with reduced immunoglobulin level B cell function and / or number of B cells include X-linked agammaglobulinemia (Bruton's disease), X-linked pediatric agammaglobulinemia, hyper IgM X-linked immunodeficiency, non-X-linked immunodeficiency with Hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia, including congenital and acquired agammaglobulinemia, adult-onset agammaglobulinemia , Late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), selective IgM deficiency, selective IgA Deficiency, selective residence IgG subclass deficiency, IgG subclass deficiency (with or without IgA), increased IgM Ig deficiency, IgG and IgA deficiency with increased IgM, antibody deficiency with normal or elevated Ig, Ig heavy chain deficiency, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), non-classifiable immune deficiency (CVID), Includes non-classifiable immunodeficiency (CVI) (acquired), and transient hypogammaglobulinemia in children.

  In a specific embodiment, vasodilatory dysfunction or a condition associated with vasodilatory schizophrenia is treated and prevented using the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention. Diagnose and / or prognose.

  Examples of congenital immunodeficiencies with reduced T cell and / or B cell function and / or number include, but are not limited to, DiGeorge Abnormal, Severe Combined Immunodeficiency (SCID) (but not limited to X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, class II MHC deficiency (deficient lymphocyte syndrome), Wiscott-Aldrich syndrome and telangiectasia ataxia), Thymic dysplasia, third and fourth pharyngeal sac syndrome, 22q11.2 deficiency, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4 + T-lymphocytopenia, dominant T cell deficiency (details) And immunodeficiencies with unknown details and cell-mediated immunity details.

  In certain embodiments, a DiGeorge abnormality or a condition associated with a DiGeorge abnormality is treated, prevented, diagnosed and / or treated with a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. Or make a prognosis.

  Other immunodeficiencies that may be treated, prevented, diagnosed and / or prognosticated using the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to: , Chronic granulomatous disease, Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, (C1, C2, C3 Complement component deficiency (including C4, C5, C6, C7, C8 and / or C9 deficiency), reticulodysplasia, thymic lymphogenesis, immunodeficiency with thymoma, severe congenital leukopenia , Dysplasia with immunodeficiency, neonatal neutropenia, shortened limb short stature, and mixed-deficiency of Nezerof syndrome with Ig

  In a preferred embodiment, the immunodeficiency and / or conditions associated with immunodeficiency listed above are treated with the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention, Prevent, diagnose and / or prognose.

  In a preferred embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used as an agent to promote immune responsiveness among individuals with immune deficiency. . In certain embodiments, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to promote immune responsiveness among individual B-cell and / or T-cell immunodeficiencies. It can be used as a drug.

  Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are useful in treating, preventing, diagnosing and / or prognosing autoimmune diseases. possible. Many autoimmune diseases are the result of inappropriate recognition of self as a foreign substance by immune cells. This inappropriate recognition results in an immune response that leads to host tissue destruction. Thus, administration of a fusion protein of the present invention and / or a polynucleotide encoding an albumin fusion protein of the present invention capable of inhibiting an immune response, particularly T cell proliferation, differentiation or chemotaxis, results in an autoimmune disease. Can be an effective treatment in preventing.

  An autoimmune disease or disorder that can be treated, prevented, diagnosed and / or prognosticated by a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention includes, but is not limited to, one or More than that, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune platelets Reduced purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, purpura (eg, Henrotch-Scoenlein purpura), autoimmune cytopenias, goodpasture syndrome, pemphigus vulgaris, myasthenia gravis Grave's disease (hyperthyroidism) and insulin resistant diabetes are included.

  Further diseases that may have autoimmune components that can be treated, prevented, and / or diagnosed by albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are not limited. But type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatoid heart disease, neuritis, uveitis ophthalmitis, multigland endocrine disorder , Reiter syndrome, stiff man syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre syndrome, insulin-dependent diabetes, and autoimmune inflammatory eye diseases.

  Additional diseases that may have autoimmune components that can be treated, prevented, and / or diagnosed by albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are not limited. Scleroderma with anti-collagen antibodies (often characterized by eg nuclear and other nuclear antibodies), mixed connective tissue (often characterized by eg extractable nuclear antigens (eg ribonucleoprotein)) Disease, polymyositis (often characterized by, for example, non-histone ANA), pernicious anemia (often characterized by antimural cells, microsomes, and endogenous factor antibodies), (often, for example, humoral and Idiopathic hydrangea characterized by cell-mediated adrenal cytotoxicity Disease, infertility (often characterized, for example, by anti-sperm antibodies), glomerulitis (often characterized by glomerular basement membrane antibodies or immune complexes), (often, for example, IgG and complement in the basement membrane) Bullous pemphigoid (characterized by the body), Sjogren's syndrome (often characterized by, for example, multiple tissue antibodies, and / or specific non-histone ANA (SS-B)), (often cell-mediated, for example) Diabetes characterized by humoral and islet cell antibodies, and adrenergic (often characterized by beta-adrenergic receptor antibodies) (including adrenergic drug resistance in asthma or cystic fibrosis) Drug resistance is included.

  Further diseases that may have autoimmune components that can be treated, prevented, and / or diagnosed by albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are not limited. Chronic active hepatitis (often characterized by, for example, smooth muscle antibodies), primary biliary cirrhosis (often characterized by, for example, mitochondrial antibodies), (often, for example, in some cases, certain Other endocrine deficiencies characterized by tissue antibodies, vitiligo (often characterized by eg melanocyte antibodies), often by eg Ig and complement in the vessel wall, and / or by low serum complement Vasculitis, often characterized by myocardial antibodies) After myocardial infarction, cardiotomy syndrome (often characterized by myocardial antibodies), urticaria (often characterized by IgG and IgM antibodies to IgE), and often characterized by IgG and IgM antibodies to IgE ) Atopic dermatitis, asthma (often characterized by IgG and IgM antibodies to IgE), and many other inflammation, granulomas, degeneration, and atrophic diseases.

  In a preferred embodiment, an autoimmune disease and disease, and / or a condition associated with the disease and disease listed above, eg, a polynucleotide encoding a fusion protein of the invention and / or an albumin fusion protein of the invention To treat, prevent, diagnose and / or prognose. In certain preferred embodiments, rheumatoid arthritis is treated, prevented, and / or diagnosed using a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention.

  In other specifically preferred embodiments, systemic lupus erythematosus is treated, prevented, and / or diagnosed with a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. In other specifically preferred embodiments, idiopathic thrombocytopenic purpura is treated, prevented, and / or diagnosed using a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. To do.

  In other specifically preferred embodiments, IgA nephropathy is treated, prevented, and / or diagnosed using an albumin fusion protein of the invention and / or a polynucleotide encoding a fusion protein of the invention.

  In a preferred embodiment, autoimmune diseases and diseases and / or conditions associated with the diseases and diseases listed above are used with the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention. Treatment, prevention, diagnosis and / or prognosis.

  In a preferred embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as immunosuppressive agent (s).

  An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention treats, prevents, prognoses, and / or diagnoses a disease, condition and / or condition of hematopoietic cells, and / or Or it may be useful in diagnosing. Treat or prevent diseases, illnesses and / or conditions associated with a decrease in certain (or many) types of hematopoietic cells, including but not limited to leukopenia, neutropenia, anemia and thrombocytopenia In order to increase the differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used. . Alternatively, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be in a specific (or many) type of hematopoietic cell, including but not limited to histiocytosis. It can be used to increase the differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, for the purpose of treating or preventing diseases, illnesses and / or conditions associated with the increase.

  Treating allergic reactions and conditions, such as asthma (especially allergic asthma) or other respiratory problems, with a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention Prevention, diagnosis and / or prognosis. Furthermore, these molecules can be used to treat, prevent, prognose and / or diagnose anaphylaxis, hypersensitivity to allergic molecules, or blood group incompatibility.

  Furthermore, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used for treating, preventing, diagnosing and / or prognosing IgE-mediated allergic reactions. . Such allergic reactions include, but are not limited to, asthma, rhinitis and atopic dermatitis. In certain embodiments, a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be used to modulate IgE concentration in vitro or in vivo.

  Furthermore, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used in the diagnosis, prognosis, prevention and / or treatment of inflammatory conditions. For example, since the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can inhibit activation, proliferation and / or differentiation of cells involved in inflammatory responses, It can be used to prevent and / or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (eg, septic shock, septicemia, or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hypertension Acute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, overproduction of cytokines (eg TNF or IL-1), respiratory diseases (eg asthma and allergies), gastrointestinal diseases (eg inflammatory) Bowel disease), cancer (eg stomach, ovary, lung, bladder, liver and milk), CNS disease (eg multiple sclerosis, ischemic brain injury and / or stroke, traumatic brain injury, neurodegenerative disease (eg Parkinson's disease and Alzheimer's disease), AIDS-related dementia, and prion disease), cardiovascular diseases (eg, athero Many arteriosclerosis, myocarditis, cardiovascular and cardiopulmonary bypass complications) and many additional diseases, conditions and diseases characterized by inflammation (eg, hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis, Sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus, diabetes, and allograft rejection).

  Because inflammation is a fundamental defense mechanism, inflammatory diseases can affect virtually any body tissue. Accordingly, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include, but are not limited to, adrenalitis, alveolitis, cholangitis, cecalitis, balanitis, blepharitis, bronchiolitis Inflammation, bursitis, carditis, cellulitis, cervicitis, cholecystitis, vocal fold inflammation, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, ear Ductitis, connective tissue inflammation, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, otitis media, laryngitis, lymphangitis, mastitis, otitis media, meningitis, myometrium Inflammation, mucositis, myocarditis, myositis, tympanitis, nephritis, neuritis, testitis, osteochondritis, otitis, pericarditis, tendonitis, peritonitis, pharyngitis, phlebitis, acute leukomyelitis, prostatitis, Pulpitis, retinitis, rhinitis, fallopianitis, scleritis, scleral choroiditis Used in the treatment of tissue-specific inflammatory diseases including scrotitis, sinusitis, spondylitis, steatitis, gastritis, synovitis, otitis, tendonitis, congenitis, urethritis and vaginitis The

  In a particular embodiment, the fusion protein of the invention and / or a polynucleotide encoding the albumin fusion protein of the invention diagnoses, prognoses, organ transplant rejection and graft-versus-host disease (GVHD), Useful for prevention and / or treatment. Organ rejection occurs by the host immune system disruption of the transplanted tissue through an immune response. Similarly, the immune response is also involved in GVHD, but in this case foreign transplanted immune cells destroy host tissue. Polypeptides, antibodies or polynucleotides of the invention, and / or agonists or antagonists thereof that inhibit immune responses, especially T-cell activation, proliferation, differentiation or chemotaxis, in preventing organ rejection or GVHD It can be an effective treatment. In certain embodiments, a polynucleotide encoding a fusion protein of the invention and / or an albumin fusion protein of the invention that inhibits an immune response, particularly T-cell activation, proliferation, differentiation or chemotaxis, It can be an effective treatment in preventing experimental allergies and hyperacute xenograft rejection.

  In other embodiments, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention includes, but is not limited to, serum disease, post-streptococcal glomerulonephritis, nodular multiple arteries. It is useful for diagnosing, prognosing, preventing and / or treating immune complex diseases, including inflammation and immune complex induced vasculitis.

  The fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used to treat, detect and / or prevent infectious agents. For example, an infectious disease is treated, detected and / or prevented by increasing the immune response, by increasing proliferation activation and / or differentiation of specific B and / or T cells. The immune response can be increased either by enhancing the existing immune response or by initiating a new immune response. Alternatively, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can also directly inhibit the infectious agent without the necessary trigger of an immune response (for applications that list infectious agents). See item etc.).

  In other embodiments, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used as vaccine adjuvants that enhance immune responsiveness to antigens. In certain embodiments, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used as adjuvants to enhance tumor-specific immune responses.

  In other specific embodiments, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used as adjuvants to enhance the antiviral immune response. Antiviral immune responses that can be enhanced using the compositions of the present invention as adjuvants include viruses and virus-related diseases or conditions described herein or known in the art. In certain embodiments, the composition of the invention is used to enhance an immune response against a virus, disease or condition selected from the group consisting of AIDS, meningitis, dengue fever, EBV and hepatitis (eg, hepatitis B). And used as an adjuvant. In another specific embodiment, the composition of the present invention is treated with HIV / AIDS, respiratory syncytial virus, dengue fever, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, Used as an adjuvant to enhance the immune response to a virus, disease or condition selected from the group consisting of rabies, Junin, Chikungunya fever, Rift Valley fever, herpes simplex and yellow fever.

  In other specific embodiments, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used as adjuvants to enhance antibacterial or antifungal immune responses. Antibacterial or antifungal immune responses that can be enhanced using the compositions of the invention as adjuvants include bacteria or fungi and bacterial or fungal related diseases or conditions described herein or known in the art. Is included. In certain embodiments, the composition of the present invention is used as an adjuvant to enhance the immune response against bacteria or fungi, diseases or conditions selected from the group consisting of tetanus, diphtheria, botulinum and type B meningitis. use.

  In another specific embodiment, the composition of the present invention is applied to Vibro cholera, Mycobacterium leprae, Salmonella typhi, Salmonella palaefi, Salmonella palaefi ), A symptom or disease-free disease selected from the group consisting of Streptococcus pneumoniae, Group B Streptococcus, Shigella, Enterotoxin-producing Escherichia coli, Enterohemorrhagic Escherichia coli and Borrelia burgdorferi Used as an adjuvant to enhance the epidemic response.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an adjuvant to enhance the anti-parasitic immune response. Anti-parasitic immune responses that can be enhanced using the compositions of the present invention as adjuvants include parasites and parasite-related diseases or conditions described herein or known in the art. In certain embodiments, the compositions of the invention are used as adjuvants to enhance the immune response against parasites. In another particular embodiment, the composition of the invention is used as an adjuvant to enhance the immune response against variants (malaria) or Leishmania.

  In other specific embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may also be produced, for example, by preventing mobilization and activation of mononuclear phagocytosis. May be used to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis.

  In another specific embodiment, an antibody for inhibiting or enhancing an immune-mediated response to an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention Used as an antigen for the production of

  In one embodiment, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is added to one or more antibodies (eg, IgG, IgA, IgM and In order to increase the amount of IgE), to induce higher affinity antibody production and immunoglobulin class switches (eg IgG, IgA, IgM and IgE) and / or to increase the immune response (eg Mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, chickens, camels, goats, horses, cows, sheep, dogs, cats, non-human primates, and humans, most preferably humans.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a B cell responsive stimulator against a pathogen.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an activator of T cells.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to increase the individual immune status prior to receiving immunosuppressive therapy. Use as

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an agent to induce higher affinity antibodies.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an agent to increase serum immunoglobulin concentration.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an agent to even accelerate solid recovery of immunodeficiency.

  In another particular embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as an agent to promote immune responsiveness in an elderly population and / or neonate To do.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is transferred to a bone marrow transplant and / or other transplantation (eg, allogeneic or xenogeneic organ transplantation). Used as an immune system enhancer before, during, or after. With respect to transplantation, the compositions of the invention may be administered before, simultaneously with and / or after transplantation. In certain embodiments, the compositions of the invention are administered after transplantation and before the start of recovery of the T-cell population. In another specific embodiment, the composition of the invention is first administered after transplantation, after initiation of T cell population recovery, but prior to full recovery of the B cell population.

  In another specific embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to promote immune responsiveness in a solid having an acquired defect of B cell function. Used as a drug to do. The condition resulting in an acquired deficiency in B cell function that can be reduced or treated by administering an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is not limited. HIV infection, AIDS, bone marrow transplantation and B cell chronic lymphocytic leukemia (CLL).

  In another specific embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is used as an agent to promote solid immune responsiveness in transient immune deficiency. use. The conditions resulting in transient immune deficiency that can be reduced or treated by administering an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include, but are not limited to, viral infection Includes recovery from (e.g. influenza), conditions related to malnutrition, recovery from infectious mononucleosis, or conditions related to stress, recovery from measles, recovery from blood transfusion, and recovery from surgery It is.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a regulator of antigen presentation by monocytes, dendritic cells and / or B cells. use. In one embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention enhances antigen presentation or abolishes the action of antigen presentation in vitro or in vivo. To. Furthermore, in related embodiments, enhancement or abolition of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.

  In another specific embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is administered to a humoral response (ie TH2 ) Use as a drug to direct the individual immune system to development.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention induces tumor growth and is therefore more sensitive to antineoplastic agents. Use as a way for. For example, multiple myeloma is a slowly dividing disease and is therefore refractory to virtually all antineoplastic regimes. If these cells were allowed to grow more quickly, their sensitivity profile could change.

  In other specific embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used as AIDS, chronic lymphocyte disease and / or general variant immunodeficiency. Used as a stimulator of B cell production in pathology.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used for the production and / or regeneration of lymphoid tissue after surgery, trauma or genetic defect. Use as a treatment for. In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used in pretreatment of bone marrow samples prior to transplantation.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may result in immune-deficiency / immunodeficiency as observed in SCID patients. Used as a gene-based therapy for genetic diseases.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is protected against parasitic diseases effective against monocytes such as Leishmania. Therefore, it is used as a method to activate monocytes / macrophages.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a method of modulating secreted cytokines attracted by the polypeptide of the invention. .

  In other specific embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention may be used as pharmaceuticals in veterinary medicine and are described herein. Used in one or more applications.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used as a method of inhibiting various aspects of an immune response against a foreign agent or itself. To do. Examples of diseases or conditions where it is desirable to inhibit certain aspects of the immune response include autoimmune diseases such as lupus, and arthritis, and diseases / illnesses associated with skin allergies, inflammation, bowel disease, injury and pathogens. included.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is administered to a self such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis. It is used as a therapy to prevent B cell proliferation and Ig secretion related to immune diseases.

  In other specific embodiments, the polypeptides, antibodies, polynucleotides and / or agonists or antagonists of the present invention, and / or polynucleotides encoding albumin fusion proteins of the present invention are treated with B and / or in endothelial cells. Or used as an inhibitor of T cell migration. This activity disrupts histological or cognate responses and is useful, for example, in disrupting immune responses and inhibiting sepsis.

  In another specific embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is treated with undefined monoclonal hypergammaglobulinemia (MGUS), Valdens Used as a treatment for chronic hypergammaglobulinemia events in diseases such as Tlem's disease, associated idiopathic monoclonal hypergammaglobulinemia and plasmacytoma.

  In other specific embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used in macrophages and the like, for example in certain autoimmune and chronic inflammation and infectious diseases. Inhibits polypeptide chemotaxis and activation of neutrophils, basophils, B lymphocytes and some T-cell subsets such as activated and CD8 cytotoxic T cells and natural killer cells May be used to Examples of autoimmune diseases are described herein and include multiple sclerosis and insulin dependent diabetes.

  In another specific embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is converted to idiopathic eosinophils, for example by preventing eosinophil production and migration. It may be used to treat increased syndrome.

  In other specific embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used to enhance or inhibit complement-mediated cell lysis.

  In other specific embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used to enhance or inhibit antibody-dependent cytotoxicity.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is treated with, for example, atherosclerosis by preventing monocyte infiltration in the arterial wall, for example. May be used to treat the disease.

  In other specific embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention may be used to treat adult respiratory distress syndrome (ARDS).

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to stimulate wound and tissue recovery, to stimulate angiogenesis, and / or Or it may be useful to stimulate the recovery of vascular or lymphatic disease or illness. Furthermore, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used to stimulate regeneration of the mucosal surface.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is congenital or acquired immune deficiency, serum immunoglobulin production deficiency, recurrent infection, And / or to diagnose, prognose, treat and / or prevent immune system failure. Further, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may be used for infection of joints, bones, skin and / or parotid glands, blood-bone infections (eg sepsis, meningitis) Septic arthritis and / or osteomyelitis), autoimmune diseases (eg those disclosed herein), inflammatory diseases and malignant neoplasms, and / or without limitation, CVID, other congenital immunodeficiencies, These infections, including HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, shingles (eg severe shingles), and / or Pneumocystis carini, It may be used to treat or prevent any disease or condition or condition associated with a disease, disorder and / or malignancy. Other diseases and conditions that may be prevented, diagnosed, prognosticated, and / or treated with a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include, but are not limited to: HIV infection, HTLV-BLV infection, lymphopenia, phagocytic bacterial anemia, thrombocytopenia and hemoglobinuria are included.

  In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is treated with a general variant immunodeficiency (“CVID”, or “acquired agammaglobulin” Are also used to treat and / or diagnose a subset of the disease.

  In a particular embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is treated with a cancer or neoplasm, including immune cells or immune tissue-related cancers or neoplasms. It may be used for diagnosis, prognosis, prevention and / or treatment. Examples of cancers or neoplasms that may be prevented, diagnosed or treated with a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include, but are not limited to, acute myeloid leukemia, Chronic myelogenous leukemia, Hodgkins disease, non-Hodgkins lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, plasmacytoma, multiple myeloma, Burkitt lymphoma, EBV-transduced disease, and / or the present specification Diseases and illnesses described elsewhere under the name “Hyperproliferative disorders” are included.

  An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention is used as a therapy to reduce cell proliferation in large B-cell lymphomas.

  The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used as a method of reducing B cell and Ig involvement associated with chronic myeloid leukemia.

In certain embodiments, the compositions of the invention are used as agents to promote immune responsiveness in B cell immunodeficient individuals, such as individuals who have undergone partial or complete splenectomy.

Blood related diseases

  An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be used to modulate hemostasis (bleeding cessation) or thrombolytic (clotting) activity. For example, by increasing hemostasis or thrombolytic activity, a polynucleotide encoding the fusion protein of the invention and / or the albumin fusion protein of the invention can be converted into a blood clotting disease, disease and / or condition (eg, fibrinogen-free blood). Disease, factor failure, hemophilia), platelet disease, disease and / or condition (eg, thrombocytopenia), or wounds that are the result of trauma, surgery or other causes. Alternatively, a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention that can reduce hemostatic or thrombolytic activity can be used to inhibit or lyse clots. These molecules may be important in the treatment or prevention of heart attacks (infarction), strokes and scarring.

  In a specific embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is added to thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atheroma May be used to prevent, diagnose, prognose and / or treat atherosclerosis, myocardial infarction, transient ischemic stroke, unstable angina. In certain embodiments, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may be peripheries as may be associated with angioplasty to prevent the development of a saphenous graft. To reduce the risk of treatment thrombosis, to reduce the risk of stroke in patients with atrial fibrillation, including non-rheumatic atrial fibrillation, to prevent embolism associated with mechanical heart valve and / or mitral valve disease May be used to reduce risk. Other uses for albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include, but are not limited to, extracorporeal devices (eg, intravenous cannulas, vascular access in hemodialysis patients) Prevention of obstruction in shunts, hemodialysis machines, and cardiopulmonary bypass machines).

  In a particular embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is isolated from the blood and / or tissue associated with the tissue (s) in which the polypeptide of the invention is expressed. Alternatively, blood forming organ diseases and conditions may be used to prevent, diagnose, prognose and / or treat.

  The fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used to modulate hematopoietic activity (blood cell formation). For example, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be converted into, for example, erythrocytes, lymphocytes (B or T cells), bone marrow cells (such as basophils, eosinophils, It may be used to increase the amount of all or a subset of blood cells such as neutrophils, mast cells, macrophages) and platelets. The ability to reduce the amount of blood cells or a subset of blood cells may be useful in the prevention, detection, diagnosis and / or treatment of anemia and leukopenia as described below. Alternatively, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may be converted into, for example, erythrocytes, lymphocytes (B or T cells), bone marrow cells (for example, basophils, eosinophils, It may be used to reduce the amount of all or a subset of blood cells such as neutrophils, mast cells, macrophages) and platelets. The ability to reduce the amount of blood cells or a subset of blood cells may be useful in the prevention, detection, diagnosis and / or treatment of leukocytes, such as, for example, eosinophilia.

  A fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be used to prevent, treat or diagnose hematopoietic dysfunction.

  Anemia is a condition in which the number of red blood cells, or the amount of hemoglobin (a protein that carries oxygen) therein, is subnormal. Anemia can be caused by excessive bleeding, decreased red blood cell production, or increased red blood cell destruction (hemolysis). An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be useful in treating, preventing and / or diagnosing anemia. Anemia that may be treated, prevented or diagnosed by the albumin fusion protein of the invention and / or a polynucleotide encoding the albumin fusion protein of the invention includes iron deficiency anemia, hypochromic anemia, microcytic anemia, yellow Disease, hereditary ironblastic anemia, idiopathic acquired ironblastic anemia, erythroblastic fistula, giant erythroblastic anemia (eg, pernicious anemia, (vitamin B12 deficiency) and folate deficiency anemia), poor regeneration Anemia, hemolytic anemia (eg, autoimmune hemolytic anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is associated with diseases including but not limited to systemic lupus erythematosus, cancer, lymphoma, chronic kidney disease and spleen enlargement It may be useful in treating, preventing and / or diagnosing anemia. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention treats anemia caused by drug treatment such as anemia associated with methyldopa, dapsone and / or sulfa drugs, prevention May be useful in doing and / or diagnosing. Further, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may be, but is not limited to, hereditary spherocytosis, hereditary elliptical erythrocytosis, glucose-6-phosphate dehydrogenase deficiency And anemia associated with abnormal red blood cell structure, including sickle cell anemia, may be useful in treating, preventing and / or diagnosing.

  The polynucleotide encoding the albumin fusion protein of the present invention and / or the albumin fusion protein of the present invention is used for hemoglobin abnormalities (for example, sickle cell anemia, hemoglobin C disease, hemoglobin SC disease, and hemoglobin E disease). May be useful in treating, preventing, and / or diagnosing the related). Further, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention treats thalassemia, including but not limited to alpha-thalassemia and beta-thalassemia major and minor forms May be useful in the prevention, prevention and / or diagnosis.

  In other embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention includes, but is not limited to, thrombocytopenia (eg, idiopathic thrombocytopenic purpura, thrombotic platelets). Reduced purpura), von Willebrand disease, hereditary platelet diseases (eg storage pool diseases such as Chediak-Higashi and Hermansky Padrack syndrome, thromboxane A2 deficiency, thrombophilia, Bernard-Soulier syndrome) Hereditary hemorrhagic telangiectasia also known as hemolytic uremic syndrome, femophilia A or factor VII deficiency and femophilia such as Christmas disease or factor IX deficiency, Randu-Osler-Weber syndrome Useful in diagnosing, prognosing, preventing, and / or treating bleeding disorders including Telangectsia), allergic purpura (Hennoch-Schönlein purpura) and disseminated intravascular coagulation syndrome possible.

  The effect of the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention on the clotting time of blood is not limited, but is limited to whole blood partial thromboplastin time (PTT) Any coagulation test known in the art may be monitored, including time (aPTT), activated clotting time (ACT), remineralized activated clotting time, or Lee-White clotting time.

  Different diseases and different drugs can cause platelet failure. Thus, in certain embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is a platelet with renal failure, leukemia, multiple myeloma, cirrhosis and systemic lupus erythematosus Acquired platelets such as platelet disorders associated with dysfunction and drug treatment including treatment with aspirin, ticolopidine, non-steroidal anti-inflammatory drugs (used for arthritis, pain and sprains), and high doses of penicillin It may be useful in diagnosing, prognosing, preventing and / or treating a failure.

  In other embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is characterized by or associated with an increase or decrease in the number of white blood cells and It may be useful in diagnosing, prognosing, preventing, and / or treating a disease. Leukopenia occurs when the number of white blood cells decreases below normal. Leukopenia includes, but is not limited to, neutropenia and lymphopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body produces an increase in the number of white blood cells during infection. Thus, leukocytosis can simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis can be an indication of a wound or other disease such as cancer. Thrombocytosis includes, but is not limited to, eosinophilia and macrophage accumulation. In certain embodiments, an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention diagnoses, prognoses, prevents, and / or prevents leukopenia Can be useful in treating. In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention diagnoses, prognoses, prevents, and prevents leukocytosis, and / Or may be useful in treating.

  Leukopenia can be a reduction in all types of white blood cells throughout the body, or a specific depletion of specific types of white blood cells. Thus, in a particular embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention diagnoses a decrease in neutrophil count, known as neutropenia Can be useful in prognosing, prognosing, preventing, and / or treating. Neutropenia that can be diagnosed, prognosticated, prevented and / or treated with an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention includes, but is not limited to, pediatric genetics Agranulocytosis, familial leukopenia, cyclic leukopenia, dietary deficiency (eg vitamin B12 deficiency or folate deficiency) or related leukopenia, drug treatment (eg penicillin treatment, sulfonamide treatment, Anticoagulant treatment, anticonvulsant drugs, antithyroid drugs, and cancer chemotherapy) or associated leukopenia, and bacterial or viral infections, allergic diseases, autoimmune diseases, an individual with an enlarged spleen (Eg Ferti syndrome, malaria and sarcoidosis), and drug treatment cash register Include leukopenia consequence of increased associated with may neutrophils destruction occurs.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention includes, but is not limited to, stress, drug treatment (eg, drug treatment with corticosteroids, cancer chemotherapy, and / or Radiation therapy), AIDS infection and / or, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infection, viral infection and / or hereditary disease (eg, DiGeorge syndrome, Wiscott Aldrich syndrome, severe mixed immunodeficiency, capillary) Diagnosing lymphocytosis, including lymphopenia (decreased number of B and / or T lymphocytes) as a result of or associated with vasodilatory ataxia, prognosticating, Useful in preventing and / or treating There can.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention includes, but is not limited to, Gaucher disease, Niemann-Pick disease, Letterer Zyve disease and Hand-Schuller Christian disease May be useful in diagnosing, prognosing, preventing, and / or treating diseases and disorders associated with macrophage counts and / or macrophage function.

  In another embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention includes, but is not limited to, idiopathic eosinophilia syndrome, eosinophilia / myalgia In diagnosing, prognosing, preventing, and / or treating diseases and illnesses related to eosinophil count and / or eosinophil function, including Syndrome and Hand-Schuller Christian disease Can be useful.

  In another embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention includes, but is not limited to, acute lymphocytic (lymphoblastic) leukemia (ALL), Acute myeloid (myelocytic, myelogenous, myeloblastic or myelomonocytic) leukemia, chronic lymphocytic leukemia (eg, B cell leukemia, T cell leukemia, Sezary syndrome, and hairy cell leukemia) ), Diagnosing leukemia and lymphoma, including chronic myeloid (myeloid, myelogenous or granular) leukemia, Hodgkins lymphoma, non-Hodgkins lymphoma, Burkitt lymphoma and mushroom mycosis Diagnosing, preventing, It can be useful in pre / or treatment.

  In other embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention includes, but is not limited to, plasma cell disease, monoclonal hypergammaglobulinemia, meaningless Diagnosing plasma cell diseases and disorders including confirmed monoclonal hypergammaglobulinemia, multiple myeloma, macroglobulinemia, Waldenstrom macroglobulinemia, cryoglobulinemia and Raynaud's phenomenon, It can be useful in prognosing, preventing, and / or treating.

  In other embodiments, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention includes, but is not limited to, polycythemia vera, relative erythrocytosis, secondary polycytosis. Treatment of myeloproliferative disorders including thrombosis, myelofibrosis, acute myelofibrosis, primary myelofibrosis, thrombocythemia (including both primary and secondary thrombocythemia), and chronic myelogenous leukemia May be useful in doing, preventing, and / or diagnosing.

  In other embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention may be useful as a pre-surgical treatment to increase blood cell production.

  In another embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention comprises migration, phagocytosis, superoxide production, neutrophils, eosinophils and macrophages. It may be useful as an agent for enhancing antibody dependent cytotoxicity.

  In another embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is useful as an agent to increase the number of circulating stem cells prior to stem cell pheresis. It can be. In another specific embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is an agent for increasing the number of circulating stem cells prior to plateletpheresis. As useful.

  In another embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is useful as an agent for increasing cytokine production.

In other embodiments, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are useful in preventing, diagnosing and / or treating primary hematopoietic diseases. It can be.

Hyperproliferative disease

  In certain embodiments, the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention can be used to treat or detect hyperproliferative diseases, including neoplasms. An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may inhibit disease growth through direct or indirect interactions. Alternatively, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can grow other cells capable of inhibiting hyperproliferative diseases.

  For example, hyperproliferative diseases can be treated by increasing the immune response, especially by increasing the antigenic nature of the hyperproliferative disease, or by amplifying, differentiating or mobilizing T-cells. This immune response may be increased by enhancing an existing immune response or by initiating a new immune response. Alternatively, reducing an immune response, such as a chemotherapeutic agent, can also be a method of treating a hyperproliferative disease.

  Examples of hyperproliferative diseases that can be treated or detected by a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include, but are not limited to, large intestine, abdomen, bone, milk, digestion Organs, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovary, thymus, thyroid), eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, Includes neoplasms that are localized in the spleen, chest, and urogenital tract.

  Similarly, other hyperproliferative diseases can also be treated or detected by the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention. Examples of such hyperproliferative diseases include, but are not limited to, acute childhood lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia in addition to neoplasms localized in the listed organ systems Leukemia, acute myeloid leukemia, adrenal cortex cancer, adult (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkins disease, adult Hodgkins lymphoma Adult lymphocytic leukemia, adult non-Hodgkins lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant tumor, anal cancer, astrocytoma, cholangiocarcinoma, bladder cancer, Bone cancer, brain stem glioma, brain tumor, breast cancer, renal pelvis and ureter cancer, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, childhood ( Primary) Cell carcinoma, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, childhood extracranial germ cell tumor Childhood Hodgkin's disease, childhood Hodgkin lymphoma, childhood visual pathway and hypothalamic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood pineal gland and supratentorial primitive neuroectodermal tumor, Childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphoblastic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine pancreas Islet cell cancer, endometrial cancer, ependymoma, epithelial cancer, esophageal cancer, Ewing sarcoma and related tumors, exocrine pancreatic cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct Cancer, eyes , Female breast cancer, Gaucher disease, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumor, germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkins disease, Hodgkins Lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, intraocular melanoma, islet cell cancer, islet cell pancreatic cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, Liver cancer, lung cancer, lymphoproliferative disorder, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary Metastatic primary cervical squamous cell carcinoma, metastatic cervical squamous cell carcinoma, multiple myeloma, multiple myeloma / plasmacytoma, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia( yellow Leukemia), myeloproliferative syndrome, sinus and nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, non-melanoma skin cancer, non-small cell lung cancer, primary metastatic Squamous cell carcinoma of the neck, oropharyngeal cancer, bone / malignant fibrosarcoma, osteosarcoma / malignant fibrous histiocytoma, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian epithelial cancer, ovarian germ cell tumor, ovary Low-grade tumor, pancreatic cancer, proteinemia, purpura, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, plasmacytoma / multiple myeloma, primary central nervous system lymphoma Primary liver cancer, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureteral cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoidosis sarcoma, Sezary syndrome, skin Small cell lung cancer, small intestine cancer, soft tissue sarcoma, cervical squamous Squamous cell carcinoma, stomach cancer, tent primitive primitive ectoderm and pineal tumor, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, renal pelvis and transitional cell carcinoma of the ureter, cellular renal pelvis and urine Transitional cancer of the duct, trophoblastic tumor, ureteral and renal pelvic cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom macro Globulinemia, Wilms tumor, and any other hyperproliferative disease are included.

  In another preferred embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is diagnosed for pre-cancerous conditions, including but not limited to the diseases described above. Used for prognosis, prevention and / or treatment, and to suppress progression to neoplastic or malignant conditions. Such use is known or expected to progress to neoplasm or cancer, particularly in the state where neoplastic cell proliferation consisting of hyperplasia, metaplasia or most particularly dysplasia occurs. (See Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79.) .

  Hyperplasia is a form of controlled cell growth that includes an increase in the number of cells in a tissue or organ without significant changes in structure or function. Hyperplastic diseases that can be diagnosed, prognosticated, prevented and / or treated with the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include, but are not limited to, vascular vesicle longitudinal Lymph node hyperplasia, vascular lymph hyperplasia with eosinophilia, atypical pigment cell hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementitious hyperplasia, congenital adrenocortical hyperplasia, congenital Sebaceous gland hyperplasia, cystic hyperplasia, milk cystic hyperplasia, denture hyperplasia, gland duct formation, endometrial hyperplasia, fibromuscular hyperplasia, localized epithelial hyperplasia, gingival hyperplasia, inflammatory fiber hyperplasia Inflammatory papillary hyperplasia, endovascular papillary endothelial hyperplasia, prostate nodular hyperplasia, nodular regenerative hyperplasia, pseudoepithelial hyperplasia, senile sebaceous hyperplasia and wart thickening.

  Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell replaces the other type of adult cell. Metaplastic diseases that can be diagnosed, prognosticated, prevented and / or treated with a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include, but are not limited to, primary myelofibrosis , Apocrine metamorphosis, atypical metaplasia, autogenous metaplasia, connective tissue metaplasia, epithelial metaplasia, interstitial metaplasia, metaplastic anemia, metaplastic deformed ossification, metaplastic polyp, myeloid metaplasia, primary Myeloid metaplasia, secondary myelogenous metaplasia, squamous metaplasia, amniotic squamous metaplasia, and symptomatic myelogenic metaplasia are included.

  Dysplasia is often a pre-stage of cancer, is mainly found in the epithelium, is an almost disordered form of non-hyperplastic cell growth, lack of homogeneity in individual cells, and lack of cellular structural orientation Is included. Dysplastic cells often have unusually large, deeply colored nuclei and display diversity. Dysplasia occurs characteristically in the presence of chronic irritation or inflammation. Non-sweaty ectoderm includes, but is not limited to, dysplastic diseases that can be diagnosed, prognosticated, prevented and / or treated with a polynucleotide encoding an albumin protein of the invention and / or an albumin fusion protein of the invention Sexual dysplasia, Protofacial dysplasia, Choking thoracic dysplasia, Atrial finger dysplasia, Bronchopulmonary dysplasia, Brain dysplasia, Cervical dysplasia, Cartilage ectodermal dysplasia, Clavicular skull dysplasia, Congenital Ectodermal dysplasia, cranial dysplasia, cranial carpal and tarsal dysplasia, cranial metaphyseal dysplasia, dentinal dysplasia, diaphyseal dysplasia, ectodermal dysplasia, enamel dysplasia , Brain-eye dysplasia, epiphyseal defect dysplasia, epiphyseal dysplasia, epiphyseal dysplasia, epithelial dysplasia, facial genital dysplasia, chin familial fibrous dysplasia, familial white curvature Formation, fibromuscular dysplasia, fibrous bone dysplasia Flowering bone dysplasia, hereditary kidney-retinal dysplasia, sweating ectodermal dysplasia, hypohidrotic ectoderm dysplasia, lymphopenia thymus dysplasia, mammary dysplasia, mandibular facial dysplasia, metaphyseal dysplasia Formation, Mondini dysplasia, monostoic fiber dysplasia, mucosal epithelial dysplasia, multiple epiphyseal dysplasia, ocular-ear spinal dysplasia, odontoid dysplasia, ocular spinal dysplasia, odontogenic Dysplasia, ocular mandibular limb dysplasia, apical cementum dysplasia, multiple fibrous bone dysplasia, pseudochondral dysplasia vertebral epidysplasia, retinal dysplasia, septal ocular dysplasia This includes chamber dysplasia.

  Additional precancerous relaxations that can be diagnosed, prognosticated, prevented and / or treated with a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention include, but are not limited to, benign growth Abnormalities (eg, benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps and esophageal dysplasia), leukoplakia, keratosis, Bowen's disease, farmer skin, sun cheilitis and actinic keratosis .

  In another embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is used to diagnose a disease associated with the tissue (s) in which the polypeptide of the present invention is expressed. And / or may be used for prognosis.

  In other embodiments, as described herein, an albumin fusion protein of the invention conjugated to a toxin or a radioactive isotope and / or a polynucleotide encoding an albumin fusion protein of the invention is provided herein. It may be used to treat cancer and neoplasms, including but not limited to those described in the book. In a further preferred embodiment, as described herein, an albumin fusion protein of the invention conjugated to a toxin or radioactive isotope and / or a polynucleotide encoding an albumin fusion protein of the invention is treated with acute bone marrow. It may be used to treat sex leukemia.

  Furthermore, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can affect apoptosis, thus preventing a number of diseases associated with increased cell survival or inhibition of apoptosis. It can be useful to treat. For example, diseases associated with inhibition of cell survival or apoptosis that can be diagnosed, prognosticated, prevented and / or treated by the polynucleotides, polypeptides, and / or agonists or antagonists of the present invention include (follicular lymphoma, Cancers containing p53 mutations, including but not limited to colorectal cancer, heart cancer, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, Such as hormone-dependent tumors including mixioma, mioma, lymphoma, endothelial tumor, osteoblastoma, giant cell tumor, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi sarcoma and ovarian cancer) Cancer, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Burchett's disease, Crohn's disease Autoimmune diseases such as polymyositis, systemic lupus erythematosus, and immune-related glomerulonephritis and rheumatoid arthritis, and viral infections (such as herpes virus, poxvirus and adenovirus), inflammation, graft-versus-host disease, acute Includes graft rejection, and chronic graft rejection.

  In a preferred embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to inhibit the growth, progression and / or metastasis of cancer, especially those listed above. use.

  Additional diseases or conditions associated with increased cell survival that can be diagnosed, prognosticated, prevented and / or treated by the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include: (Including but not limited to, acute leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia)), and chronic leukemia ( Leukemia (including chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia)), leukocytosis, lymphoma (eg Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia , Heavy chain disease, and without limitation, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, Sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, Squamous cell cancer, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchial cancer, kidney cell Cancer, liver cancer, cholangiocarcinoma, choriomas, seminoma, fetal cancer, Wilm tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, star Such as cell tumor, medulloblastoma, craniopharyngioma, ependymoma, pineal gland, amgioblastoma, acoustic neuroma, oligodendroglioma, manangioma, melanoma, neuroblastoma and retinoblastoma Progression of malignancies and related diseases such as solid tumors including sarcomas and cancer and / or Others, including the transition.

  AIDS, (Alzheimer's) is a disease associated with increased apoptosis that can be diagnosed, prognosticated, prevented and / or treated by a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. Disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or previous related disease), multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, bile Cirrhosis, Burcett's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis), myelodysplasia (such as aplastic anemia), graft Host-host disease, ischemic injury (eg, caused by myocardial infarction, stroke, and reperfusion injury Liver injury (eg hepatitis-related liver injury, ischemia / reperfusion injury, cholestosis (bile duct injury) and liver cancer), toxin-induced liver disease (such as caused by alcohol), septic Includes shock, epidemics, and anorexia.

  There is no limitation on hyperproliferative diseases and / or diseases that can be diagnosed, prognosticated, prevented and / or treated by the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention. But the liver, abdomen, bones, milk, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eyes, head and neck, nervous system (central and peripheral), Included are neoplasms that are localized in the lymphatic system, pelvis, skin, soft tissue, spleen, chest and urogenital tract.

  Similarly, other hyperproliferative diseases can also be diagnosed, prognosticated, prevented and / or treated by the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention. Examples of such hyperproliferative disorders include, but are not limited to, hypergammaglobulinemia, lymphoproliferative disorders, proteinoidemia, in addition to neoplasms localized in the organ system listed above. , Purpura, sarcoidosis, Sezary syndrome, Waldenstrom macroglobulinemia, Gauche disease, histiocytosis, and other hyperproliferative diseases.

  Another preferred embodiment is the use of a polynucleotide encoding an albumin fusion protein of the invention to inhibit abnormal cell division by gene therapy using the invention and / or protein fusions or fragments thereof. Use.

  Accordingly, the present invention provides a method for treating a cell hyperproliferative disease by inserting a polynucleotide encoding an albumin fusion protein of the present invention into an abnormally proliferating cell, The polynucleotide suppresses the expression.

  Another embodiment of the present invention is a method of treating a cell proliferative disorder in an individual comprising the administration of one or more active gene copies of the present invention to an abnormally proliferating cell or group of cells. I will provide a. In a preferred embodiment, the polynucleotide of the present invention is a DNA construct comprising a recombinant expression vector that is effective in expressing a DNA sequence encoding said polynucleotide. In another preferred embodiment of the invention, a DNA construct encoding the fusion protein of the invention is inserted into a cell to be treated with a retrovirus, or more preferably an adenoviral vector (according to the reference). G J. Nabel, et. Al., PNAS 1999 96: 324-326, which is incorporated herein by reference). In the most preferred embodiment, the viral vector is incomplete, non-proliferating cells are not transduced, and only proliferating cells are transduced. Furthermore, in a preferred embodiment, expression of the encoded protein product is carried out with the polynucleotide of the present invention alone or in combination with other polynucleotides or fused into a proliferating cell. In order to induce, it can be regulated via an external stimulus (ie, magnetic, specific small molecule, chemical or drug administration, etc.) that acts upon a promoter upstream of the polynucleotide. As such, the beneficial therapeutic effects of the present invention may be clearly modified based on the external stimulus (ie, to increase, decrease or inhibit the expression of the present invention).

  The polynucleotide of the present invention may be useful in suppressing the expression of oncogenes or antigens. “Suppressing the expression of oncogenes” means suppression of gene transcription, degradation of gene transcript (pre-message RNA), inhibition of splicing, destruction of messenger RNA, suppression of post-translational modification of protein, destruction of protein Or intended to inhibit the normal function of the protein.

  For topical administration to abnormally proliferating cells, the polynucleotides of the invention may be used in, but not limited to, transfection, electrophoresis, cellular microinjection, or excipients such as liposomes, lipofectin, Alternatively, it may be administered as a naked polynucleotide or by any method known to one of skill in the art, including other methods described throughout this specification. The polynucleotides of the invention include, but are not limited to, retroviral vectors (Gilboa, J. Virology 44: 845 (1982); Hocke, Nature 320: 275 (1986); Wilson, et al., Proc. Natl. Acad. Sci.U.S.A. 85: 3014), vaccine virus systems (Chakrabarty et al., Mol. Cell Biol. 5: 3403 (1985), or other effective DNA delivery systems known to those skilled in the art (Yates) et al., Nature 313: 812 (1985)) These references are for illustration only and are incorporated by reference. Retroviruses known to those skilled in the art, or (described elsewhere in the art or elsewhere herein) to specifically deliver or transfect cells that are spare non-dividing cells It is preferred to use an adenoviral delivery system (such as), since host DNA replication is required to integrate retroviral DNA, the retrovirus lacks the retroviral genes necessary for its life cycle. Using such retroviral delivery systems for the polynucleotides of the present invention targets the genes and constructs to abnormally proliferating cells and is non-dividing. Save normal cells.

  The polynucleotides of the present invention may be delivered directly to a cell proliferative disease / disease site in internal organs, body cavities, etc. by use in an imaging device used to direct the injection needle directly to the disease site. The polynucleotides of the invention may also be administered to the disease site at the time of surgical intervention.

  A “cell proliferative disorder” is any benign or malignant, any one or any of an organ, cavity or body part characterized by a single or multiple local overgrowth of cells, populations of cells, or tissues Means any human or animal disease or condition affecting the combination.

  Any amount of the polynucleotide of the invention may be administered so long as to have a biological inhibitory effect on the growth of the treated cells. Furthermore, two or more polynucleotides of the present invention can be administered simultaneously to the same site. “Biologically inhibit” means partial or total growth inhibition, as well as a decrease in the rate of cell growth or increase. Biological inhibition / time can be determined by assessing the effect of the polynucleotides of the invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell culture, or others known to those skilled in the art. It can be determined by any method.

  Furthermore, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used alone, as a protein fusion, or as described elsewhere in this specification. In combination with a polypeptide, it is useful in inhibiting angiogenesis of proliferating cells or tissues, either directly or indirectly. In the most preferred embodiment, the anti-angiogenic effect can be achieved indirectly via inhibition of tumor specific cells such as hematopoiesis, tumor associated macrophages (Joseph IB, et al., Incorporated by reference). al., J Natl Cancer Inst, 90 (21): 1648-53 (1998)).

  The fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention may be useful in inhibiting proliferating cells or tissues through induction of apoptosis. These fusion proteins and / or polynucleotides include, for example, tumor necrosis factor (TNF) receptor-1, CD95 (Fas / APO-1), TNF-receptor-related apoptosis mediator protein (TRAMP), and TNF-related apoptosis-inducing ligand. Activation of death-domain receptors, such as (TRAIL) receptor-1 and -2, can act directly or indirectly to induce apoptosis of proliferating cells and tissues (herein by reference). See Schulze-Osthoff K, et.al., Eur J Biochem 254 (3): 439-59 (1998), incorporated). Furthermore, in other preferred embodiments of the invention, these fusion proteins and / or polynucleotides are either alone or in combination with small molecule drugs or adjuvants, such as apotonin, galectins, thioredoxins, anti-inflammatory proteins. However, apoptosis may be induced through other mechanisms, such as in the activation of other proteins that activate apoptosis or through stimulating the expression of these proteins (eg, reference Mutat Res 400 (1-2): 447-55 (1998), Med Hypotheses.50 (5): 423-33 (1998), Chem Biol Interact. Apr 24; 111-112: 23-34 1998), J Mol Med.76 (6): 402-12 (1998), Int J Tissue React; 20 (1): 3-15 see (1998)).

  The polynucleotide encoding the fusion protein of the present invention and / or the albumin fusion protein of the present invention is useful in inhibiting metastasis of proliferating cells or tissues. Inhibition is either as a direct result of administration of these albumin fusion proteins and / or polynucleotides, or by activating the expression of proteins known to inhibit metastasis, such as alpha 4 integrins. (See, for example, Curr Top Microbiol Immunol 1998; 231: 125-41, incorporated herein by reference). Such therapeutic effects of the present invention can be achieved either alone or in combination with small molecule drugs or adjuvants.

  In another embodiment, the present invention binds a composition comprising an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention bound by an albumin fusion protein of the invention. A method of delivering to a targeted cell expressing a related or related polypeptide is provided. The albumin fusion proteins of the present invention may be related to heterologous polypeptides, heterologous nucleic acids, toxins or prodrugs via hydrophobic, hydrophilic, ionic and / or covalent bonds.

The albumin fusion proteins of the present invention can respond directly to proliferative antigens and immunogens, such as when immune responses are vaccinated, or immune responses to said antigens and immunogens (eg chemokines) Useful in enhancing the immunogenicity and / or antigenicity of proliferating cells or tissues indirectly, such as in activating the expression of proteins known to enhance .

Kidney disease

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used for treating, preventing, diagnosing and / or prognosing diseases of the renal system. Kidney diseases that can be diagnosed, prognosticated, prevented and / or treated with the compositions of the present invention include, but are not limited to, renal failure, nephritis, renal vascular disease, metabolic and congenital renal failure, renal urinary tract Diseases, autoimmune diseases, sclerosis and necrosis, electrolyte imbalance and kidney cancer are included.

  Kidney diseases that can be diagnosed, prognosticated, prevented and / or treated with the composition of the present invention include, but are not limited to, acute renal failure, chronic renal failure, atheroembolic renal failure, end stage renal disease, renal inflammatory disease (Eg, acute glomerulonephritis, post-infection glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I and II, mesangial proliferating glomeruli Nephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, post-pustular nephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis, interstitial nephritis and streptococcal infection Glomerulonephritis), renal vascular disease (eg renal infarction, atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal perfusion, renal retinopathy, renal ischemia-reperfusion, kidney Arterial embolism, and renal artery stenosis), and kidney disease that results from urinary tract disease (eg, pyelonephritis, hydronephrosis, urolithiasis (kidney stone disease, nephrolithiasis), reflux nephropathy, Urinary tract infection, urinary retention, and acute or chronic unilateral obstructive urinary tract disease).

  In addition, the compositions of the present invention may be used to treat metabolic and congenital diseases of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, tubular acidosis, renal diabetes, nephrogenic diabetes, cystinuria, Fanconi syndrome, renal fibrotic bone tissue formation (renal rickets), Hartnup disease, Barter syndrome, Riddle syndrome, polycystic kidney disease, medullary cystic disease, medullary cancellous kidney, Alport syndrome, nail-patella syndrome, Congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy) and renal autoimmune diseases (eg, systemic lupus erythematosus (SLE) ), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis), prognosis, prevention and / or Others can be used to treat.

  Compositions of the invention can be used to treat renal sclerosis or necrosis (eg, glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), Renal cancer (eg, nephroma, adrenal gland, nephroblastoma, renal cell carcinoma, transitional cell carcinoma, renal adenocarcinoma, squamous cell carcinoma, Wilm cancer), and electrolyte imbalance (eg, Nephrocalcinosis, abscess, edema, hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia And hyperphosphatemia) can be used for diagnosis, prognosis, prevention and / or treatment.

The composition of the present invention includes, but is not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter injection, biolistic injection, particle accelerator, gel sponge depot, other commercially available depot materials, Administration may be performed using any method known in the art, including isotonic pumps, oral or suppository individual pharmacological formulations, decanting and topical application during surgery, aerosol delivery. Such methods are known in the art. The compositions of the invention may be administered as part of a therapeutic, as described in more detail below. Methods for delivering the polynucleotides of the invention are described in more detail herein.

Cardiovascular disease

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is used for treating, preventing, diagnosing cardiovascular diseases including but not limited to peripheral arterial diseases such as limb ischemia, And / or may be used for prognosis.

  Cardiovascular diseases include, but are not limited to, cardiovascular abnormalities such as arterial-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformation, congenital heart defects, pulmonary artery closure and scimitar syndrome. Congenital heart defects include, but are not limited to, aortic constriction, trilobular heart, coronary anomaly, crossed heart, right thoracic heart, patent ductus arteriosus, Epstein malformation, Eisenmengel complex, hypoplastic left heart syndrome, Left thoracic heart, tetralogy of Fallot, transposition of large vessels, right ventricular origin of both large vessels, tricuspid atresia, residual arterial duct, and aortopulmonary septal defect, endocardial bed defect, rutenbach Includes cardiac septal defects such as Char syndrome, trilogy of Fallot, and ventricular ventricular septal defects.

  Cardiovascular diseases also include but are not limited to arrhythmia, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), cardiac aneurysm, cardiac arrest Congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve disease, myocardial disease Disease, myocardial ischemia, epicardial fluid, pericarditis (including stenosis and tuberculosis), endocardial emphysema, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia Heart diseases such as cardiovascular pregnancy complications, scimitar syndrome, cardiovascular syphilis and cardiovascular tuberculosis are included.

  Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes syndrome, leg block, sinoatrial block, QT prolongation syndrome, accessory contraction, LGL syndrome, This includes Mahaim-type early excitability syndrome, Wolf-Parkinson-White syndrome, sinus syndrome, tachycardia and ventricular fibrillation. Tachycardia includes but is not limited to paroxysmal tachycardia, supraventricular tachycardia, tachyarrhythmic ventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic Included are tachycardia, sinoatrial node reentry tachycardia, sinus tachycardia, ventricular arrhythmia, and ventricular tachycardia.

  Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic stenosis, heart murmur, aortic valve prolapse, mitral prolapse, tricuspid prolapse, mitral regurgitation, mitral stenosis, pulmonary artery closure Pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid insufficiency, and tricuspid stenosis.

  Cardiomyopathy includes, but is not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic stenosis, subpulmonary stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fiber elasticity Disease, endocardial fibrosis, Keynes syndrome, myocardial reperfusion and myocarditis.

  Myocardial ischemia includes but is not limited to coronary heart disease such as angina pectoris, coronary aneurysm, coronary atherosclerosis, coronary artery thrombosis, coronary spastic angina, myocardial infarction and stunned myocardium included.

  Cardiovascular diseases also include aneurysms, vascular dysplasia, hemangiomatosis, bacterial hemangiomatosis, Hippel-Lindau disease, Klippel-Trenonnay-Weber syndrome, Sturge-Weber syndrome, vasomotor edema, aortic disease, Takayasu's arteritis, aortitis, lurich syndrome, arterial occlusive disease, arteritis, arteritis, nodular polyarteritis, cerebrovascular disorder, diabetic vascular disorder, diabetic retinopathy, embolism, thrombosis, limb Erythema pain, hemorrhoids, hepatic venous occlusive disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary venous occlusive disease, Raynaud's disease, CREST syndrome, retinal venous occlusion, scimitar syndrome, above Vena cava syndrome, telangiectasia, telangiectasia ataxia, hereditary hemorrhagic telangiectasia, varicocele, varicose vein, varicose ulcer Vasculitis and include venous insufficiency.

  Aneurysms include, but are not limited to, dissecting aneurysms, pseudoaneurysms, infectious aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms and iliac aneurysms .

  Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, moyamoya disease, renal artery occlusion, renal artery occlusion, And obstructive thromboangiitis.

  Cerebrovascular disease includes but is not limited to carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, cerebral embolism and thrombosis, Carotid artery thrombosis, sinus thrombosis, Barenbury syndrome, intracerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, (including transient) cerebral ischemia, subclavian Arterial steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar circulation failure.

  Embolism includes, but is not limited to, air embolism, amniotic fluid embolism, cholesterol embolism, Brewthau syndrome, fat embolism, pulmonary embolism, thromboembolism. Thrombosis includes, but is not limited to, coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Barenbury syndrome, and thrombophlebitis.

  Ischemic diseases include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndrome, precompartment syndrome, myocardial ischemia, reperfusion injury, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's syndrome, Churg-Strauss syndrome, mucocutaneous lymph node syndrome, obstructive thromboangiitis, hypersensitivity vasculitis, Shoenrain-Henok purpura, allergic Dermatovasculitis and Wegener's granulomatosis are included.

The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may be, but is not limited to, direct needle injection at the delivery site, intravenous injection, local administration, catheter injection, biolistic injection, Known in the art, including particle accelerators, gel-like sponge depots, other commercially available depot materials, isotonic pumps, oral or suppository individual pharmacological formulations, decanting and topical application during surgery, aerosol delivery Any of the methods may be used. Such methods are known in the art. Methods for delivering the polynucleotides of the invention are described in more detail herein.

Respiratory disease

  The albumin fusion protein of the present invention and / or the polypeptide encoding the albumin fusion protein of the present invention is used for treating, preventing, diagnosing and / or prognosing a respiratory disease and / or disease. Also good.

  Respiratory diseases and conditions include, but are not limited to, nasal vestitis, non-allergic rhinitis (eg, acute rhinitis, chronic rhinitis, atopic rhinitis, vasomotor rhinitis), nasal polyps and sinusitis, juvenile blood vessels Fibromas, nasal cancer and juvenile papilloma, vocal cord polyps, nodules (singer nodules), contact ulcers, vocal cord paralysis, laryngeal cysts, pharyngitis (eg viruses and bacteria), tonsillitis, tonsillitis, parapharynx Gap abscess, laryngitis, laryngeal cyst and throat cancer (eg nasopharyngeal cancer, condylar cancer, laryngeal cancer), lung cancer (eg squamous cell carcinoma, small cell (buckwheat cell) cancer, large cell carcinoma And adenocarcinoma), allergic diseases (eosinophilic pneumonia, hypersensitivity pneumonia (eg exogenous allergic alveolitis, allergic interstitial pneumonia, organic pneumoconiosis, allergic bronchopulmonary aspergill) Disease, asthma, Wagner granulomatosis, granulomatous vasculitis, Goodpasture syndrome), pneumonia (eg, bacterial pneumonia (eg, Streptococcus pneumoniae, Staphylococcus aureus), gram-negative bacteria Pneumonia (eg caused by Klebsiella and Pseudomas spp), Mycoplasma pneumoniae pneumonia, Hemophilus influenza pneumonia, Legionella pneumophila (legionella disease), and influenza )) Is included.

Additional diseases and disorders of the respiratory system include but are not limited to bronchitis, polio (acute leukomyelitis), croup, respiratory syncytial virus infection, epidemic parotitis, infectious erythema (fifth disease) , Idiopathic rash, progressive rubella encephalitis, three-day measles and subacute sclerosing panencephalitis), fungal pneumonia (eg, histoplasmosis, coccidioidomycosis, blastosis, microbial infection in patients with severe immunosuppression ( For example, cryptococcosis caused by Cryptococcus neoformans, aspergillosis caused by Aspergillus spp., Candidiasis caused by Candida, and mucormycosis), Pneumocystis carinii (pneumocystis pneumonia), Mycoplasma and Chlamydia spp.), Opportunistic pneumonia, nosocomial pneumonia, chemical pneumonia, and aspiration pneumonia, pleural diseases (eg pleurisy, pleural effusion and pneumothorax (eg simple idiopathic pneumothorax, combined idiopathic pneumothorax, tension pneumothorax) )), Obstructive airway disease (eg asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational lung disease (eg silicosis, black lung disease (coal miner pneumoconiosis), Asbestosis, beryllium poisoning, occupational asthma, pneumonia and benign pneumoconiosis), invasive lung disease (eg, pulmonary fibrosis (eg, fibrotic alveolitis, conventional interstitial pneumonia), idiopathic pulmonary fiber) Disease, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, histocytosis X (eg, Letterer-Sieve disease, hand-Schuller-Christian disease, eosinophilic meat Blastoma), idiopathic pulmonary hemosiderinosis, sarcoidosis and alveolar proteinosis), acute alveolar protein syndrome (eg also called adult respiratory distress syndrome), edema, pulmonary embolism, bronchitis (eg virus, bacteria), Bronchiectasis, pulmonary diastolic failure, lung abscesses (eg, caused by Staphylococcus aureus or Legionella pneumophila) and cystic fibrosis are included.

Anti-angiogenic activity

  The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is that in which inhibitory effects predominate Rastinejad et al. , Cell 56: 345-355 (1989). In rare cases where neovascularization occurs under normal physiological conditions such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is strongly controlled and spatial and temporal extent Is determined. Under conditions of pathogenic angiogenesis that characterize individual tumor growth, these controls lose control. Uncontrolled angiogenesis becomes pathological and maintains many neoplastic and non-neoplastic progressions. A number of severe diseases are dominated by abnormal neovascularization, including solid tumor growth and metastasis, arthritis, some types of eye diseases, and psoriasis. For example, Moses et al. , Biotech. 9: 630-634 (1991); Folkman et al. , N.M. Engl. J. et al. Med. 333: 1757-1763 (1995); Auerbach et al. , J. et al. Microvasc. Res. 29: 401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. et al. Optalmol. 94: 715-743 (1982); and Folkman et al. , Science 221: 719-725 (1983). In many pathogenic conditions, the process of angiogenesis is involved in the disease state. For example, significant data has been accumulated suggesting that solid tumor growth is dependent on angiogenesis. Folkman and Klagsbrun, Science 235: 442-447 (1987).

  The present invention provides for the treatment of diseases or disorders associated with neovascularization by the administration of the fusion proteins of the invention and / or polypeptides encoding the albumin fusion proteins of the invention. Malignant and metastatic conditions that can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the present invention include, but are not limited to, malignant tumors, solid cancers, and those described herein or in the art. Known cancers are included (for review of such diseases, see Fishman et al., Medicine, 2d Ed., JB Lippincott Co., Philadelphia (1985)). Accordingly, the present invention includes administering to an individual in need thereof a therapeutically effective amount of an albumin fusion protein of the invention and / or a polypeptide encoding an albumin fusion protein of the invention. A method of treating angiogenesis-related diseases and / or diseases is provided. For example, a fusion protein of the invention and / or a polypeptide encoding an albumin fusion protein of the invention may be used in a variety of additional ways to therapeutically treat a cancer or tumor. Cancers that may be treated with a fusion protein of the invention and / or a polypeptide encoding an albumin fusion protein of the invention include, but are not limited to, prostate, lung, breast, ovary, stomach, pancreas, larynx, Esophagus, testicles, liver, parotid gland, bile duct, large intestine, rectum, neck, uterus, endometrium, liver, bladder, solid tumors including thyroid cancer, primary tumors and metastases, melanoma, glioblastoma, Kaposi sarcoma , Leiomyosarcoma, non-small cell lung cancer, colorectal cancer, solid cancer including advanced malignant tumors, and blood derived tumors such as leukemia. For example, a fusion protein of the invention and / or a polypeptide encoding an albumin fusion protein of the invention may be administered topically to treat cancers such as skin cancer, head and neck tumors, breast cancer, and Kaposi sarcoma. May be delivered in

  In yet another aspect, a fusion protein of the invention and / or a polypeptide encoding an albumin fusion protein of the invention is used to treat a superficial form of bladder cancer, for example by intravesical administration. May be. The albumin fusion protein of the invention and / or the polypeptide encoding the albumin fusion protein of the invention may be delivered directly into or around the tumor site via injection or catheter. Of course, those skilled in the art will appreciate that the appropriate mode of administration may vary according to the cancer to be treated. Other modes of delivery are discussed herein.

  An albumin fusion protein of the invention and / or a polypeptide encoding an albumin fusion protein of the invention may be useful in treating other diseases other than cancer that involve angiogenesis. These diseases include, but are not limited to, benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachoma and purulent granulomas, atheromatous plaques, ocular neovascular diseases such as diabetic retinopathies, premature infant retinopathies, Macular degeneration, corneal transplant rejection, angiogenic glaucoma, posterior lens fibroproliferation, rubeosis, retinoblastoma, ocular uveitis and pterygium (abnormal vascular proliferation), rheumatoid arthritis, psoriasis, delayed wound healing, uterus Endometriosis, Angiogenesis, Arachnoid granule, Abnormal hypertrophic wound (Keloid), Unadherent fracture, Scleroderma, Trachoma, Vascular adhesion, Myocardial neovascularization, Coronary collateral, Cerebral collateral, Arteriovenous malformation, Ischemic Limb angiogenesis, Osler-Weber syndrome, plaque neovascularization, telangiectasia, hemophilia joint, hemangiofibroma, fibromuscular dysplasia, wound granulation, c It contains over's disease and atherosclerosis.

  For example, in one aspect of the invention, the method comprises administering to the abnormally enlarged wound and keloid the albumin fusion protein of the invention and / or the polypeptide encoding the albumin fusion protein of the invention. Provided to treat abnormal hypertrophy wounds and keloids.

  Within one embodiment of the present invention, the fusion protein of the present invention and / or the polypeptide encoding the albumin fusion protein of the present invention can be treated with an abnormally hypertrophic wound and keloid in order to prevent the progression of these injuries. Inject directly. This treatment is particularly valuable in prophylactic treatment of conditions known to result in abnormal hypertrophy and development of keloids (eg burns), preferably after the proliferative phase is time for progression (early Approximately 14 days after the wound) begins before abnormal hypertrophy and keloid development. As mentioned above, the present invention also provides methods for treating ocular neovascular diseases including, for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathies, posterior lens fibroproliferation and macular degeneration. Also provide.

  Further, the ocular diseases involved in neovascularization that can be treated with the albumin fusion protein of the present invention and / or the polypeptide encoding the albumin fusion protein of the present invention include, but are not limited to, neovascular glaucoma, diabetes Retinopathy, retinoblastoma, posterior lens fibroproliferation, uveitis, prematurity retinopathy, macular degeneration, corneal transplant neovascularization, and other eye inflammatory diseases, eyes associated with choroidal or iris neovascularization Tumors and diseases. For example, Waltman et al. , Am. J. et al. Ophthal. 85: 704-710 (1978) and Gartner et al. , Surv. Ophthal. 22: 291-312 (1978).

  Accordingly, in one aspect of the invention, a therapeutically effective amount of a compound (eg, encoding a fusion protein of the invention and / or an albumin fusion protein of the invention) that inhibits blood vessel formation. A method is provided for treating ocular neovascular diseases such as corneal neovascularization (including corneal transplant neovascularization) comprising administering a polypeptide to the cornea. Simply put, the cornea is a tissue that usually lacks blood vessels. However, under certain pathological conditions, capillaries can extend from the limbal corneal vascular plexus into the cornea. If the cornea becomes vascularized, it becomes cloudy, resulting in a reduction in the patient's visual field. When the cornea is completely turbid, complete vision loss occurs. For example, corneal infections (eg trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (eg transplant rejection and Stevens-Johnson syndrome), alkaline burns, trauma, inflammation (of any cause) A wide variety of diseases, including toxicity, nutritional deficiencies, and as a complication of wearing contact lenses, result in corneal neovascularization.

  Within certain preferred embodiments of the present invention may be prepared for topical administration in saline (in combination with any preservatives and antimicrobial agents commonly used in eye preparations), It may be administered in the form of eye drops. Solutions or suspensions may be prepared in its pure form and administered several times a day. Alternatively, an anti-angiogenic composition prepared as described above may also be administered directly to the cornea. Within a preferred embodiment, an anti-angiogenic composition is prepared with a mucoadhesive polymer that binds to the cornea. Within further embodiments, an anti-angiogenic factor or anti-angiogenic composition may be used as an additive to conventional steroid therapy. Topical treatment may also be useful prophylactically in corneal disorders, which are known to have a high potential to induce an angiogenic response (such as chemical burns). In these instances, perhaps in combination with a steroid, treatment may be initiated immediately to help prevent subsequent complications.

  Within other embodiments, the compounds described above may be injected directly into the corneal matrix by an ophthalmologist under microscopic guidance. The preferred site of injection may vary in the form of individual lesions, but the goal of administration is to place the composition in the anterior part of the vasculature (ie between the blood vessel and the normal cornea). Scattered). In most cases, the method includes limbal corneal injection to “protect” the cornea from progressive blood vessels. This method can also be utilized immediately after corneal injury to prevent corneal neovascularization prophylactically. In this situation, the substance can be injected into the limbal cornea that extends between the corneal disorder and its potentially unwanted limbic blood supply. Such a method may also be used in a similar manner to prevent capillary infiltration of the transplanted cornea. Only a few sustained injections of the sustained release form are required per year. Steroids can also be added to the injection solution to reduce the inflammation that is the result of the injection itself.

  Within another aspect of the invention, the method comprises administering to a patient's eye a therapeutically effective amount of an albumin fusion protein of the invention and / or a polypeptide encoding an albumin fusion protein of the invention. Methods are provided for treating neovascular glaucoma, and the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye to treat an early form of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber corner. Within other embodiments, the compound may also be placed in any location so that the compound is continuously released into the aqueous humor. Within another aspect of the invention, the method comprises administering to a patient a therapeutically effective amount of a fusion protein of the invention and / or a polypeptide encoding an albumin fusion protein of the invention. A method for treating proliferative diabetic retinopathies is provided and the formation of blood vessels is inhibited.

  In a particularly preferred embodiment of the invention, proliferative diabetic retinopathy is injected into the aqueous humor or vitreous for the purpose of increasing local concentrations of polynucleotides, polypeptides, antagonists and / or agonists in the retina. Can be treated. This treatment is preferably initiated before becoming severe enough to require photocoagulation.

  In another embodiment of the invention, a method for treating post-lens fibroproliferation is provided. The method comprises administering to a patient's eye a therapeutically effective amount of an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention, thereby preventing angiogenesis. Is included. The compound may be administered locally by intravitreal injection and / or intraocular injection.

  Further, disorders that can be treated using the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include hemangiomas, arthritis, psoriasis, angiofibromas, arteriosclerotic plaques, wound healing Delay, granulation, hemophilic joints, hypertrophic scars, non-adhesive fractures, Osler-Weber syndrome, purulent granulomas, scleroderma, trachoma, and vascular adhesions, but are not limited to these.

  Further, disorders and / or conditions that can be treated, prevented, diagnosed, and / or prognosticated using the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention. , Solid tumors, hematologic tumors such as leukemia, tumor metastasis, Kaposi's sarcoma, benign tumors (eg hemangiomas, acoustic neuromas, neurofibromas, trachoma, and purulent granulomas), rheumatoid arthritis, psoriasis, ocular neovascular diseases (Eg, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplant rejection, angiogenic glaucoma, post-lens fibroplasia, lebeosis, retinoblastoma, uveitis), delayed wound healing, endometrium , Angiogenesis, granulation, hypertrophic scar (keloid), non-adhesive fracture, scleroderma, trachoma, vascular adhesion, myocardial neovascularization, coronary motion Side branch, vascular side branch, arteriovenous malformation, ischemic limb angiogenesis, Osler-Weber syndrome, plaque neovascularization, telangiectasia, hemophilic joint, angiofibroma, fibromuscular dysplasia, wound Granulation, Crohn's disease, atherosclerosis, contraceptives by preventing angiogenesis necessary for embryo implantation and performing controlled menstruation, cat scratch disease (Rochel minora quintosa), ulcer (Helicobacter pylori (Helicobacter) pylori)), bartonellosis, and bacterial hemangioma include, but are not limited to, diseases with angiogenesis as pathological consequences.

  In one embodiment of the birth control method, an amount of the compound sufficient to inhibit embryo implantation is administered before or after sexual intercourse and fertilization is performed, thereby effective birth control methods, A “morning after” method is provided. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can also be used for the control of menstruation, or administered as a peritoneal cavity wash or for peritoneal transplantation in the treatment of endometriosis Can be.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be incorporated into a suture to prevent suture granulomas.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used in a variety of surgical procedures. For example, in one embodiment of the invention, the composition (eg, in the form of a spray or membrane) is used to isolate the surrounding normal tissue from the malignant tissue and / or the disease is spread to the surrounding tissue. Can be used to cover or spray an area prior to tumor removal. In other embodiments of the invention, the composition (eg, in the form of a spray) is applied via endoscopic surgery to cover the tumor or to inhibit angiogenesis at the site of interest. Can be delivered. In yet another embodiment of the present invention, a surgical mesh coated with the anti-angiogenic composition of the present invention may be used for any procedure where a surgical mesh would be used. For example, in one embodiment of the invention, an anti-angiogenic composition during abdominal cancer resection surgery (eg after colon resection) to provide structural support and release anti-angiogenic factors Surgical mesh containing can be used.

  In a further embodiment of the invention, a method of treating a site where a tumor has been resected is provided. This includes administering an albumin fusion protein of the invention and / or a polynucleotide encoding the albumin fusion protein of the invention to the resected margin of the tumor after resection, whereby the cancer at the site is Local recurrence and neovascularization are prevented. In one embodiment of the invention, the anti-angiogenic compound is administered directly to the site of tumor resection (eg, wiping the brushed margin with an anti-angiogenic compound, brushing, or otherwise coating the surface). Apply by covering). Alternatively, the anti-angiogenic compound may be incorporated into a known surgical glue prior to administration. In a particularly preferred embodiment of the invention, the anti-angiogenic compound is administered after hepatectomy for the malignant tumor and neurosurgery.

  In one embodiment of the invention, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may be used to excise various tumors including, for example, breast tumors, colon tumors, brain tumors, and liver tumors. Can be administered to the limbus. For example, in one embodiment of the present invention, the anti-angiogenic compound may be administered to the relevant part of the tumor of the nervous system after resection, thereby preventing the formation of new blood vessels in the relevant part.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may also be administered together with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include anti-invasive factors, retinoic acid and its derivatives, paclitaxel, suramin, tissue inhibitors of metalloproteinase-1, metalloproteinase-2 tissue inhibitors, type 1 plasminogen activity Activating factor inhibitors, type 2 plasminogen activator inhibitors, and various lightweight “d group” transfer metals.

  Lightweight “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum. Such transition metal species form a transition metal complex. Suitable complexes of the above transition metalomas include oxo transition metal complexes.

  Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl sulfate, including vanadyl acetylacetonate, and vanadyl sulfate hydrates such as vanadyl sulfate monohydrate and vanadyl sulfate trihydrate.

  Representative examples of the tungsten complex and the molybdenum complex include oxo complexes. Suitable oxo tungsten complexes include tungstate complexes and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxomolybdenum complexes include molybdate complexes, molybdenum oxide complexes, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrate, sodium molybdate and its hydrate, potassium molybdate and its hydrate. Suitable molybdenum oxides include molybdenum (IV) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

Various other angiogenic factors can also be used in the present invention. Representative examples include platelet factor 4, protamine sulfate, sulfated chitin derivatives (made from snow crab shells) (Murata et al., Cancer Res. 51: 22-26, 1991), sulfated polysaccharide peptidoglycan complex (SP -PG) (the action of this compound is increased by the presence of steroids such as estrogens and tamoxifen citrate), staurosporine, modulators of matrix metabolism (eg proline analogues, cishydroxyproline, d, L -3,4-dehydroproline, thiaproline, alpha, alpha-dipyridyl, aminopropionitrile fumarate, 4-propyl-5- (4-pyridinyl) -2 (3H) -oxazolone, methotrexate, mitoxantrone, heparin, Interferon macroglob Serum, ChIMP-3 (Pavloff et al., J. Bio. Chem. 267: 17321-17326, (1992)), chymostatin (Tomkinson et al., Biochem J. 286: 475-480, (1992)), Cyclodextrin tetradecasulfate, eponemycin, camptothecin, fumagillin (Ingber et al., Nature), sodium gold thiomalate (GST) (Matsubara and Ziff, J. Clin. Invest. 79: 1440-1446, (1987)), Anti-collagenase serum, alpha 2-antiplasmin (Holmes et al., J. Biol. Chem. 262 (IV): 1659-1664 (1987)), bisantren ( National Cancer Institute, Robenzalit disodium (N- (2) -carboxyphenyl-4-chloroanthronyl disodium or CCA) (Takeuchi et al., Agents Actions 36: 312-316) (1992)), metalloproteinase inhibitors such as thalidomide, Angostotic steroids, AGM-1470, carboxamidotriazole, and BB94.

Disease at the cellular stage

  For the purpose of increasing cell survival or inhibiting apoptosis, wherein the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for treatment, prevention, diagnosis, and / or determination of prognosis. Related diseases include cancer (follicular lymphoma, carcinoma caused by p53 mutation, and hormone-dependent tumors (colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glia) Blastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelial tumor, osteoblastoma, giant cell tumor, osteosarcoma, chondrosarcoma, adenoma, breast cancer, Prostate cancer, Kaposi's sarcoma, and ovarian cancer), autoimmune disease (multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Gött's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, and immune-related glomerulonephritis and rheumatoid arthritis), and viral infections (herpes virus, varicella virus, and secondary virus), inflammation, transplantation These include unilateral host disease, acute transplant rejection, and chronic transplant rejection.

  In a preferred embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention inhibits the growth, progression and / or metastasis of cancer, particularly the cancers listed above. Used for.

  Additional diseases or conditions associated with increased cell survival that can be treated or detected using the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include malignant tumors Progression and / or metastasis, and acute, such as leukemia (including acute lymphocytic leukemia, acute myeloid leukemia (including myeloblasts, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia)) Leukemia and chronic leukemia (such as chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom Macroglobulinemia, heavy chain disease, and solid tumors (fibrosarcoma, viscosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, spine Tumor, hemangiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer , Prostate cancer, squamous cell cancer, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary cancer, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, bronchial cancer , Renal cell cancer, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal cancer, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelium Sex cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma Related diseases such as, but not limited to, sarcomas and cancer), neuroblastoma, and retinoblastoma. But it is not limited to, et al.

Examples of diseases associated with increased apothesis that can be treated, prevented, diagnosed, and / or prognosticated using the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention. ADDS, neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration, and brain tumors or related diseases mentioned above), autoimmune diseases (multiple sclerosis, Sjogren) Syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, and immune-related glomerulonephritis and rheumatoid arthritis), myelodysplastic syndrome (such as aplastic anemia) , Graft-versus-host disease, ischemic injury (caused by myocardial infarction, stroke and reperfusion injury Disorders, etc.), liver damage (eg, liver damage related to hepatitis, ischemia / reperfusion injury, cholestasis (bile duct damage) and liver cancer), toxin-induced liver disease (such as alcohol-induced disease), Examples include, but are not limited to, septic shock, cachexia, and anorexia.

Wound healing and epithelial cell proliferation

  According to a further embodiment of the present invention, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention promotes epithelial cell proliferation for therapeutic purposes, eg, wound healing purposes, and basal Methods are provided for use in stimulating keratinocytes and promoting hair follicle production and skin wound healing. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is a surgical wound, excision wound, deep wound including dermis and epidermis damage, ocular tissue wound, dental tissue wound, Oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous ulcer ulcers, heat exposure or chemical burns, and other abnormal wound healing conditions (uremia, malnutrition vitamin deficiencies And systemic treatment with steroids, radiation therapy, and complications related to neoplastic therapeutic agents and antimetabolites, etc.) are clinically useful in promoting healing of wounds. The albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can be used to promote skin recovery after skin loss.

    The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is used to increase the adhesion of the skin graft to the wound bed and to promote re-epithelialization from the wound bed can do. The types of transplants (pieces) that can use the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention to promote attachment to a wound bed are shown below. Autograft, artificial skin, allograft, autologous skin transplant, autologous epidermal graft, avascular graft, Blair-Brown graft, bone graft, embryonic tissue graft, dermal graft, delayed graft, skin graft, epidermal graft , Fascia graft, full-thickness skin graft, xenograft, xenograft, allograft, proliferative transplant, superficial graft, reticular graft, mucosal graft, orier-tailsch graft, omental graft, scissor graft, stem Grafts, penetration grafts, intermediate skin grafts, split skin grafts. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to promote skin strength growth and to improve the appearance of aged skin.

  The fusion proteins of the present invention and / or polynucleotides encoding albumin fusion proteins of the present invention are also believed to cause changes in hepatocyte proliferation and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. ing. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention are sebaceous cells, hair follicles, hepatocytes, type II lung cells, mucin producing goblet cells, and other epithelial cells, and skin. Can promote the growth of epithelial cells, such as their progenitor cells, contained within the lung, liver and gastrointestinal tract. An albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may be capable of promoting proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can also be used to reduce the side effects of intestinal toxicity resulting from radiation, chemotherapy treatment, or viral infection. sell. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may have a cytoprotective effect on the mucous membrane of the small intestine. The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may also be capable of promoting the healing of mucositis (oral tumor) resulting from chemotherapy and viral infection.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can further be completely regenerated in the case of full-thickness skin defects including burns and split-thickness skin defects (ie, hair follicles, sweat glands, and It can also be used to treat other skin defects such as sebaceous gland regeneration) and psoriasis. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is epidermolysis bullosa (open and painful blistering frequently caused by defects in adhesion of the epidermis to the underlying dermis) Can also be used for the purpose of treating by accelerating epithelialization of those injuries. Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention also treat gastric and duodenal ulcers, more rapid mucosal lining, and glandular and duodenal mucosal lining It can also be used to help heal through regeneration. Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis are diseases that cause mucosal surface destruction of the small or large intestine, respectively. Therefore, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can promote regeneration of the mucosal surface to promote more rapid healing and prevent progression of inflammatory bowel disease Can be used. Treatment with a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention was expected to have a significant effect on the production of mucus in the gastrointestinal tract, was ingested, or Can be used to protect the intestinal mucosa from harmful substances associated with surgery. The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used to treat diseases associated with underexpression.

  Furthermore, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention can be used to prevent and cure lung damage due to various pathological conditions. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention promotes proliferation and differentiation and promotes alveolar and bronchiolar epithelial repair to prevent acute or chronic lung injury It can be prevented or treated. For example, pulmonary emphysema that leads to progressive loss of alveoli, such as causing bronchiolar epithelium and alveolar necrosis, and inhalation damage, i.e., airway burns and burns, are a It can be effectively treated with drugs or antagonists. The fusion protein of the invention, and / or the polynucleotide encoding the albumin fusion protein of the invention can also be used to promote type II lung cell proliferation and differentiation, although it may cause premature infant respiratory distress syndrome and Can aid in the treatment or prevention of diseases such as pulmonary hyamenopathy such as bronchopulmonary dysplasia.

    The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may promote hepatocyte proliferation and differentiation, thereby causing fulminant liver failure caused by cirrhosis, etc. It can be used to alleviate or treat liver disorders caused by liver diseases and conditions, viral hepatitis and toxic substances (ie, acetaminophen, carbon tetrahololide and other liver toxins known in the art).

Furthermore, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment or prevention of diabetes mellitus pathogenesis. The fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is suitable for patients with newly diagnosed type 1 and type II diabetes who have some islet cell function remaining. It can be used to maintain islet function for the purpose of alleviating, delaying or preventing persistent signs of disease. In addition, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used as an aid in islet cell transplantation in order to improve or promote the function of islet cells.

Nervous effects and diseases of the nervous system

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosis and / or treatment of diseases, disorders, injuries or injuries of the brain and / or nervous system. Nervous system injuries that can be treated using the compositions of the invention (eg, the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention) include axotomy or neuronal damage. Examples include, but are not limited to, nervous system damage and diseases that result in either reduction or degeneration or demyelination. Nervous system lesions that can treat patients (human and non-human mammalian patients) according to the methods of the present invention include the following lesions in either the central nervous system (including spinal cord, brain) or the peripheral nervous system: , But not limited to them. (1) Ischemic lesions, such as cerebral infarction or cerebral ischemia, or spinal cord infarction or spinal cord ischemia, where oxygen deficiency in a part of the nervous system results in nerve cell damage or death. (2) Traumatic lesions including lesions that cut off part of the nervous system or physical trauma such as compression injury or that accompany surgery. (3) A malignant lesion in which part of the nervous system is destroyed or damaged by a malignant tissue that is either a malignant tumor related to the nervous system or a malignant tumor derived from a non-neural tissue. (4) As a result of infection, for example, infection by human immunodeficiency virus, herpes zoster virus or herpes simplex virus, or abscess associated with Lyme disease, tuberculosis or syphilis destroys or injures part of the nervous system Infectious lesions. (5) A portion of the nervous system as a result of a degenerative process (including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS)) Degenerative lesions that are destroyed or damaged. (6) Nutritional disorders or metabolic disorders (vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholic amblyopia, Marquia-Ferva-Bignami disease (primary corpus callosum degeneration), and alcohol-dependent cerebellar degeneration Lesions associated with nutritional disorders or nutritional damage, which include, but are not limited to, that destroy or injure parts of the nervous system. (7) Neurological lesions associated with systemic diseases such as, but not limited to, diabetes (diabetic neuropathy, bell palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis. (8) Lesions caused by toxic substances such as alcohol, lead, or certain neurotoxins. (9) Demyelinating diseases (including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or multiple etiologies, progressive multifocal leukoencephalopathy, and Hashichuo-myelin lysis) Demyelinating lesions in which part of the nervous system is destroyed or damaged.

  In one embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to protect cells of the nervous system from the damaging effects of hypoxia. In a further preferred embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to protect cells of the nervous system from the damaging effects of brain hypoxia. According to this embodiment, the composition of the invention is used to treat or prevent nervous system cell damage associated with cerebral hypoxia. In one non-exclusive embodiment of the invention, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention treats neuronal cell damage associated with cerebral ischemia or Used to prevent. In another non-exclusive embodiment of the present invention, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention treats or prevents damage to nervous system cells associated with cerebral infarction. Used to do.

  In another preferred embodiment, the albumin fusion protein of the present invention and / or a polynucleotide encoding the albumin fusion protein of the present invention is used to treat or prevent neuronal cell damage associated with stroke. . In certain embodiments, the albumin fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used to treat or prevent brain nervous system cell damage associated with stroke.

  In another preferred embodiment, the albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to treat or prevent nervous system cell damage associated with a heart attack. The In certain embodiments, albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention are used to treat or prevent brain nervous system cell damage associated with a heart attack.

  The composition of the present invention effective for treating or preventing nervous system injury can be selected by a biological activity test for promoting survival or differentiation of nerve cells. Without being limited thereto, for example, compositions of the present invention that elicit any of the following effects may be effective according to the present invention. (1) Increase in survival time of neurons in culture with or without hypoxia or hypoxia conditions, (2) Increase in germination of neurons in culture or in vivo, (3) Culture Increased production of neuronal cell-related molecules, such as choline acetyltransferase or acetylcholinesterase, for motor neurons in the body or in vivo, or (4) reduction of symptoms of neuronal dysfunction in vivo. The above effects can be measured by any method known in the art. In preferred but non-limiting embodiments, increasing neuronal cell survival is achieved by methods described herein, or other methods known in the art (eg, Zhang et al., Proc Natl Acad Sci USA 97: 3637-42 (2000) or Arakawa et al, J. Neurosci., 10: 3507-15 (1990)). Increased neuronal sprouting can be achieved by methods known in the art, for example, Pestronk et al. , Exp. Neurol. , 70: 65-82 (1980), or Brown et al. , Ann. Rev. Neurosci. 4: 17-42 (1981). Increased production of neuronal cell-related molecules can be measured depending on the molecule being measured using techniques known in the art, such as biological assays, enzyme measurements, antibody binding, Northern blot analysis and the like. Motor neuron dysfunction can then be measured by assessing the physical expression of the motor neuron disorder, such as weakness, motor neuron conduction velocity, or functional disorder.

  In certain embodiments, motor neuron disorders that can be treated according to the present invention include motor neurons as well as other disorders that selectively affect nerve cells such as amyotrophic lateral sclerosis. Examples include, but are not limited to, infarctions, infections, toxin exposure, trauma, surgical damage, degenerative diseases or malignant tumors that may affect nervous system components. Also, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, childhood muscular atrophy, juvenile muscular atrophy, childhood progressive bulbar palsy (Fazio-Londo syndrome), polio and post-polio syndrome And hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease).

  Furthermore, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may play a role in neuronal survival, synapse formation, conductivity, neuronal differentiation, and the like. Thus, a composition of the invention (including a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention) is associated with a disease or injury (learning and / or cognitive impairment) associated with these roles. Can be used for diagnosis and / or treatment or prevention of, but not limited to. The compositions of the present invention may also be effective in the treatment or prevention of neurodegenerative disease states and / or behavioral injuries. Such neurodegenerative disease states and / or behavioral injuries include Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, Behavioral changes include, but are not limited to, learning disorders, ALS, psychosis, autism, and disturbances in eating, sleep patterns, balance, and perception. Furthermore, the compositions of the invention may also play a role in the treatment, prevention and / or detection of developmental or sexually related injuries associated with the developing embryo.

  Furthermore, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is useful for protecting cells of the nervous system from diseases, injuries, disorders or injuries associated with cerebrovascular disorders. It is possible. Cerebrovascular disorders include carotid artery disease (eg carotid artery thrombosis, carotid artery stenosis, or moyamoya disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebrum Arterial disease, cerebral embolism and cerebral thrombosis (eg carotid artery thrombosis, venous sinus thrombosis, or Wallenberg syndrome), cerebral hemorrhage (eg epidural hematoma, subdural hematoma, or subarachnoid hemorrhage), cerebral infarction , Cerebral ischemia (eg, transient cerebral ischemia, subclavian artery steel syndrome, or vertebrobasilar artery circulatory failure), vascular dementia (eg, multiple infarcts), periventricular leukomalacia, and blood vessels Include, but are not limited to, sexual headache (eg, cluster headache or migraine).

  According to a further aspect of the invention, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used for therapeutic purposes, for example to promote proliferation and / or differentiation of neural cells The method to do is obtained. Therefore, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for treatment and / or detection of neurological diseases. Furthermore, the fusion proteins of the invention and / or the polynucleotides encoding the albumin fusion proteins of the invention can be used as labels or detectors for certain nervous system diseases or disorders.

  Examples of neurological diseases that can be treated or detected using the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include metabolic brain diseases (phenyl ketones such as maternal phenylketonuria) Brain disease such as urinary disease, pyruvate carboxylase deficiency, birubinate dehydrogenase complex deficiency, Wernicke encephalopathy, cerebral edema), cerebellar tumor (subventricular tumor, choroid plexus tumor, etc., hypothalamus Brain tumors such as tumors, supratent tumors and canavan disease), cerebellar ataxia (spinal cerebellar degeneration such as telangiectasia ataxia), cerebellar comotor disorders, Fleetrich ataxia, Machado-Joseph disease Cerebellar diseases such as olive bridge cerebellar atrophy), cerebellar tumors such as sub-tentacle tumors, panaxonal encephalitis (encephalitis), etc. Sexual cerebral sclerosis, globoid cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis, and the like.

  Further neurological diseases that can be treated or detected using the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include cerebrovascular disorders (carotid artery thrombosis, carotid stenosis and Carotid artery disease including moyamoya disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, cerebral thrombosis and cerebral thrombosis (carotid artery thrombosis, vein) Sinus thrombosis and Wallenberg syndrome), cerebral hemorrhage (eg epidural hematoma, subdural hematoma and subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (transient cerebral ischemia, subclavian artery steel syndrome and vertebral brain) Vascular dementia such as multiple infarct dementia, periventricular leukomalacia, vascular headache (such as cluster headache and migraine).

  Further neurological diseases that can be treated or detected using the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include dementia such as AIDS dementia syndrome, Alzheimer's disease and Creutzfeldt.・ Presenile dementia such as Jacob disease, senile dementia such as Alzheimer's disease and progressive supranuclear palsy, vascular dementia such as multiple infarct dementia, encephalitis (periaxial axonitis, epidemic encephalitis, Japan) Including encephalitis, St. Louis encephalitis, viral encephalitis such as tick-borne encephalitis and West Nile fever), acute disseminated encephalomyelitis, uveal meningitis syndrome, post-encephalitis Parkinson's disease and subacute sclerosing panencephalitis Membranous encephalitis, cerebral softening such as periventricular leukomalacia, general epilepsy (including infantile seizures, absence seizures, MERRF syndrome) (Including myoclonic epilepsy, tonic-clonic seizures), complex partial epilepsy, partial epilepsy such as frontal and temporal lobe epilepsy, post-traumatic epilepsy, epilepsy such as status epilepticus such as persistent partial epilepsy, and hallelfor Den-Spatz syndrome.

  Additional neurological diseases that can be treated or detected using the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include hydrocephalus such as Dandy-Walker syndrome and normal pressure hydrocephalus Hypothalamus, including hypothalamic tumors, brain malaria, sleep seizures including weakness seizures, medullary leukomyelitis, pseudobrain tumors, Rett syndrome, Reye syndrome, thalamic diseases, brain toxoplasmosis, intracranial tuberculosis and Zelveger syndrome Central nervous system infections such as AIDS dementia syndrome, brain abscess, subdural abscess, equine encephalomyelitis, Venezuelan equine encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, encephalomyelitis such as visna and cerebral malaria Is mentioned.

  Further neurological diseases that can be treated or detected using the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include meningitis such as arachnoiditis, lymphocytic choroid Bacterial meningitis, including aseptic meningitis, such as viral meningitis, including meningitis, hemofirsus meningitis), Listeria meningitis, meningococcal meningitis, (Waterhouse Friedrichsen syndrome, pneumococcal meningitis and tuberculous meningitis), cryptococcal meningitis, fungal meningitis such as subdural exudate, meningoencephalitis such as uveal meningitis syndrome, transverse Myelitis such as myelitis, neurosyphilis such as spinal cord fistula, acute gray leukomyelitis including poliomyelitis and post-polio syndrome, prion disease (Kreuzfeld-Jakob disease, bovine spongiform encephalopathy, Rusutoman-Straussler-Scheinker syndrome, rickets, such as scrapie), and cerebral toxoplasmosis, and the like.

  Further neurological diseases that can be treated or detected using the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include brain tumors including cerebellar tumors such as sub-tent tumors, choroid plexus Tumors, ventricular tumors such as hypothalamic and supratent tumors, neoplasia of the meninges, spinal cord neoplasms including epidural neoplasms, demyelinating diseases such as canavan disease, Generalized sclerosis including adrenoleukodystrophy, perixonal encephalitis, spherical cell leukotrophy, metachromatic leukotrophy, allergic meningitis, necrotizing hemorrhagic Encephalomyelitis, Progressive multifocal leukoencephalopathy, Multiple sclerosis, Hashi center myelin lysis, Transverse myelitis, Optic myelitis, Scrapie, Spondylolisthesis, Chronic fatigue syndrome, Bisuna, High-pressure nerve syndrome Spinal cord neoplasms such as meningopathy, congenital myasthenia, spinal cord disorders such as amyotrophic lateral sclerosis, spinal muscular atrophy such as Weldnig-Hoffmann disease, spinal cord compression, epidural neoplasm , Syringomyelia, spinal cord fistula, stiff man syndrome, mental retardation such as Angelman syndrome, catless syndrome, Delang syndrome, Down syndrome, gangliosidosis such as GM1 gangliosidosis, Sandhoff disease, Tay-Sachs disease, heartnap Diseases, homocystinuria, Lawrence Moon Bardhi-Bedre syndrome, Lesch-Nyhan syndrome, maple syrup syndrome, fucosidosis and other mucolipidosis, neuronal ceroid lipofuscinosis, ocular brain kidney syndrome, maternal phenylketonuria and other phenyl ketones Urinary disease, Prader-Willi syndrome, Rett syndrome, ruby Stein-Teiby syndrome, tuberous sclerosis, WAGR syndrome, neurological abnormalities such as total forebrain, anencephaly including hydroencephalopathy, Arnold Chiari malformation, cerebral hernia, meningocele, meningocele, cystic spine And neural tube defects such as vertebral canal fusion abnormalities such as latent spina bifida.

  Further neurological diseases that can be treated or detected using the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include hereditary Charcot-Marie disease, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia, motor sensory neuropathy including Weldnig-Hoffmann disease, inherited sensory autonomic neuropathy such as congenital analgesia and familial autonomic ataxia, and agnosia including Gerstman syndrome Neurological symptoms, amnesia such as retrograde amnesia, apraxia, neurogenic bladder, weakness, hearing loss, partial hearing loss, hearing loss including loudness and tinnitus, aphasia, aphasia Language disorders including amnesic aphasia, Broka aphasia and Wernicke aphasia, acquired dyslexia, developmental language disorders Speech disorders such as speech impairment including dyslexia, amnesic aphasia, aphasia including Broka aphasia and Wernicke aphasia, communication disorders such as dysarthria, dysarthria, reverberant language, speechlessness, and stuttering, aphasia and hoarseness Disability, Angelman Syndrome, Ataxia, Athetosis, Chorea, Ataxia, Hypofunction, Motor Distress, Myoclonus, Chick, Torticollis, and Tremor, Muscles such as Stiffman Syndrome, Muscle Spasticity Muscular hypertension such as stiffness of the face, facial palsy including ear shingles, stomach palsy, hemiplegia, double vision, Duane syndrome, eye muscle palsy such as Homer syndrome, chronic progressive extraocular muscular palsy such as Keynes syndrome, Pairs such as medullary palsy, tropical spastic paraparesis, Brown-Secart syndrome, limb paralysis, respiratory paralysis, and vocal cord paralysis Numbness, paralysis, paralysis such as phantom limbs, taste disorders such as taste dysfunction and taste dysfunction, visual acuity such as amblyopia, blindness, color blindness, double vision, half-blindness, dark spots, and low vision, Klein-Levin syndrome Sleep disorders such as hypersomnia including sleep insomnia and sleepwalking, convulsions such as opening disorders, coma, prolonged vegetative state, syncope and unconsciousness such as dizziness, congenital myotonia, amyotrophic lateral sclerosis , Lambert-Eaton myasthenia syndrome, motor neuron disease, spinal muscular atrophy, muscular atrophy such as Charcot-Marie disease and Weldnig-Hoffmann disease, post-polio syndrome, muscular dystrophy, myasthenia gravis, atrophic myotonia , Such as myotonics, nemarine myopathy, familial periodic paralysis, multiple paramyoclonus, tropical spastic paraparesis, and stiff man syndrome Autonomic nerve diseases such as muscle disease, extremity pain, neuropathic amyloidosis, Addie syndrome, Barre-Leu syndrome, familial autonomic ataxia, Horner syndrome, reflex sympathetic dystrophy syndrome, and Shy-Drager syndrome Peripheral neurological diseases, acoustic neuropathies such as acoustic neuroma including neurofibromatosis type II, facial neurological diseases such as facial neuralgia, Merkelson-Rozental syndrome, amblyopia, nystagmus, oculomotor disorders including oculomotor paralysis, Duane Syndrome, Homer Syndrome, Kenes Syndrome including Chronic Progressive Extraocular Paralysis, Esotropia, Esotropia and Esotropia, Oculomotor Palsy, Optic Atrophy including Hereditary Optic Atrophy, Optic Nerve Drusen, Optic Nerve Optic neuritis such as myelitis, papilledema, trigeminal neuralgia, vocal cord paralysis, optic neuromyelitis and spinal curvature Optic nerve diseases such as demyelinating diseases, diabetic neuropathy, such as brain diseases, and diabetes foot, such as.

Further neurological diseases that can be treated or detected using the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention include nerve compression syndromes such as carpal tunnel syndrome, and tarsal canal Syndrome, thoracic outlet syndrome such as cervical syndrome, ulnar nerve compression syndrome, burning pain, cervical arm neuralgia, facial neuralgia, and trigeminal neuralgia, experimental allergic neuritis, optic neuritis, polyneuritis, multiple occurrences Hereditary radiculitis, neuritis such as radiculitis, such as polyradiculitis, Charcot-Marie disease, hereditary optic atrophy, refsum disease, hereditary spastic paraplegia, and Weldnih Hoffmann disease Motor sensory neuropathy, congenital analgesia and familial autonomic ataxia, Poems syndrome, sciatica, taste sweating, and Hereditary sensory autonomic neuropathy, including tetany, and the like.

Endocrine disorders

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat, prevent or prevent disorders and / or diseases related to hormonal imbalance and / or disorders or diseases of the endocrine system, It can be used for diagnosis and / or prognosis determination.

  Hormones secreted from the endocrine glands regulate physical development, sexual function, metabolism, and other functions. Disorders can be divided into two categories: hormonal production disorders and the inability of tissues to respond to hormones. The causes of these hormonal imbalances or endocrine diseases, disorders, or conditions are hereditary, somatic (such as cancer and some autoimmune diseases are acquired (eg, chemotherapy, trauma or toxins), Or it can be contagious. Furthermore, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention may be used as labels or detectors for specific diseases or disorders associated with endocrine systems and / or hormonal imbalances. it can.

  Endocrine and / or hormonal imbalances, including uterine motility disorders, include pregnancy and labor complications (eg, premature birth, late pregnancy, spontaneous miscarriage, delayed or stopped labor, and menstrual cycle Disorders and / or diseases such as, but not limited to, dysmenorrhea and endometriosis.

  Examples of disorders and / or diseases of endocrine system and / or hormonal imbalance include disorders and / or diseases of pancreas such as diabetes mellitus, diabetes insipidus, congenital pancreatic insufficiency, pheochromocytoma / islet cell tumor syndrome, For example, Addison's disease, corticosteroid deficiency, masculinized disease, hirsutism, Cushing syndrome, hyperaldosteronism, adrenal gland disorders and / or diseases such as pheochromocytoma, such as hyperpituitarism, hypopituitarism, Pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, pituitary disorders and / or diseases such as giantism, hyperthyroidism, hypothyroidism, plumer disease, Graves disease (Toxic diffuse goiter), toxic nodular goiter, thyroiditis (Hashimoto thyroiditis, subacute granulomatous thyroiditis and indolent lymphocytic thyroiditis) Pendred syndrome, myxedema, cretinism, hyperthyroidism, impaired thyroid hormone binding, thymic dysplasia, thyroid Hurtle cell tumor, thyroid malignant tumor, thyroid cancer, medullary thyroid cancer, Non-limiting examples include thyroid disorders and / or diseases, such as parathyroid disorders and / or diseases such as hyperparathyroidism, hypoparathyroidism, hypothalamic disorders and / or diseases.

  Furthermore, endocrine and / or hormonal imbalance disorders and / or diseases may include testicular or ovarian disorders and / or diseases including cancer. Other testicular or ovarian disorders and / or diseases further include, for example, ovarian cancer, polycystic ovary syndrome, Kleinfelter syndrome, disappeared testicular syndrome (bilateral adolescent), congenital Leydig cell deficiency, testicular retention, Noonan syndrome, myotonic dystrophy, testicular capillary hemangioma (benign), testicular neoplasm, and new testicle.

  Further, endocrine and / or hormonal imbalance disorders and / or diseases include, for example, polyendocrine hypofunction syndrome, pheochromocytoma, neuroblastoma, multiple endocrine adenoma, and endocrine tissue disorders and / or Can be mentioned.

In another embodiment, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is expressed in a tissue expressing a therapeutic protein corresponding to the therapeutic protein of the albumin protein of the present invention. It can be used for diagnosis, prognosis, prevention, and / or treatment of related endocrine diseases and / or disorders.

Genital disorders

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for diagnosis, treatment or prevention of diseases and / or disorders of the reproductive system. Genital disorders that can be treated by the compositions of the present invention include genital damage, infections, neoplastic diseases, birth defects, and diseases or diseases that result in complications of infertility, pregnancy, labor, or childbirth. Disability and postpartum problems include, but are not limited to.

  Reproductive system disorders and / or diseases include testicular atrophy, testicular feminization, testicular retention (unilateral and bilateral), testicular disease, ectopic testes, epididymis and testicularitis (generally, Eg due to infections such as gonorrhea, mumps, tuberculosis and syphilis), testicular torsion, nodular vasculitis, germ cell tumors (eg seminoma, fetal cancer, teratocarcinoma, villi Tumors, yolk sac tumors, and teratomas), stromal tumors (eg Leydig cell tumors), testicular aneurysms, blood masses, varicocele, semen aneurysms, inguinal hernias and sperm proliferative disorders (eg ciliary immobility syndrome, asperm) Testicular diseases and / or disorders, including erythematosus, asthenozoospermia, azoospermia, spermatopenia, and teratospermia).

  Genital system disorders also include acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatic myxia, chronic prostatitis-like syndrome, granulomatous prostatitis Also included are disorders of the prostate, such as marakoplasty, benign prostatic hyperplasia or benign prostatic hyperplasia, and prostate neoplastic diseases including adenocarcinoma, transitional cell carcinoma, ductal carcinoma and squamous cell carcinoma.

  Furthermore, the composition of the present invention comprises glans foreskinitis, obstructive dry balditis, phimosis, incarcerated phimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydial infection, mycoplasma, trichomonas, HIV, Inflammatory disorders such as ADDS, Reiter's Syndrome, Acupuncture Condyloma, Flat Condyloma, and Nacreous Penile Papules, Urethral abnormalities such as hypospadias, urethral fissures, and uncut pallets, Kelar Red Thickness, Bowen's disease, Bowen-like Penile cancer, penis, including papulosis, bushyke-Levenstein giant condyloma and precancerous lesions including wart cancer, squamous cell carcinoma, intraepithelial cancer, wart cancer, and disseminated penile cancer Tumor diseases of the urethra, including urethral cancer, urethral carcinoma, and prostate urethral cancer, and persistent erectile dysfunction, Peyronie's disease, erectile dysfunction and impotence Including erectile disorders, such as scan, diagnose penis and urethra of the disorder or disease, it can be used for treatment and / or prophylaxis.

  Furthermore, diseases and / or disorders of the vas deferens include vasculitis, CBAVD (congenital bilateral defect of the vas deferens). Furthermore, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is a seminal vesicle disease and / or disorder comprising cysticosis, congenital crawl diarrhea, and polycystic kidney disease. Can be used for diagnosis, treatment, and / or prevention.

  Other male reproductive system disorders and / or diseases include, for example, Klinefelter syndrome, Young's syndrome, premature semen, diabetes mellitus, cystic fibrosis, Carl Tajuner syndrome, high fever, multiple sclerosis, and gynecomastia It is done.

  Further, the polynucleotide of the present invention, the fusion protein, and / or the polynucleotide encoding the albumin fusion protein of the present invention are fungal vaginitis, Candida vaginitis, herpes simplex virus, flexible lower arm, inguinal granuloma, inguinal lymph Granulomatosis, scabies, human papilloma virus, vaginal trauma, vulva hemorrhage, glandular disease, chlamydial vaginitis, gonorrhea, trichomonia vaginitis, warts, syphilis, infectious molluscum, atrophic vaginitis, paget Disease, sclerotic lichen, lichen planus, vulvar pain, toxic shock syndrome, vaginal spasticity, vulvovaginitis, vestibular inflammation, and squamous hyperplasia, clear cell carcinoma, basal cell carcinoma, black May be used for diagnosis, treatment, and / or prevention of vaginal and vulvar diseases and / or disorders, including neoplastic diseases such as tumors, bartholin adenocarcinoma, and vulvar intraepithelial neoplasia Kill.

  Uterine disorders and / or diseases include dysmenorrhea, retrograde uterus, endometriosis, uterine fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing syndrome, hydatidiform mole, Usherman syndrome, premature Menopause, sexual prematurity, uterine polyps, malformed uterine bleeding (eg, due to abnormal hormonal signals) and neoplastic diseases such as adenocarcinoma, keiomyosarcoma and sarcoma. Furthermore, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is a divertic uterus, septum uterus, simple unilateral uterus, unilateral uterus having non-cavity trace horn, Diagnosis, treatment, and / or prevention of congenital uterine abnormalities, such as a single uterus having a communicating hollow trace angle, a single uterus having a communicating hollow trace angle, an arcuate uterus, a fully overlapping uterus and a T-shaped uterus Can also be used as a label or detector.

  Ovarian diseases and / or disorders include anovulation, polycystic ovary syndrome (Stein-Lebental syndrome), ovarian cyst, hypoovarian function, ovarian gonadotropin insensitivity, ovarian androgen overproduction, right ovary Venous syndrome, amenorrhea, hirsutism, and ovarian cancer (primary and secondary cancerous growth, Sertoli-Leydig tumor, ovarian endometrioid cancer, ovarian serous papillary adenocarcinoma, ovarian mucous gland And ovarian Krukenbergoma, including but not limited to).

  Cervical diseases and / or disorders include cervicitis, chronic cervicitis, mucinous purulent cervicitis, cervical dysplasia, cervical polyp, nabot cyst, cervical erosion, cervical incompetence, and Cervical neoplasms (including, for example, cervical cancer, squamous metaplasia, squamous cell carcinoma, adenosquamous carcinoma cell carcinoma, and columnar cell tumor).

  In addition, reproductive system diseases and / or disorders may include early abortion, late miscarriage, spontaneous miscarriage, artificial miscarriage, therapeutic miscarriage, imminent miscarriage, miscarriage miscarriage, incomplete miscarriage, complete miscarriage, habitual miscarriage, miscarriage miscarriage, and infection Miscarriage and stillbirth such as miscarriage, ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, hyperamniotic fluid, HELLP syndrome, early placenta detachment, preplacental place, pregnancy prevention Pregnancy disorders and / or diseases, including preeclampsia, eclampsia, gestational herpes, and urticaria of pregnancy. Furthermore, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may be used for heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, hypertension, anemia. , Kidney disease, infection (eg rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HTV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis Chronic active hepatitis, cirrhosis, primary biliary cirrhosis, asthma, systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendix, ovarian cyst, gallbladder disease, and intestinal obstruction, It can be used for diagnosis, treatment, and / or prevention of diseases that can make pregnancy difficult.

  Complications related to pregnancy and labor include premature water breaks, premature birth, late pregnancy, premature pregnancy, extremely slow labor, fetal distress (eg, abnormal heart rate (fetus or mother), respiratory problems, and abnormalities Placenta), shoulder dystocia, umbilical cord prolapse, amniotic fluid embolism, and abnormal uterine bleeding. Is mentioned.

  In addition, postpartum diseases and / or disorders include endometritis, myometrium, parauterine connective tissueitis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, Saphenous vein thrombosis, mastitis, cystitis, postpartum hemorrhage, and intrauterine varus.

Other disorders and / or diseases of the female reproductive system that can be diagnosed, treated, and / or prevented by albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention include, for example, Examples include Turner Syndrome, Pseudo-Hemi-Yang, Premenstrual Syndrome, Pelvic Infection, Pelvic Congestion (Vascular Dilation), Insensitivity, Anorgasmia, Sexual Pain, Tubal Rupture, and Menstrual Pain.

Infection

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment or detection of pathogenic bacteria. For example, an infection can be treated by an increased immune response, particularly an increase in B cell and / or T cell proliferation and differentiation. The immune response can be increased by increasing an existing immune response or by initiating a new immune response. Alternatively, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can directly suppress pathogenic bacteria without necessarily inducing an immune response.

  A virus is an example of a pathogen that can cause a disease or condition that can be treated or detected by an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention. Examples of viruses include, but are not limited to, the following DNA viruses and RNA viruses and viridae. Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Calciviridae, Sarcoviridae, Coronaviridae, Dengue virus, EBV, HTV, Flaviviridae, Hepadnavirus Family (hepatitis), herpesviridae (cytomegalovirus, herpes simplex, herpes zoster, etc.), mononegavirus (eg, paramyxoviridae, measles virus, rhabdoviridae), orthomyxoviridae (eg, influenza virus) A, influenza virus B, and parainfluenza), papilloma virus, papoviridae, parvoviridae, picornaviridae, poxviridae (such as smallpox or cowpox), reoviridae (eg, rotau) Luz), the retrovirus family (HTLV-L, HTLV-H, lentivirus), and Togaviridae (for example ruby-genus). Various diseases or symptoms that can be caused by viruses entering these families include, but are not limited to: Arthritis, bronchiolitis, respiratory syncytial virus, encephalitis, eye infection (eg, conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, chronic, acute, delta), Japanese encephalitis B , Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (eg AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, cold, acute gray Myelitis (polio), leukemia, rubella, sexually transmitted diseases, skin diseases (eg Kaposi, warts), and viremia. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat or detect any of these symptoms or diseases. In certain embodiments, the fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention treat meningitis, dengue virus, EBV, and / or hepatitis (eg, hepatitis B). Used to do. In a further specific embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is used to treat patients who are unresponsive to one or more other commercially available hepatitis vaccines. Can be used to treat. In a further specific embodiment, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can be used to treat AIDS.

  Similarly, bacterial and fungal pathogens that can cause a disease or condition that can be treated or detected by the albumin fusion protein of the invention and / or a polynucleotide encoding the albumin fusion protein of the invention include the following Gram-negative bacteria and Examples include, but are not limited to, gram positive bacteria, bacteriophages, and fungi. Actinomyces (eg, Nocardia), Acinetobacter, Cryptococcus neoformans, Aspergillus, Bacillus (eg, Bacillus anthracis), Bacteroides (eg, Bacteroides fragilis), Blastomices, Bordetella, Borrelia, Borrelia burgdorferi), Brucella, Candida, Campylobacter, Chlamydia, Clostridium (e.g. Clostridium botulinum, Clostridium perfingens, Clostridium perbium, Clostridium cobne, Bacteria) dipteriae), cryptococcus, dermatomycosis, E. coli (eg, enterotoxigenic and enterohemorrhagic E. coli), enterobacter (eg, Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella ( For example, Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serasia, Yersinia, Shigella, Eridiperosrix, Haemophilus (eg, Haemophilus influenzae B), Helicobacter, Legionella ph. , Leptospira, Listeria monocytogenes (eg Listeria monocytogenes), Mycoplasma, The genus Icobacterium (for example, Mycobacterium leprae and Mycobacterium tuberculosis), the genus Vibrio (for example, Vibrio cholerae), the Neisseria family (for example, Neisseria gonorthea, Neisseria meridae) Rickettsiaceae, spirochetes (eg, Treponema spp., Leptospira spp., Borrelia spp.), Shigella species, staphylococci (eg, Staphylococcus aureus), meningococcus, pneumococci and streptococci (eg, Streptococcus) s Peneumonia) and group A, group B, and group C streptococci), and urea plasma. Diseases or conditions that can be caused by these bacterial, parasitic, and fungal groups include, but are not limited to: Antibiotic resistant infection, bacteremia, endocarditis, sepsis, eye infection (eg conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea, opportunistic infection (eg AIDS related infection) ), Peritonitis, prosthesis-related infections, caries, Reiter's disease, respiratory infections such as whooping cough or empyema, sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid fever, food poisoning, legionellosis, chronic and Acute inflammation, erythema, yeast infection, typhoid, pneumonia, gonorrhea, meningitis (eg, type A and B meningitis), chlamydial infection, syphilis, diphtheria, leprosy, brucellosis, peptic ulcer, anthrax Abnormalities of spontaneous production, pneumonia, lung infection, ear infection, hearing loss, blindness, coma, discomfort, vomiting, chronic diarrhea, Crohn's disease, colitis, bacterial phlebitis, infertility, pelvic infection, candidiasis, paratuberculosis , Yui , Lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted disease, skin diseases (eg cellulitis, dermatomycosis), toxemia, urinary tract infection, wound wound infection, nosocomial infection. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to treat or detect any of these symptoms or diseases. In certain embodiments, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is for treating tetanus, diphtheria, botulism, and / or type B meningitis. used.

  Furthermore, the parasitic pathogen (its infectious disease) that causes a disease or symptom that can be treated, prevented, and / or diagnosed by the fusion protein of the present invention and / or a polynucleotide encoding the albumin fusion protein of the present invention includes the following: But are not limited to these. Amebiasis, babesiosis, coccidiosis, cryptosporidiosis, binuclear amebiasis, gonorrhea, ectoparasite infection, rumble flagellate disease, helminthiasis, leishmaniasis, schistosomiasis, tyrelosis, toxoplasmosis, Trypanosomiasis, and Trichomonas and spores (eg, Plasmodium virax, Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale). These parasites can cause, but are not limited to, various diseases or symptoms described below. Scabies, tsutsugamushi disease, eye infections, gastrointestinal diseases (eg, dysentery, rumble flagellate disease), liver disease, lung disease, opportunistic infections (eg AIDS related), malaria, pregnancy complications, and toxoplasmosis. The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used for the treatment, prevention, and / or diagnosis of these symptoms or diseases. In certain embodiments, the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention are used for the treatment, prevention, and / or diagnosis of malaria.

For the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention, a method of administering an effective amount of the albumin fusion protein of the present invention to a patient, or removing a cell from the patient and It is possible to adopt a method of supplying the polynucleotide of the present invention to the modified cell and returning the modified cell to the patient (ex vivo treatment). Furthermore, the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used as an antigen in a vaccine for eliciting an immune response against infection.
Regeneration

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used to differentiate, proliferate and attract cells to induce tissue regeneration (Science 276: 59-87 (1997)). Tissue regeneration may include birth defects, trauma (wounds, burns, incisions, or ulcers), aging, disease (eg, osteoporosis, osteoarthritis, periodontal disease, liver failure), surgery including cosmetic surgery Can be used to repair, replace, or protect tissue damage due to fibrosis, reperfusion injury, or systemic cytokine damage.

  Tissues that can be regenerated using the present invention include organs (eg, pancreas, liver, gastrointestinal tract, kidney, skin, endothelium), muscles (smooth muscle, skeletal muscle, or myocardium), vasculature (blood vessels and lymph vessels). ), Nerves, hematopoietic system, and skeletal (bone, cartilage, tendon, and ligament) tissues. Regeneration preferably occurs without scarring or with reduced scarring. Regeneration may also include angiogenesis.

    Furthermore, the fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may increase the regeneration of tissues that are difficult to heal. For example, increased tendon / ligament regeneration may accelerate recovery time after injury. The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention can also be used prophylactically in an attempt to avoid damage. Specific diseases that can be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament abnormalities. Additional examples of tissue regeneration of wounds that do not heal include pressure ulcers and ulcers associated with vascular failure, surgical damage, and trauma.

Similarly, nerve and brain tissue can also be regenerated by using the fusion proteins of the invention and / or polynucleotides encoding the albumin fusion proteins of the invention to differentiate and proliferate neurons. Diseases that can be treated using this method include central and peripheral neurological diseases, neurological disorders, or mechanical and traumatic disorders (eg spinal cord injury, head trauma, cerebrovascular disease, and stroke). Is mentioned. In particular, diseases related to peripheral nerve injury, peripheral neuropathy (eg caused by chemotherapy or other medical treatment), local neuropathy, and central nervous system diseases (eg Alzheimer's disease, Parkinson's disease, Huntington) Disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome) can all be treated using an albumin fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention.

Gastrointestinal disorders

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may be used to treat inflammatory diseases and / or conditions, infectious diseases, cancer (eg, intestinal tumor (small intestine carcinoid tumor, small intestine It can be used to treat, prevent, diagnose, and / or prognose gastrointestinal diseases, including ulcers such as non-Hodgkin lymphoma, small intestinal lymphoma), and peptic ulcers.

  Gastrointestinal disorders include dysphagia, swallowing pain, esophageal inflammation, peptic esophagitis, gastric reflux, submucosal fibrosis and benign stenosis, Mallory-Weiss lesions, leiomyoma, lipoma, epidermoid cancer, adenocarcinoma, Gastric retention disorder gastroenteritis, stomach atrophy, gastric cancer, gastric polyp, malignant anemia, autoimmune disorders, pyloric stenosis, gastritis (bacterial gastritis, viral gastritis, eosinophilic gastritis, stress-induced gastritis, chronic Erosive, atrophic, plasma cells, and giant hypertrophic gastritis (Menetrie's disease)), and peritoneal diseases (eg, chypoperitonitis, intraperitoneal hemorrhage, mesenteric cysts, mesenteric lymphadenitis, mesentery) Vaso-occlusion, panniculitis, neoplasm, peritonitis, pneumoperitoneum, subphrenic abscess).

  Gastrointestinal disorders also include malabsorption syndrome, swelling, irritable bowel syndrome, glucose intolerance, celiac disease, duodenal ulcer, duodenal inflammation, tropical sprue, Whipple disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, times Intestinal obstruction, Meckel's diverticulosis, multiple diverticula, failure of complete circulation of the small and large intestines, lymphoma, and fungal and parasitic diseases (eg, traveler's diarrhea, typhoid and paratyphoid fever, cholera, roundworm (Ascariasis) liimbricoides), worms (Ancylostoma dudenale), nematodes (Enterobius vermicularis), tapeworms (Taenia saginata, Echinococcus granulosus, Diphylumbosp. ium) infection with, and also the small intestine diseases associated with such.

  Liver diseases and / or disorders include intrahepatic cholestasis (Alagille syndrome, biliary cirrhosis), fatty liver (alcoholic fatty liver, Reye syndrome), hepatic vein thrombosis, hepatic lens nuclear degeneration, hepatomegaly , Hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), cirrhosis (alcoholic cirrhosis, biliary cirrhosis, and experimental cirrhosis), alcoholic liver Diseases (fatty liver, hepatitis, cirrhosis), parasitic diseases (hepatic cystosis, cirrhosis, amebic liver abscess), jaundice (hemolytic jaundice, hepatocellular jaundice, and cholestatic jaundice), cholestasis Stagnation, portal hypertension, hepatomegaly, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune hepatitis, hepatitis B, hepatitis C, hepatitis D, drug-induced hepatitis), Toxic hepatitis, human viral hepatitis (type A liver , Hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varicose veins, hepatic encephalopathy, Primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangioma, gallstones, liver failure (hepatic encephalopathy, acute liver failure), and liver neoplasm [(with angiomyolipoma, calcification Liver metastasis, cystic liver metastasis, epithelial tumor, fibrosis hepatocellular carcinoma, localized nodular hyperplasia, liver adenoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, Liver cancer, hepatic hemangioendothelioma, mesenchymal hamartoma, liver mesenchymal tumor, nodular regenerative hyperplasia, benign liver tumor {hepatic cyst (simple cyst, polycystic liver disease, hepatobiliary cystadenoma) , Common bile duct cyst), mesenchymal tumor (mesenchymal hamartoma, childhood hemangioendothelioma, hemangioma, hepatic purpura, lipoma Inflammatory pseudotumor, mixed), epithelial tumor {bile duct epithelium (bile duct hamartoma, ductal adenoma), hepatocytes (adenoma, localized nodular hyperplasia, nodular regenerative hyperplasia)}}, malignant liver tumor ( Hepatocellular carcinoma, hepatoblastoma, hepatocellular carcinoma, cholangiocarcinoma, cholangiocarcinoma, cystadenocarcinoma, vascular tumor, angiosarcoma, Kaposi sarcoma, angiosarcoma, other tumors, fetal sarcoma, fibrosarcoma Leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous cell carcinoma, primary lymphoma)], hepatic purpura, hematopoietic polyphyllosis, hepatic polyphyllosis (acute intermittent porphyrin) Symptom, late cutaneous porphyria), Zellweger syndrome.

  Pancreatic diseases and / or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (pancreatic adenocarcinoma, cystadenocarcinoma, islet cell tumor, gastrinoma, and glucagon-producing tumor, Cystic neoplasms, islet cell tumors, pancreatoblastomas, and other pancreatic diseases (eg, cystic fibrosis, cysts (pancreatic pseudocyst, pancreatic fistula, dysfunction)).

  Gallbladder diseases include gallstones (cholelithiasis and common bile duct stones), post cholecystectomy syndrome, gallbladder diverticulosis, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucous cysts.

  Colonic diseases and / or disorders include antibiotic-induced colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal diseases (eg, fissure, Hemorrhoids), colon disease [colitis, colon neoplasia {colon cancer, adenomatous colon polyp (eg villous adenoma), colon cancer, colorectal cancer}, colon diverticulitis, colon diverticulosis, giant colon (Hirsch spring) Disease, toxic megacolon), sigmoid colon disease (rectocolitis, neoplasia of sigmoid colon)], constipation, Crohn's disease, diarrhea (infant diarrhea, dysentery), duodenal disease (duodenal tumor, duodenal obstruction, duodenum) Ulcer, duodenalitis), enteritis (total enteritis), HIV-related bowel disease, ileal disease (ileal tumor, ileitis), immunoproliferative small bowel disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal closure, Parasitic disease (Anisaki , Barantidium disease, Blast cystis infection, Cryptosporidium disease, Binuclear amebiasis, Amoeba dysentery, Rumble flagellate disease, Intestinal fistula (rectal fistula), Intestinal tumor (cecal neoplasm, colon neoplasm, duodenal tumor) Ileal tumor, intestinal polyp, jejunal neoplasm, rectal neoplasm), intestinal obstruction (imported leg syndrome, duodenal obstruction, impacted feces, intestinal pseudoobstruction (cecal volvulus), intussusception), intestinal perforation, intestinal polyp (colon) Polyp, Gardner syndrome, Poitz-Jegers syndrome), jejunal disease (neoplasm of the jejunum), malabsorption syndrome (blind snare syndrome, celiac disease, lactose intolerance, short bowel syndrome, tropical sprue, whipple disease), Mesenteric vascular occlusion, intestinal wall cystoid emphysema, protein-losing bowel disease (intestinal lymphangiectasia), rectal disease (anal disease, fecal incontinence, hemorrhoids, proctitis, rectal fistula, rectal prolapse, rectal herni ), Peptic ulcer (duodenal ulcer, peptic esophagitis, bleeding, perforation, gastric ulcer, Zollinger-Ellison syndrome), post-gastrectomy syndrome (dumping syndrome), gastric disease (eg, hydrochloric acid deficiency, duodenal gastric reflux (bile reflux) Gastric vestibular telangiectasia, gastric fistula, gastric opening obstruction, gastritis (atrophic or hypertrophic gastritis), gastric paralysis, gastric dilatation, gastric diverticulum, gastric neoplasm (gastric cancer, gastric polyp, gastric gland) Cancer, hyperplastic gastric polyps), gastric rupture, gastric ulcer, gastric volvulus), tuberculosis, visceral drooping, vomiting (eg, vomiting, nausea during pregnancy, postoperative nausea and vomiting), and hemorrhagic colitis It is done.

Further gastrointestinal diseases and / or disorders include gastric wall rupture, sputum (eg, biliary fistula, esophageal fistula, gastrostoma, intestinal fistula, pancreatic fistula), neoplasm (eg, bile duct neoplasia, esophageal gland) Cancer, esophageal tumors such as squamous cell carcinoma of the esophagus, gastrointestinal tumors, pancreatic adenocarcinoma, pancreatic mucinous cystic tumors, pancreatic tumors such as pancreatic cystic tumors, pancreatic blastomas, and peritoneal tumors), esophageal diseases (eg , Bullous disease, candidiasis, glycogenic epidermis thickening, ulceration, Barrett's esophageal varices, atresia, cyst, diverticulum (eg, Zenker's diverticulum), sputum (eg, tracheoesophageal fistula), gastrointestinal motility abnormalities (eg, , Crest syndrome, dysphagia, achalasia, convulsion, gastroesophageal reflux), neoplasm, perforation (eg, Bourhafe syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (eg, hiatal hernia), And gastroenteritis (eg, cholera disease, norwalk virus infection), bleeding (eg, vomiting, melena, peptic ulcer, bleeding), gastric neoplasm (gastric cancer, gastric polyp, gastric adenocarcinoma, gastric cancer)), hernia Gastrointestinal diseases such as congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, navel hernia, abdominal wall hernia) and bowel disease (eg, caecal disease (appendicitis, cecal neoplasm)).

Chemotaxis

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may have chemotactic activity. Chemotactic molecules are located in specific parts of the body (such as sites of inflammation, infection, or hyperproliferation) at cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, Attracts or mobilizes epithelial cells and / or endothelial cells). The recruited cells can then repel and / or heal certain tumors or abnormalities.

  An albumin fusion protein of the present invention and / or a polynucleotide encoding an albumin fusion protein of the present invention may increase the chemotactic activity of certain cells. As a result, these chemotactic molecules can be used to treat inflammation, infections, hyperproliferative disorders, or any immune system disorder by increasing the number of cells that are directed to specific sites in the body. . For example, chemotactic molecules can be used for the purpose of treating tissue wounds and other trauma by attracting immune cells to the site of injury. The chemotactic molecules of the present invention can also attract fibroblasts that can be used for wound healing.

It is also contemplated that a fusion protein of the invention and / or a polynucleotide encoding an albumin fusion protein of the invention may inhibit chemotactic activity. These molecules can also be used to treat diseases. Therefore, the fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be used as an inhibitor of chemotaxis.

Binding assay

  The albumin fusion proteins of the invention may be used to screen for molecules that bind to the therapeutic protein portion of the fusion protein, or for molecules that bind the therapeutic protein portion of the fusion protein. The binding of the fusion protein and molecule can activate (agonist), increase, inhibit (antagonist) or decrease the activity of the fusion protein or bound molecule. Examples of such molecules include antibodies, oligonucleotides, proteins (eg receptors) or small molecules.

  Preferably, the molecule is closely related to a natural ligand, such as a fragment or ligand, or a natural substrate, ligand, structural or functional mimetic of a therapeutic protein portion of a fusion protein of the invention (Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991)). Similarly, a molecule can be a natural receptor to which a therapeutic protein portion of an albumin fusion protein of the invention binds, or at least one fragment of a receptor that can be bound by a therapeutic protein portion of an albumin fusion protein of the invention (eg, active Closely related). In either case, the molecule can be rationally designed using known techniques.

  Preferably, screening for these molecules includes producing suitable cells that express the albumin fusion protein of the invention. Preferred cells include cells from mammals, yeast, Drosophila or E. coli.

  The assay may simply test the binding of the candidate compound to the albumin fusion protein of the invention, where binding is detected by a label or in an assay that includes competition with a labeled competitor. Furthermore, the assay may test whether the candidate compound results in a signal produced by binding to the fusion protein.

  Alternatively, the assay can be performed using cell-free regulators, fusion proteins / molecules bound to an individual support, chemical libraries or natural product mixtures. The assay also comprises the steps of mixing the candidate compound with a solution containing the albumin fusion protein, measuring the fusion protein / molecular activity or binding, and comparing the fusion protein / molecular activity or binding to a standard. It can simply be included.

  Preferably, an ELISA assay can measure fusion protein levels or activity in a sample (eg, a biological sample) using monoclonal or polyclonal antibodies. The level or activity of the albumin fusion protein can be measured either by binding the antibody directly or indirectly to the albumin fusion protein or by comparing the substrate to the albumin fusion protein.

  Further, receptors to which the therapeutic protein portion of the albumin fusion protein of the present invention binds can be synthesized by, for example, ligand panning and FACS sorting ((Coligan, et al., Current Protocols in Immun., 1 (2), Chapter 5, (1991 For example, if the therapeutic protein portion of the fusion protein corresponds to FGF, expression cloning, polyadenylated RNA, responsive to albumin fusion protein can be identified. CDNA libraries made from this RNA may be prepared and used from cells, eg, NIH3T3 cells known to contain multiple receptors for FGF family proteins, and SC-3 cells. Can be separated into pools and used to transfect COS cells or other cells that are not responsive to the albumin fusion protein.After labeling the transfected cells growing on glass slides, Exposure to an albumin fusion protein of the invention The albumin fusion protein can be labeled by a variety of methods including iodination or introduction of a recognition site for a site-specific protein kinase.

  After immobilization and incubation, slides are subjected to automated radiographic analysis. Positive pools are identified, subpools are prepared, retransfected using repeated subpools and rescreening steps, and finally a single clone encoding the expected receptor is produced.

  As another approach for receptor identification, the labeled albumin fusion protein can be photoaffinity linked to a cell membrane or extract preparation that expresses the receptor molecule for the therapeutic protein component of the albumin fusion protein of the present invention. The material may be analyzed by PAGE analysis and exposed to X-ray film. The labeled complex containing the fusion protein receptor is removed, broken into peptide fragments, and subjected to protein microsequencing. Using a sequence of amino acids obtained from microsequencing, a set of degenerate oligonucleotide probes is designed to select a cDNA library and identify the gene encoding the expected receptor.

  Further, gene-shuffling, motif-shuffling, exon-shuffling, and / or codon-shuffling (synonymously referred to as “DNA shuffling”) techniques for fusion protein activity, and / or albumin fusion protein of the invention May be used to modulate the activity of the therapeutic protein moiety or albumin component, thereby effectively producing agonists and antagonists of the albumin fusion proteins of the present invention. Generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten. , P.M. A. , Et al. Curr. Opinion Biotechnol. 8: 724-33 (1997); Harayama, S .; Trends Biotechnol. 16 (2): 76-82 (1998); Hansson, L .; O. , Et al. , J. et al. Mol. Biol. 287: 265-76 (1999); and Lorenzo, M .; M.M. and Blasco, R.A. See Biotechniques 24 (2): 308-13 (1998). Each of these patents and publications is incorporated by reference. In one embodiment, alteration of the polynucleotide encoding the albumin fusion protein of the invention, and thus alteration of the albumin fusion protein encoded thereby, may be accomplished by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into the desired molecule by homologous or site-specific recombination. In another embodiment, the polynucleotide encoding the albumin fusion protein of the present invention, and thus the albumin fusion protein encoded thereby, is subjected to random mutagenesis, random nucleotide insertion by error-prone PCR before recombination. Or you may change by applying to another method. In other embodiments, one or more components, motifs, segments, portions, domains, fragments, etc. of the albumin fusion proteins of the invention are combined with one or more components of one or more heterologous molecules. , Motifs, segments, parts, domains, fragments and the like. In a preferred embodiment, the heterologous molecule is a family member. In a further preferred embodiment, the heterologous molecule is, for example, platelet derived growth factor (PDGF), insulin-like growth factor (IGF-I), transducing growth factor (TGF) -alpha, epidermal growth factor (EGF), fibroblasts Growth factor (FGF), TGF-beta, Bone morphogenetic protein (BMP) -2, BMP-4, BMP-5, BMP-6, BMP-7, Activin A and B, Decapentapregic (dpp), 60A , OP-2, dorsaline, growth differentiation factor (GDF), nodar, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TFG-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF) ) Is a growth factor.

  Other preferred fragments are biologically active fragments of the therapeutic protein portion and / or albumin component of the albumin fusion proteins of the invention. Biologically active fragments are those that exhibit similar, but not necessarily identical, activity to the therapeutic protein portion and / or albumin component of the albumin fusion protein of the invention. The biological activity of the fragment may include an improved desired activity or a decreased unwanted activity.

Furthermore, the present invention provides methods for screening compounds to identify those that modulate the activity of an albumin fusion protein of the present invention. An example of such an assay is mixing mammalian fibroblasts, the albumin fusion protein of the invention, and the compound to be screened and ³ [H] thymidine under cell culture conditions where fibroblasts normally grow. It is included. A control assay may be performed in the absence of the compound to be screened to determine whether the compound stimulates proliferation by measuring the uptake of ³ [H] thymidine in each case. It may be compared to the amount of fibroblast proliferation in the presence. The amount of fibroblast proliferation is measured by liquid scintillation chromatography, which measures ³ [H] thymidine incorporation. Both agonist and antagonist compounds can be identified by this procedure.

  In other methods, mammalian cells or membrane modulators expressing receptors for the therapeutic protein component of the fusion protein of the invention are incubated with the labeled fusion protein of the invention in the presence of the compound. The ability of the compound to enhance or inhibit this interaction can then be measured. Alternatively, the compound is essentially a fusion protein that measures the response of a known second messenger system following the interaction of the compound to be screened with the receptor, binds the compound to the receptor, and elicits a second messenger response. Measure to determine if is. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion exchange or phosphoinositide hydrolysis.

  All of these above assays can be used as diagnostic or prophylactic markers. Discovered molecules used in these assays are used to treat diseases in patients by activating or inhibiting fusion proteins / molecules or to produce specific results (eg, vascular proliferation) Is possible. Furthermore, assays can discover agents that can inhibit or enhance the production of albumin fusion proteins of the present invention from suitably engineered cells or tissues.

Accordingly, the present invention includes an albumin fusion of the present invention comprising the steps of (a) incubating a candidate binding compound with an albumin fusion protein of the present invention and (b) determining whether binding has occurred. A method of identifying a compound that binds to a protein is included. Further, the present invention provides (a) incubating a candidate compound with an albumin fusion protein of the present invention, and (b) assaying biological activity, and (b) altering the biological activity of the fusion protein. A method of identifying an agonist / antagonist comprising the step of determining whether to.

Targeted delivery

  In another embodiment, the present invention provides a method of delivering a composition to targeted cells expressing a receptor for a component of an albumin fusion protein of the present invention.

  As discussed herein, the fusion protein of the invention binds to a heterologous polypeptide, heterologous nucleic acid, toxin, or prodrug via hydrophilic, hydrophobic, ionic and / or covalent interactions. Also good. In one embodiment, the invention provides a method for the specific delivery of a composition of the invention to a cell by administering a fusion protein of the invention (including an antibody) conjugated to a heterologous polypeptide or nucleic acid. I will provide a. In one example, the present invention provides a method for delivering a therapeutic protein to a target cell. In other examples, the invention relates to single-difference nucleic acids (eg, antisense or ribozymes), or double-stranded nucleic acids (eg, DNA that can be integrated into the genome of a cell, or that can be replicated episomally and transcribed. ) In a targeted cell.

  In another embodiment, the present invention provides for cell-specificity by administering an albumin fusion protein of the invention (eg, a polypeptide of the invention or an antibody of the invention) together with a toxin or cytotoxic prodrug. Provides a method for complete destruction (eg, destruction of tumor cells).

“Toxins” are endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or in the surface of cells that cause cell death under defined conditions Or refers to a compound that binds and activates any molecule or enzyme not normally present above. Toxins that may be used in accordance with the methods of the present invention include, but are not limited to, radioisotopes known in the art, such as antibodies that bind to the native or derived endogenous cytotoxic effector system. (Or its complement immobilization containing portion), thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera Compounds such as toxins are included. “Cytotoxic prodrug” means a non-toxic compound that is converted to a cytotoxic compound by enzymes normally present in the cell. Cytotoxic prodrugs that may be used in accordance with the methods of the present invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agents, etoposide or mitomycin C phosphate derivatives, cytosine arabinoside, daunorubicin, And phenoxyacetamide derivatives of doxorubicin.

Drug screening

  Screen for molecules that alter the activity of the albumin fusion proteins of the invention, or of the polynucleotides encoding these fusion proteins, of the albumin fusion proteins of the invention, or proteins corresponding to therapeutic protein portions of albumin fusion proteins Further utilization is contemplated. Such methods include contacting the fusion protein with a selected compound that is expected to have antagonist or agonist activity, and assaying the activity of the fusion protein after binding.

  The present invention is particularly useful for binding fragments thereof using the albumin fusion proteins of the present invention or in any of a variety of drug screening techniques. The albumin fusion protein used in such tests may be bound to a solid support, expressed on the cell surface, free in solution, or localized within the cell. One method of drug screening utilizes prokaryotic or eukaryotic host cells stably transduced with a recombinant nucleic acid expressing an albumin fusion protein. Drugs are screened against such transduced cells or supernatants obtained from culturing such cells in competitive binding assays. For example, the formation of a complex between the agent being tested and the albumin fusion protein of the invention may be measured.

  Thus, the present invention provides a method of screening for drugs or any other agent that affects the activity mediated by the albumin fusion protein of the present invention. These methods involve contacting the drug with an albumin fusion protein or fragment thereof of the present invention, and of the complex between the drug and albumin fusion protein or fragment thereof by methods well known in the art. Assaying for presence. In such competitive binding assays, the screened agent is typically labeled. After incubation, free drug is separated from that present in bound form, and the amount of free or unconjugated label is a measure of the ability of a particular drug to bind to the albumin fusion protein of the invention.

  Other techniques for drug screening provide high-throughput screening for compounds with effective binding affinity for the albumin fusion proteins of the present invention, 1984, incorporated by reference herein. This is described in greater detail in European Patent Specification No. 84/03564, issued September 13. Briefly, a number of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or other surfaces. The peptide test compound is reacted with the albumin fusion protein of the invention and washed. The bound peptide is then detected by methods known in the art. Purified albumin fusion protein may be coated directly onto the plate for use in the above drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on a solid support.

The present invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the albumin fusion proteins of the present invention specifically compete with test compounds for binding to albumin fusion proteins or fragments thereof. . In this manner, the antibody is used to detect the presence of any peptide that shares one or more antigenic epitopes with the albumin fusion protein of the invention.

Binding peptides and other molecules

  The present invention also contemplates screening methods for identifying polypeptides and non-polypeptides that bind to the albumin fusion proteins of the present invention, and binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the albumin fusion proteins of the present invention. Such agonists and antagonists can be used in accordance with the present invention in the therapeutic embodiments discussed in detail below.

  The method includes contacting the albumin fusion protein of the present invention with a number of molecules and identifying a molecule that binds to the albumin fusion protein.

  The step of contacting the albumin fusion protein of the present invention with a large number of molecules can be carried out in a number of ways. For example, it may be contemplated that the albumin fusion protein is immobilized on a solid support and a solution of multiple molecules is contacted with the immobilized polypeptide. Such a procedure is an affinity matrix consisting of the immobilized albumin fusion protein of the present invention and is similar to the affinity chromatography step. Molecules with selective affinity for albumin fusion proteins can then be purified by affinity sorting. The nature of the solid support, the steps for adhesion of the albumin fusion protein to the solid support, the solvent, and the conditions for affinity isolation or selection have been primarily followed and are well known to those skilled in the art.

  Alternatively, multiple polypeptides may be separated into essentially separate fractions that include a subset of the polypeptides, or individual. For example, multiple polypeptides can be separated by gel electrophoresis, column chromatography, or similar methods known to those skilled in the art for polypeptide separation. Individual polypeptides can be produced by transduced host cells in a manner such that they are expressed on or around their outer surface (eg, recombinant phage). In order to determine if there is a selective affinity interaction between the individual isolates and then between the albumin fusion protein and the individual clones, optionally in the presence of an inducer that must be required for expression, A “probe” is possible with the albumin fusion protein of the present invention. Prior to contacting the albumin fusion protein with each fraction containing the individual polypeptides, the polypeptides can first be transferred to a solid support for further convenience. Such a solid support can simply be part of a filter membrane, such as consisting of nitrocellulose or nylon. In this manner, positive clones can be identified from a population of transduced host cells in an expression library having a DNA construct encoding a polypeptide with selective affinity for an albumin fusion protein of the invention. Furthermore, the amino acid sequence of a polypeptide having selective affinity for the albumin fusion protein of the present invention can be directly determined by conventional methods, and the coding sequence of the DNA encoding the polypeptide is frequently and more convenient. Can be determined. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, microsequencing techniques may be used. Sequencing techniques include mass spectrometry.

  Wash off any unbound polypeptide from the mixture of albumin fusion protein of the invention and a number of polypeptides before attempting to measure or detect the presence of a selective affinity interaction in a particular state It is desirable to do. Such a washing step is particularly desirable when the albumin fusion protein or multiple polypeptides of the present invention are bound to a solid support.

  Numerous molecules provided according to this method can be used in a variety of live, such as random or recombinant peptide or non-peptide libraries that can be screened against molecules that specifically bind to an albumin fusion protein of the invention. May be provided by a rally. Many libraries are known in the art and can be used, such as, for example, chemical synthesis libraries, recombination (eg, phage display libraries) and libraries based on in vitro translation. An example of a chemically synthesized library is described by Fodor et al. , Science 251: 767-773 (1991); Houghten et al. , Nature 354: 84-86 (1991); Lam et al. , Nature 354: 82-84 (1991); Medynski, Bio / Technology 12: 709-710 (1994); Gallop et al. , J. et al. Medicinal Chemistry 37 (9): 1233-1251 (1994); Ohmeyer et al. , Proc. Natl. Acad. Sci. USA 90: 10922-10926 (1993); Erb et al. , Proc. Natl. Acad. Sci. USA 91: 11142-11426 (1994); Houghten et al. , Biotechniques 13: 412 (1992); Jaywickreme et al. , Proc. Natl. Acad. Sci. USA 91: 1614-1618 (1994); Salmon et al. , Proc. Natl. Acad. Sci. USA 90: 11708-11712 (1993); PCT Specification No. WO 93/20242, and Brenner and Lerner, Proc. Natl. Acad. Sci. USA 89: 5381-5383 (1992).

  An example of a phage display library is described in Scott et al. , Science 249: 386-390 (1990); Devlin et al. , Science, 249: 404-406 (1990); Christian et al. , 1992, J. et al. Mol. Biol. 227: 711-718 1992); Lenstra, J. et al. Immunol. Meth. 152: 149-157 (1992); Kay et al. , Gene 128: 59-65 (1993); and PCT Publication No. WO 94/18318, Aug. 18, 1994.

  Libraries based on in vitro translation include, but are not limited to, PCT Specification No. WO 91/05058, August 18, 1991, and Mattheakis et al. , Proc. Natl. Acad. Sci. USA 91: 9022-9026 (1994).

  As an example of a non-peptide library, a benzodiazepine library (see, eg, Bunin et al., Proc. Natl. Acad. Sci. USA 91: 4708-4712 (1994)) can be adapted for use. . A peptoid library (Simon et al., Proc. Natl. Acad. Sci. USA 89: 9367-9371 (1992)) can also be used. Another example of a library that can be used wherein the amide functionality in the peptide is permethylated to form a chemically transduced recombinant library is described by Ostresh et al. (Proc. Natl. Acad. Sci. USA 91: 11138-11142 (1994)).

  Various non-peptide libraries useful in the present invention are excellent. For example, Ecker and Crooke (Bio / Technology 13: 351-360 (1995) describes benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs among the chemical species that form the basis of various libraries. , Beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubans, xanthines, amine imides, and oxazolones.

    Non-peptide libraries can be broadly classified into two types, decorative monomers and oligomers. The decorative monomer library uses a relatively simple scaffold structure to which various functional groups are added. Often the scaffold is a molecule with known useful pharmacological activity. For example, the scaffold can be a benzodiazepine structure.

  Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in a way that creates a new form, depending on the order of the monomers. Carbamates, pyrrolinones and morpholinos are monomer units used. Peptoids, peptidomimetic oligomers whose side chains are not alpha carbon but bind alpha amino acids form the basis for other versions of non-peptide oligomer libraries. The first non-peptide oligomer library utilized a single type of monomer and thus contained a repeating backbone. Modern libraries use one or more monomers, which adds flexibility to the library.

  Library screening can be accomplished by any of a variety of commonly known methods. For example, the following reference disclosing screening of peptide libraries, Parmley et al. , Adv. Exp. Med. Biol. 251: 215-218 (1989); Scott et al,. Science 249: 386-390 (1990); Fowles et al. BioTechniques 13: 422-427 (1992); Oldenburg et al. , Proc. Natl. Acad. Sci. USA 89: 5393-5397 (1992); Yu et al. Cell 76: 933-945 (1994); Staudt et al. , Science 241: 577-580 (1988); Bock et al. , Nature 355: 564-566 (1992); Tuerk et al. , Proc. Natl. Acad. Sci. USA 89: 6988-6992 (1992); Ellington et al. , Nature 355: 850-852 (1992); all Ladner et al. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346; Rebar et al. , Science 263: 671-673 (1993); and PCT Patent Specification No. WO 94/18318.

  In certain embodiments, screening to identify molecules that bind to an albumin fusion protein of the present invention is performed by contacting a member of the library with an albumin fusion protein of the present invention immobilized on a solid phase, and albumin This can be done by recovering library members that bind to the fusion protein. An example of such a screening method, referred to as “panning”, is described in Parmley et al. Gene 73: 305-318 (1988); Fowlkes et al. BioTechniques 13: 422-427 (1992); PCT Specification No. WO 94/18318 and in the references cited herein.

  In other embodiments, a two-hybrid system for screening interacting proteins in yeast (Fields et al., Nature 340: 245-246 (1989); Chien et al., Proc. Natl. Acad. USA 88: 9578-9582 (1991)) can be used to identify molecules that specifically bind to a polypeptide of the invention.

  Where the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The phrase “biased” means that the method of generating the library is manipulated to limit the resulting population of molecules, in this case one or more parameters governing peptide diversity. Used herein to mean.

  Thus, an accurate random peptide library will produce a population of peptides where the probability of finding a particular amino acid at that position of the peptide is the same for all 20 amino acids. However, a bias is introduced into the library, for example, by specifying that lysine occurs every fifth amino acid or that the decapeptide library at positions 4, 8, and 9 is immobilized containing only arginine. Is possible. Obviously, many types of bias are contemplated and the invention is not limited to any particular bias. In addition, the present invention contemplates certain types of peptide libraries, such as those that utilize phage display peptide libraries and constructs capable of cloning DNA containing lambda phage vectors containing DNA inserts.

  As described above, in the case of a binding molecule that is a polypeptide, the polypeptide has about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably about 6 to about It may have about 22 amino acids. In other embodiments, the binding peptide has a range of 15-100 amino acids, or 20-50 amino acids.

Selected binding polypeptides can be obtained by chemical synthesis or recombinant expression.

Other activities

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be transformed into ischemic tissue due to various disease states such as thrombosis, atherosclerosis, and other cardiovascular conditions. May be utilized in treatments to stimulate the revascularization of The albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention may also be utilized to stimulate angiogenesis and limb regeneration, as discussed above.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention is also mitogenic for various cells of different origin, such as fibroblasts and skeletal muscle cells, and thus It may be used to treat wounds, burns, post-surgical tissue regeneration, and wounds due to ulcers, as it promotes regeneration or replacement of damaged or diseased tissue.

  Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention may also be identified to stimulate neuronal proliferation and such as Alzheimer's disease, Parkinson's disease, and AIDS-related complications It may be utilized to treat and prevent neuronal injury that occurs in other neurological or neurodegenerative conditions. Albumin fusion proteins of the invention and / or polynucleotides encoding albumin fusion proteins of the invention may also have the ability to stimulate chondrocyte proliferation, thus enhancing bone and periodontal regeneration and tissue It may be utilized to aid in transplantation or bone grafting.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may also be used to prevent skin aging by sunburn by stimulating keratinocyte proliferation.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may also be utilized to prevent hair loss. Similarly, when the albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention is used in combination with other cytokines, it stimulates the proliferation and differentiation of hematopoietic stem cells and bone marrow cells. May be used for

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may also be utilized to maintain an organ prior to transplantation or to support primary tissue cell culture. Good. The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may also be utilized to induce tissue of mesoderm origin to differentiate in early embryos.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may also increase or decrease embryonic stem cell differentiation or proliferation in addition to the hematopoietic lineage, as discussed above. .

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention may also comprise body length, weight, hair color, eye color, skin, adipose tissue ratio, pigmentation, size, And may be used to adjust mammalian characteristics, such as shape (eg, cosmetic surgery). Similarly, an albumin fusion protein of the present invention and / or a polynucleotide encoding an albumin fusion protein of the present invention can be used to alter mammalian metabolism that affects catabolism, anabolism, energy processing, utilization and storage. May be used to adjust.

  The albumin fusion protein of the present invention and / or the polynucleotide encoding the albumin fusion protein of the present invention can be converted into biorhythm, caricadic rhythm, depression (including depression), violent epilepsy, pain resistance, fertility ( To alter the mental or physical state of a mammal by affecting hormone or endocrine levels, appetite, sexual impulses, memory, stress or other cognitive characteristics (preferably by activin or inhibin-like activity) May be used.

  The albumin fusion protein of the invention and / or the polynucleotide encoding the albumin fusion protein of the invention may also increase storage capacity, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components May be used as a food additive or preservative to reduce or reduce.

  The applications relisted above are used in a wide variety of hosts. Such hosts include, but are not limited to, humans, murines, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, micro-pigs, chickens, goats, cows, sheep, dogs , Cats, non-human primates and humans. In certain embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In a preferred embodiment, the host is a mammal. In the most preferred embodiment, the host is a human.

  While the present invention has been described generally, the same will be more readily understood with reference to the following examples, which are provided for purposes of illustration and are not intended to be limiting.

Without further description, it will be appreciated that, using the foregoing description and the following illustrative examples, one of ordinary skill in the art can make and use the changes detected in the present invention to implement the claimed method. . Accordingly, the following practical examples particularly point out preferred embodiments of the invention and are not to be construed as limiting the remainder of the disclosure in any way.

Example
Example 1 Production of pScNHSA and pScCHSA

  The vectors pScNHSA (ATCC accession number PTA-3279) and pScCHSA (ATCC accession number PTA-3276) are derivatives of pPPC0005 (ATCC accession number PTA-3278) and encode therapeutic proteins or fragments or variants thereof. Is used as a cloning vector, inserted adjacent to the polynucleotide encoding serum albumin “HSA” and within the translation frame. pScCHSA may be used to produce therapeutic protein-HSA fusions, while pScNHSA may be used to produce HSA-therapeutic protein fusions.

Production of pScCHSA: Albumin fusion with albumin site C-terminus for therapeutic protein Vector to facilitate cloning of DNA encoding therapeutic protein N-terminus relative to DNA encoding mature albumin protein Was made by altering the nucleic acid sequence encoding the chimeric HSA signal peptide in pPPC0005 to include XhoI and ClaI restriction sites.

  First, the unique XhoI and ClaI sites (located 3 'of the ADH1 terminator sequence) are eliminated by digesting pPPC0005 with XhoI and ClaI, filling the sticky ends with T4 DNA polymerase, and religating the blunt end. Thus, pPPC0006 is manufactured.

Second, the XhoI and ClaI restriction sites, the nucleic acid sequence encoding the signal peptide of HSA (kex2 chimera from HSA leader and mating factor alpha “MAF”) in pPPC0006 using two rounds of PCR. Modify in. In the first round of PCR, amplification with the primers shown as SEQ ID NO: 36 and SEQ ID NO: 37 was performed. The primer, whose sequence is shown as SEQ ID NO: 36, comprises a nucleic acid sequence encoding part of the signal peptide sequence of HSA, the kex2 site from the mating factor alpha leader sequence, and the amino-terminal part of the mature form of HSA. Including. Four point mutations were introduced into the sequence to create XhoI and ClaI sites at the junction of the chimeric signal peptide and the mature form of HSA. These four mutations are underlined in the sequence shown below. In pPPC0005, the nucleotides at these four positions 5 ′ to 3 ′ are T, G, T and G.
5'-GC C T C GA G AAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG A TTTAAAGATTTGGG-3 '( SEQ ID NO: 36) and 5'-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAATAAGC-3' (SEQ ID NO: 37). A second round of PCR was then performed using the upstream flanking primer, 5′-TACAAACTTAAGAGTCCCAATTTAGC-3 ′ (SEQ ID NO: 38) and the downstream flanking primer 5′-CACTTCTCTAGAGTGGTTTCATATGTTCTT-3 ′ (SEQ ID NO: 39). The resulting PCR product was then purified and digested with Afl III and XbaI and ligated into the same site in pPPC0006 to produce pScCHSA. The resulting plasmid has modified XhoI and ClaI sites within a single sequence. The presence of the XhoI site creates a single amino acid change at the end of the signal sequence from LDKR to LEKR. The change from D to E indicates that the nucleic acid comprising a polynucleotide encoding a therapeutic protein of an albumin fusion protein having a 5 ′ SalI site and a 3 ′ ClaI site (compatible with the XhoI site) is expressed in the XhoI and ClaI of pScCHSA. When ligated into the site, it is not present in the final albumin fusion protein expression plasmid. SalI to XhoI ligation restores the original amino acid sequence of the signal peptide sequence. DNA encoding the therapeutic portion of the albumin fusion protein may be inserted after the Kex2 site (Kex2 is cleaved at the end of the signal peptide after the dibasic amino acid sequence KR) and before the ClaI site. .

Production of pScNHSA: an albumin fusion protein with an albumin site N-terminus for the therapeutic protein. A vector to facilitate cloning of the DNA encoding the therapeutic protein portion C-terminus relative to the DNA encoding the mature albumin protein. Was made by adding three 8 base pair restriction sites to pScCHSA. AscI, FseI and PmeI restriction sites were added between Bsu36I and HindII at the end of the nucleic acid sequence encoding the mature HSA protein. This was done through the use of two complementary synthetic primers containing the underlined AscI, FseI and PmeI restriction sites (SEQ ID NO: 40 and SEQ ID NO: 41). 5′-AAGCTGCCCTTAGGCTTATAATAA GGCGCGGCCGGCCGGCCGTTTAAAC TAAGCTTAATTCT-3 ′ (SEQ ID NO: 40) and 5′-AGATATAGCTTA GTTTAAAACGGCCGGCCGGCGCGCGCCTTTATATAAGCTTAAGC These primers were annealed, digested with Bsu36I and HindIII, and ligated into the same site in pScCHSA to produce pScNHSA.

Example 2: Production of a general construct for yeast transduction

The vectors pScNHSA and pScCHSA are used as cloning vectors in which a polynucleotide encoding a therapeutic protein or fragment or variant thereof is inserted adjacent to a pornucleotide encoding mature human serum albumin “HSA”. May be. pScCHSA may be used to produce a therapeutic protein-HSA fusion, while pScNHSA may be used to produce an HSA-therapeutic protein fusion.
Production of albumin fusion constructs containing HSA-therapeutic protein fusion products

By encoding DNA encoding a therapeutic protein (eg, the sequence shown in SEQ ID NO: X or known in the art) (eg, by adding a restriction site, coding for a seamless fusion) PCR amplification may be used with primers that facilitate production of the fusion construct, such as by encoding a linker sequence. For example, a 5 ′ primer that adds a polynucleotide encoding the last 4 amino acids of the mature form of HSA (and including the Bsu36I site) on the 5 ′ end of DNA encoding the therapeutic protein. And a 3 ′ primer can be designed that adds a STOP codon and an appropriate cloning site on the 3 ′ end of the therapeutic protein coding sequence. For example, a forward primer used to amplify DNA encoding a therapeutic protein may have the sequence 5′-aagctG CCTTAGG CTTA (N) ₁₅ -3 ′ (SEQ ID NO: 42) underlined with a Bsu36I site. In these cases, the nucleotide encodes the last 4 amino acids of the mature HSA protein (ALGL), and (N) ₁₅ is identical to the first 15 nucleotides encoding the therapeutic protein of interest. Similarly, the reverse primer used to amplify DNA encoding a therapeutic protein is
Sequence 5′-GCGCGCGTTTAAACGGCGCGGCC GGCGCGCC TATTTA (N) ₁₅ -3 ′ (SEQ ID NO: 43)
The italicized sequence (TTTAAAC) is the PmeI site, the double underlined sequence is the FseI site, the single underlined sequence is the AscI site, and the boxed nucleotides are two The reverse complement of the tandem stop codon, (N) ₁₅ is identical to the reverse complement of the last 15 nucleotides encoding the therapeutic protein of interest. Once the PCR product is amplified, it is cut with one of Bsu36I and (AscI, FseI, or PmeI) and ligated into pScNHSA.

The presence of the XhoI site in the HSA chimeric leader sequence creates a single amino acid change at the end of the chimeric signal sequence, ie, the HSA-kex2 signal sequence from LDKR (SEQ ID NO: 44) to LEKR (SEQ ID NO: 45). Is produced.
Production of albumin fusion constructs containing gene-HSA fusion products

Similar to the method described above, DNA encoding a therapeutic protein may be PCR amplified using the following primers. A polynucleotide encoding the last three amino acids of the HSA leader sequence, a 5 ′ primer to add DKR, and a therapeutic protein on the 5 ′ end of the DNA containing the SalI site and encoding the therapeutic protein A 3 ′ primer that adds a polynucleotide encoding the first few amino acids of mature HSA containing a ClaI site on the 3 ′ end of the encoding DNA. For example, the forward primer used to amplify DNA encoding a therapeutic protein has the sequence 5′-aggagc gtcGAC AAAAGA (N) ₁₅ -3 ′ (SEQ ID NO: 46), where the underlined sequence is A SalkI site, in which case the nucleotide encodes the last 3 amino acids (DKR) of the HSA leader sequence and (N) ₁₅ is the first 15 nucleotides encoding the therapeutic protein of interest and Are the same. Similarly, the reverse primer used to amplify the DNA encoding the therapeutic protein may have the sequence 5′-CTTTAAAATCG ATGAGCCAACCTCACTCTTGTGTGCATC (N) ₁₅ -3 ′ (SEQ ID NO: 47), where italics The sequence (ATCGAT) is a ClaI site, the underlined nucleotide is the reverse complement of DNA encoding the first 9 amino acids of the mature form of HSA (DAHKSEVAH, SEQ ID NO: 48), and (N) ₁₅ is the subject Is the same as the last 15 nucleotide reverse complement of the therapeutic protein. Once the PCR product is amplified, it is cut with SalI and ClaI and ligated into pScCHSA digested with XhoI and ClaI. For example, invertase “INV” (Swiss-Prot Access P00724), mating factor alpha (Genbank Accession AAA18405), MPIF (Geneseq AAF82936), fibrin B (Fibulin B) (Swiss-Prot Access class P142Sw) Different signal or leader sequences are desirable, such as Accession P10909), Insulin-Like Growth Factor-Binding Protein 4 (Swiss-Prot Accession P22692), and substitution of the HSA leader sequence Known in the technical field By standard methods, it can be subcloned into a suitable vector.
Yeast S. Production of an albumin fusion construct adaptable for expression in S. cerevisiae

A NotI fragment containing DNA encoding either an N-terminal or C-terminal albumin fusion protein produced from pScNHSA or pScCHSA may then be cloned into the NotI site of pSAC35 with a LEU2 selectable marker. The resulting vector was then transformed into yeast S. Used for transduction of cerevisiae expression system.

Example 3: Yeast General expression in cerevisiae

Expression vectors that are compatible with yeast expression are known in the art, such as lithium acetate transduction, electroporation, or Sambrook, Fritsch, and Maniatis. 1989. "Molecular Cloning: A Laboratory Manual, 2 nd edition", volumes 1-3, and Ausubel et al. 2000. Massachusetts General Hospital and Harvard Medical School “Current Protocols in Molecular Biology”, volumes 1-4, by other methods. It can be transduced into cerevisiae. The expression vector is transformed into S. cerevisiae by transduction. Introduced into S. cerevisiae strains DXY1, D88 or BXP10 and individual transducts, for example in 10 mL YEPD (1% w / v yeast extract, 2% w / v peptone, 2% w / v dextrose) at 30 ° C. And can be harvested in the stationary phase 60 hours after growth. The supernatant is recovered by clarification at 3000 g for 10 minutes.

pSAC35 (see Sleep et al., 1990, Biotechnology 8:42 and FIG. 3) contains, in addition to the LEU2 selectable marker, a whole yeast 2 μm plasmid that provides replication function, a PRB1 promoter, and an ADH1 termination signal .
Example 4

Yeast S. General purification of albumin fusion protein expressed from albumin fusion in S. cerevisiae

  In preferred embodiments, the albumin fusion proteins of the invention comprise mature forms of HSA fused to either the N- or C-terminus of the mature form of the therapeutic protein or portion thereof (eg, the treatments listed in Table 1). Mature form of therapeutic protein, or mature form of therapeutic protein shown in Table 2 as SEQ ID NO: Z). In one embodiment of the invention, the albumin fusion protein of the invention further comprises a signal sequence directed to the nascent fusion polypeptide in the secretory pathway of the host used "for expression". In a preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature albumin fusion protein is secreted directly into the culture medium. The albumin fusion proteins of the invention are preferably, but not limited to, MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor protein 4, and mutations including but not limited to chimeric HSA / MAF leader sequences A heterologous signal sequence (eg, a non-native signal sequence of a particular therapeutic protein), including a human HSA leader sequence, or other heterologous signal sequence known in the art. The signal sequences listed in Table 2 and / or the signals listed in “Fusion protein expression” and / or “Further methods of recombinant and synthetic production of albumin fusion proteins” as described herein above. An arrangement is particularly preferred. In a preferred embodiment, the fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and / or variants, are also encompassed by the present invention.

  As described above, albumin fusion proteins expressed in yeast can be purified on a small scale on a Dyax peptide affinity column as follows. Supernatants from yeast expressing albumin fusion protein are dialyzed against 3 mM phosphate buffer pH 6.2, 20 mM NaCl and 0.01% Tween 20 to reduce volume and remove dye. The solution is then filtered through a 0.22 μm instrument. Place the filtrate on a Dyax peptide affinity column. The column is eluted with 100 mM Tris / HCl, pH 8.2 buffer. The peak fraction containing the protein is collected, concentrated 5 times, and analyzed on SDS-PAGE.

The following methods can be used for large scale purification. The supernatant in excess 2 L is dialyzed and concentrated to 500 mL in 20 mM Tris / HC1 pH 8.0. The concentrated protein solution is placed on a pre-equilibrated 50 mL DEAE-Sepharose Fast Flow column, the column is washed, and the protein is washed with a linear NaCl gradient from 0 to 0.4 M NaCl in 20 mM Tris / HCl, pH 8.0. Elute with. Fractions containing protein are pooled and adjusted to pH 6.8 with 0.5 M sodium phosphate (NaH ₂ PO ₄ ). A final concentration of 0.9 M (NH ₄ ) ₂ SO ₄ is added to the protein solution and the entire solution is loaded onto a pre-equilibrated 50 mL Butyl650S column. The protein is eluted with a linear gradient of ammonia sulfate ((0.9 to 0 M (NH ₄ ) ₂ SO ₄ ). The fraction containing albumin fusion is reconstituted with 10 mM Na ₂ HPO ₄ / citrate buffer pH 5 Dialyze against .75 and place on a 50 mL pre-equilibrated SP-Sepharose Fast Flow column Elute the protein with a 0-0.5 M NaCl linear gradient Combine the fractions containing the protein of interest The buffer is changed by Amicon concentrator to 10 mM Na ₂ HPO ₄ / citric acid pH 6.25, conductivity is <2.5 mS / cm. On a modified Q-Sepharose high performance column, washing the column and washing the protein with a linear NaCl gradient from 0 to 0.15 M NaCl. The eluted. Purified protein then can be formulated to particular buffer solution components by buffer exchange.

Example 5: General construct production for mammalian cell transfection
Production of albumin fusion constructs adaptable for expression in mammalian cell lines

  The albumin fusion construct can be expressed in an expression vector for use in mammalian cell culture systems. DNA encoding the therapeutic protein is cloned into a mammalian expression vector, N-terminal or C-terminal to HSA, by standard methods known in the art (eg, PCR amplification, restriction digestion, and ligation) Is possible. Once the expression vector is constructed, transfection into the mammalian system can proceed. Suitable vectors are known in the art including, but not limited to, for example, pC4 vectors and / or vectors available from Lonza Biologics, Inc. (Portsmouth, NH).

  DNA encoding human serum albumin was cloned into a pC4 vector suitable for mammalian culture systems to produce plasmid pC4: HSA (ATCC accession number PTA-3277). The vector has a dihydrofolate reductase, “DHFR”, which allows selection in the presence of methotrexate.

  The pC4: HSA vector is suitable for the expression of albumin fusion proteins in CHO cells. For expression, it is preferred that the fragment containing or consisting of the DNA encoding the albumin fusion protein is subcloned into other expression vectors in other mammalian cell culture systems. For example, a fragment comprising or consisting of DNA encoding a mature albumin fusion protein can be subcloned into other expression vectors, including but not limited to any mammalian expression vector described herein. May be.

In a preferred embodiment, the DNA encoding the albumin fusion construct is obtained by Lonza Biologics, Inc. (Portsmouth, NH) by procedures known in the art for expression in NS0 cells. Subcloned into the provided vector.
Production of albumin fusion constructs containing HSA-therapeutic protein fusion products

Using pC4: HSA (ATCC accession number PTA-3277), an albumin fusion construct can be produced such that the therapeutic protein moiety is C-terminal to the mature albumin sequence. For example, DNA encoding a fragment or variant therapeutic protein between the Bsu 36I and Asc I restriction sites of the vector can be cloned. During cloning into Bsu 36I and Asc I, the same primer design (SEQ ID NOs: 42 and 43) used to clone into the yeast vector system may be used (see Example 2). )
Production of albumin fusion constructs containing gene-HSA fusion products

Using pC4: HSA (ATCC accession number PTA-3277), an albumin fusion construct can be produced to clone the therapeutic protein site to the N-terminus of the mature albumin fusion sequence. For example, DNA encoding a therapeutic protein with its unique signal sequence between the Bam HI (or Hind III) and Cla I sites of pC4: HSA can be cloned. When cloning into either Bam HI or Hind III, a Kozak sequence (CCGCCCACC ATG , SEQ ID NO: 49) is preferably included in front of the translation initiation codon from which the DNA encoding the therapeutic protein can be cloned. . If the therapeutic protein does not have a signal sequence, the DNA encoding the therapeutic protein may be cloned between Xho I and Cla I of pC4: HSA. When using the XhoI site, the following 5 ′ (SEQ ID NO: 50) and 3 ′ (SEQ ID NO: 51) exemplified PCR primers may be used.
_{5'-CCGCCG CTCGAG GGGTGTGTTTCGTCGA (N)} 18 -3 '( SEQ ID NO: 50)
5′-AGTCCC ATCGAT GAGCAACCTCACTCTTGTGTGCATC (N) ₁₈ -3 ′ (SEQ ID NO: 51)

In the 5 ′ primer (SEQ ID NO: 50), the underlined sequence is the XhoI site, and the XhoI site and the DNA following the XhoI site encode the last 7 amino acids of the native human serum albumin leader sequence. In SEQ ID NO: 50, “(N) ₁₈ ” is the same DNA as the first 18 nucleotides encoding the therapeutic protein of interest. In the 3 ′ primer (SEQ ID NO: 51), the underlined sequence is a ClaI site, and the ClaI site and the subsequent DNA are reverse complements of the DNA encoding the first 10 amino acids of the mature HSA protein (SEQ ID NO: 1). Is the body. In SEQ ID NO: 51, “(N) ₁₈ ” is the reverse complement of DNA encoding the last 18 nucleotides encoding the therapeutic protein of interest. With these two primers, the therapeutic protein of interest can be PCR amplified, the PCR product purified, digested with XhoI and ClaI restriction enzymes, and cloned into the XhoI and ClaI sites in the pC4: HSA vector.

If other leader sequences are desired, the native albumin leader sequence can be replaced with a chimeric albumin leader, ie, an HSA-kex2 signal peptide, or other leader by standard methods known in the art. (For example, one skilled in the art can PCR-amplify other leaders in the conventional manner and maintain the reading frame while subcloning the PCR product into an albumin fusion construct instead of the albumin leader).

Example 6: General expression in mammalian cell lines

Albumin fusion constructs produced in expression vectors compatible with expression in mammalian cell lines are calcium phosphate precipitation, lipofectamine, electroporation, or known in the art and / or Sambrook, Fritsch, and Maniatis . 1989. "Molecular Cloning: A Laboratory Manual, 2 nd edition" and in Ausubel et al. 2000. Suitable cell lines can be transfected by other transfection methods as described in Massachusetts General Hospital and Harvard Medical School “Current Protocols in Molecular Biology”, volumes 1-4. The transfected cells are then sorted by the presence of a sorting reagent determined by a selectable marker in the expression vector.

  The pC4 expression vector (ATCC Accession No. 209646) is a derivative of the plasmid pSV2-DHFR (ATCC Accession No. 37146). pC4 is a strong promoter, Long Sarcoma Virus Long Terminal Repeats (LTR) (Cullen et al., March 1985, Molecular and Cellular Biology, 438-447 Mega). Virus (CytoMegaloVirus) "CMV"-contains a fragment of the enhancer (Boshart et al., 1985, Cell 41: 521-530). The vector also contains the 3 'intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster oocytes (“CHO”) cells or other cell lines lacking an active DHFR gene are used for transfection. Transfection of albumin fusion constructs in pC4 into CHO cells by methods known in the art allows the expression of albumin fusion proteins in CHO cells, followed by cleavage of the leader sequence and into the supernatant. Secretion continues. The albumin fusion protein is then further purified from the supernatant.

pEE. The 121 expression vector is a derivative of pEE6 provided by Lonza Biologics, Inc. (Portsmouth, NH) (Stephens and Cockett, 1989, Nucl. Acids Res. 17 : 7110). This vector consists of the human cytomegalovirus major metaphase early gene, the promoter of “hCMV-MIE”, the enhancer and the complete 5′-untranslated region (International Patent Specification No. WO 89/01036), upstream of the sequence of interest, And the glutamine synthase gene (Murphy et al., 1991, Biochem J. 227: 277-279; Bebbington et al., For the purpose of sorting transfected cells in media containing selective and methionine sulfoximine. , 1992, Bio / Technology 10: 169-175, and US Pat. No. 5,122,464). Albumin fusion proteins in NS0 cells by transfection of the albumin fusion construct made in pEE12.1 into NS0 cells by methods known in the art (International Patent Specification No. WO86 / 05807). Expression, followed by cleavage of the leader sequence and secretion into the supernatant. The albumin fusion protein is then further purified from the supernatant using techniques described herein or known in the art.

  The expression of the albumin fusion protein may be analyzed, for example, by SDS-PAGE and Western blot, reverse phase HPLC analysis, or other methods known in the art.

  Stable CHO and NS0 cell lines transfected with albumin fusion constructs are produced by methods known in the art (eg, Lipofectamine transfection), eg, with the DiHyDroFolate Reductase “DHFR” gene as a selectable marker The vectors are screened for growth with 100 nM methotrexate or in the absence of glutamine. Expression levels are tested, for example, by immunoblotting first with anti-HSA serum as the first antibody or second with serum containing an antibody directed against the therapeutic protein portion of the albumin fusion protein as the first antibody. Is possible.

Expression levels are tested by immunoblot detection with anti-HSA serum as the primary antibody. A specific production rate can be determined via an ELISA, where the capture antibody can be a monoclonal antibody to the therapeutic protein portion of the albumin fusion and the detection antibody is a monoclonal anti-HSA-biotinylated antibody (or vice versa). Determined by horseradish peroxidase / streptavidin binding and analysis according to manufacturer's protocol.

Example 7: Expression of albumin fusion protein in mammalian cells

  The albumin fusion protein of the present invention can be expressed in mammalian cells. A typical mammalian expression vector contains a promoter element that mediates the initiation of transcription of mRNA, a protein coding sequence, and signals necessary for termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and interrupted sequences flanked by donors and acceptors for RNA splicing. Highly effective transcription is achieved with early and late promoters from SV40, long terminal repeats (LTR) from retroviruses, such as RSV, HTLVI, HIVI, and cytomegalovirus (CMV) early promoters. However, cellular elements (eg, human actin promoter) can also be used.

  Suitable expression vectors for use in practicing the present invention include, for example, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109). ), Vectors such as pCMVSPort 2.0, and pCMVSPort 3.0. Mammalian host cells that can be used include, but are not limited to, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, qual QC1-3 cells, mouse L cells and Chinese hamster oocyte (CHO) cells are included.

  Alternatively, the albumin fusion protein can be expressed in a stable cell line containing a polynucleotide encoding an albumin fusion protein integrated within a chromosome. Co-transfection with a selectable marker such as DHFR, gpt, neomycin or hygromycin allows the identification and isolation of the transfected cells.

  The transfected polynucleotide encoding the fusion protein can also be amplified to express large amounts of the encoded fusion protein. DHFR (dihydrofolate reductase) markers are useful for developing cell lines with hundreds or thousands of copies of the gene of interest (eg, Alt et al., J. Biol. Chem. 253: 1357-1370). (1978); Hamlin et al., Biochem. Et Biophys. Acta, 1097: 107-143 (1990); see Page et al., Biotechnology 9: 64-68 (1991)). Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227: 277-279 (1991); Bebbington et al., Bio / Technology 10: 169-175 (1992)). Using these markers, mammals are grown in selective media to select the most resistant cells, these cell lines contain the amplified gene (s) integrated into the chromosome. Chinese hamster oocytes (CHO) and NSO cells are often used for protein production.

  Derivatives of plasmid pSV2-dhfr (ATCC accession number 37146), expression vectors pC4 (ATCC accession number 209646) and pC6 (ATCC accession number 209647) are the strong promoter (LTR) of Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) + CMV-enhancer fragment (Boshart et al., Cell 41: 521-530 (1985)). For example, multiple cloning sites including restriction enzyme cleavage sites BamHI, XbaI and Asp718 facilitate cloning of the gene of interest. Vectors also include the 3 'intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under the control of the SV40 early promoter.

  In particular, for example, plasmid pC6 is digested with appropriate restriction enzymes and dephosphorylated with bovine intestinal phosphate by procedures known in the art. The vector is then isolated from a 1% agarose gel.

  A polynucleotide encoding an albumin fusion protein of the invention is made using techniques known in the art and the polynucleotide is amplified using PCR techniques known in the art. If a naturally occurring signal sequence is used to produce the fusion protein of the invention, the vector does not require a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, WO 96/34891).

  The amplified fragment encoding the fusion protein of the invention is isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with the appropriate restriction enzymes and purified again on a 1% agarose gel.

  The amplified fragment encoding the albumin fusion protein of the invention is then digested with the same restriction enzymes and purified on a 1% agarose gel. The isolated fragment and dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transduced and the bacteria are identified to contain the fragment inserted into plasmid pC6, for example using restriction enzyme analysis.

Chinese hamster oocytes lacking an active DHFR gene are used for transfection. 5 μg of the expression plasmid pC6 or pC4 is cotransfected with 0.5 μg of the plasmid pSVneo using lipofection (Felner et al., Supra). Plasmid pSV2-neo contains the neo gene from Tn5 which encodes an enzyme conferring resistance to a group of antibiotics including a dominant selectable marker, G418. Cells are seeded in alpha minus MEM containing 1 g / mg G418. Two days later, the cells are trypsinized and plated in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM containing 10, 25 or 50 ng / ml methotrexate + 1 mg / ml G418. After about 10-14 days, single colonies are trypsinized and plated in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentration of methotrexate are then transferred to new 6-well plates containing higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 μM. Expression of the desired fusion protein is analyzed, for example, by SDS-PAGE and Western blot, or by reverse phase HPLC analysis.

Example 8: General purification of albumin fusion proteins expressed from albumin fusion constructs in mammalian cell lines

  In a preferred embodiment, the albumin fusion protein of the invention comprises a mature form of a therapeutic protein or portion thereof (eg, a mature form of a therapeutic protein listed in Table 1, or a treatment shown in Table 2 as SEQ ID NO: Z) A mature form of the HSA fused to either the N- or C-terminus of the mature protein). In one embodiment of the invention, the albumin fusion protein of the invention comprises a signal sequence directed to a nascent fusion polypeptide in the secretory pathway of the host used for expression. In a preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature albumin fusion protein is secreted directly into the culture medium. The albumin fusion protein of the present invention is preferably, but not limited to, MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, mutant HSA comprising but not limited to a chimeric HSA / MAF leader sequence Includes a heterologous signal sequence (eg, a non-native signal sequence of a particular therapeutic protein), including a leader sequence, other heterologous signal sequences known in the art. Signal sequences listed in Table 2 and / or listed in “Fusion protein expression” and / or “Further methods of recombinant and synthetic production of albumin fusion proteins” as described hereinabove. A signal sequence is particularly preferred. In a preferred embodiment, the fusion protein of the invention further comprises an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and / or variants, are also encompassed by the present invention.

Albumin fusion proteins from mammalian cell line supernatants are purified according to different protocols, depending on the expression system used.
Purification from CHO and 293T cell lines

For purification of albumin fusion protein from CHO cell supernatant or from single transfected 293T cell supernatant, initial capture with anionic HQ resin using sodium phosphate buffer and phosphate gradient elution, Subsequent affinity chromatography on a Blue Sepharose FF column using salt gradient elution can be included. Blue Sepharose FF removes major BSA / fetuin contaminants. Further purification on the Poros PI 50 resin with a phosphate gradient can remove and reduce endotoxin contaminants and albumin fusion protein concentrations.
Purification from NS0 cell line

  Purification of albumin fusion protein from NS0 cell supernatant may include Q-Sepharose anion exchange chromatography, followed by step-elution SP-Sepharose purification, subsequent-step elution Phenyl-650M purification, and final dialysis .

The purified protein may then be formulated by buffer exchange.

Example 9: Bacterial expression of albumin fusion protein

A polynucleotide encoding an albumin fusion protein of the present invention containing a bacterial signal sequence is amplified using PCR oligonucleotide primers corresponding to the 5 ′ and 3 ′ ends of the DNA sequence to synthesize the insert. . The primer used to amplify the polynucleotide encoding the insert is preferably a restriction such as BamHI and XbaI at the 5 'end of the primer to clone the amplification product into the expression vector. Including sites. For example, BamHI and XbaI correspond to restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector consists of antibiotic resistance (Amp ^r ), bacterial complex origin (ori), IPTG-regulatable promoter / operator (P / O), ribosome binding site (RBS), 6-histidine tag (6-His), And encodes a restriction enzyme cloning site.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector, maintaining the reading frame initiated in bacterial RBS. The ligation mixture is then used to express E. coli strain M15 / rep4 (Qiagen, Inc.) containing multiple copies of plasmid pREP4 that expresses the lacl repressor and confers kanamycin resistance (Kan ^r ). Is transduced. Transductants are identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired structure are grown overnight (O / N) in liquid culture in LB medium containing both Amp (100 ug / ml) and Kan (25 ug / ml). The O / N culture is used to inoculate a large culture at a ratio of 1: 100 to 1: 250. Cells are grown to an optical density 600 (OD ⁶⁰⁰ ) between 0.4 and 0.6. IPTG (isopropyl-BD-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacl repressor and purifying the P / O leading to increased gene expression.

  Cells are grown for an additional 3-4 hours. Cells are then harvested by centrifugation (6000 × g, 20 minutes). Cell pellets are solubilized by stirring in chaotropic reagent 6 mol guanidine HCl, preferably in 8-mer urea and 2-mercaptoethanol at a concentration of 0.14 M or more at 4 ° C. for 3-4 hours (eg, Burton et al., Eur. J. Biochem. 179: 379-387 (1989))). Cell debris is removed by centrifugation and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA)” affinity resin column (available from Qiagen supra). A protein containing a 6 × His tag binds to Ni-NTA resin with high affinity and can be purified by a simple one-step procedure (see The QIA expressionist (1995) QIAGEN, Inc., supra for details. ).

  Briefly, the supernatant is loaded onto the column in 6M guanidine-HCl, pH 8. The column is first washed with 10 volumes of 6M guanidine-HCl, pH 8, then washed with 10 volumes of 6M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6M guanidine-HCl, pH 5.

  The purified protein is then denatured by dialyzing against phosphate buffered saline (PBS) or 50 mM Na-acetic acid, pH 6 buffer + 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on a Ni-NTA column. Exemplary conditions are as follows. Reconstitution using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl pH 7.4 with protease inhibitors. The restoration should be performed over a period of 1.5 hours or more. After re-denaturation, the protein is eluted by the addition of 250 mM imidazole. Imidazole is removed by a final dialysis step against PBS or 50 mM sodium acetate pH 6 buffer + 200 mM NaCl. The purified protein is stored at 4 ° C or frozen at -80 ° C.

  In addition to the expression vectors described above, the present invention further comprises pHE4a (ATCC Accession No. 209645, 1998 2), comprising a phage operator and promoter element operably linked to a polynucleotide encoding an albumin fusion protein of the present invention. And an expression vector called pHE4a (ATCC accession number 209645, accepted on February 25, 1998). This vector has 1) as a selection marker, the neomycin phosphotransferase gene, 2) the origin of E. coli replication, 3) the T5 phage promoter sequence, 4) the two lac operator sequences, 5) the Shine-Delgarno sequence, and 6) the lactose operon repressor. Contains the gene (laclq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). Produced by synthesizing promoter and operator sequences.

  Restrict the DNA with NdeI and XbaI, BamHI, XhoI, or Asp718, run the restricted product on a gel, and isolate a larger fragment (the stuffer fragment is approximately 310 base pairs) Should be inserted into pHE4a. DNA inserts are described herein using PCR primers with restriction sites for NdeI (5 ′ primer) and XbaI, BamHI, XhoI, or Asp718 (3 ′ primer), or in the art In accordance with the known PCR protocol. The PCR insert is gel purified and restricted with a compatible enzyme. The insert and vector are ligated according to standard protocols.

The modified vector may be replaced by the above protocol in order to express the protein in a bacterial system.

Example 10: Isolation of selected cDNA clones from commissioned samples

  Many of the albumin fusion constructs of the present invention were contracted to the ATCC as shown in Table 3. The albumin fusion construct has the following expression vector: yeast S. cerevisiae. It may comprise any one of cerevisiae expression vector pSAC35, mammalian expression vector pC4, or mammalian expression vector pEE12.1.

  pSAC35 (Sleep et al., 1990, Biotechnology 8:42), pC4 (ATCC accession number 209646; Cullen et al., Molecular and Cellular Biology, 438-447 (1985); Boshart et al. 41: 52. (1985)), and pEE12.1 (Lonza Biologics, Inc.); Stephens and Cockett, Nucl. Acids Res. 17: 7110 (1989); International Publication No. WO89 / 01036; , Biochem J. 227: 277-279 (1991); ington et al., Bio / Technology 10: 169-175 (1992); US Pat. No. 5,122,464; International Publication No. WO86 / 05807) A vector is an ampicillin resistance gene for growth in bacterial cells. including. These vectors and / or albumin fusion constructs containing them can be obtained using Stratagene XL-1 Blue (Stratagene Cloning Systems, Inc.), using techniques described in the art such as Hanahan. 11011 N. Torrey Pines Road, La Jolla, CA, 92037), may be plated on Luria-Broth agar plates containing 100 mg / mL ampicillin and grown overnight at 37 ° C.

  The deposit material in the sample assigned the ATCC deposit number quoted in Table 3 for any such albumin fusion construct may also include one or more additional albumin fusion constructs, each with a different albumin fusion. It encodes a protein. Thus, deposits that share the same ATCC deposit number include at least one albumin fusion construct identified in the corresponding column of Table 3.

Two approaches can be used to isolate a particular albumin fusion construct from a contracted sample of plasmid DNA cited for the albumin fusion construct in Table 3.
Method 1: Screening

First, an albumin fusion construct is prepared using a method known in the art, using a polynucleotide probe corresponding to SEQ ID NO: X for each construct ID in Table 1 and a sample of the deposited plasmid DNAs. May be isolated directly by screening. For example, a specific polynucleotide of 30-40 nucleotides may be synthesized using an Applied Biosystems DNA synthesizer according to the reported sequence. Oligonucleotides can be labeled with ³² P-γ-ATP, for example using T4 polynucleotide kinase, and purified according to routine methods. (For example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY (1982)). Albumin fusion constructs from the ATCC contract are those provided by the vector supplier as suggested above (as in XL-1 Blue (as in Stratagene), or the references cited above. Transduce suitable hosts using techniques known to those skilled in the art, such as in publications or patents, to a concentration of about 150 transductants (colony) per plate (appropriate Plate on 1.5% agar plates containing a selection reagent, such as ampicillin These plates are routine methods for bacterial colony screening (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit. , (1989), Cold Spring Harbor Laboratory Press, according to pages 1.93 to 1.104), or those skilled in other known techniques and screened using a nylon membrane.
Method 2: PCR

Alternatively, two primers of 17-20 nucleotides that hybridize the DNA encoding the albumin fusion protein to, for example, the deposited albumin fusion constructs 5 ′ and 3 ′ of the DNA encoding the albumin fusion protein May be amplified from a sample of a contracted albumin fusion construct having SEQ ID NO: X. The polymerase chain reaction is performed under routine conditions in a 25 μl reaction mixture containing, for example, 0.5 ug of the above cDNA template. A simple reaction mixture is 1.5-5 mM MgCl ₂ , 0.01% (w / v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 units of Taq polymerase. is there. 35 cycles of PCR (94 ° C for 1 minute denaturation, 55 ° C for 1 minute annealing, 72 ° C for 1 minute extension) are performed in a Perkin-Elmer Cetus automated temperature circulator. The amplified product is analyzed by agarose gel electrophoresis, and a DNA band of the expected molecular weight is extracted and purified. The PCR product is confirmed to be the selected sequence by subcloning and sequencing the DNA product.

  A variety of methods are available for the identification of 5 'or 3' non-coding portions of genes that may not be present in the deposited clone. These methods include, but are not limited to, filter probing, clonal enrichment using specific probes, and protocols similar or identical to 5 'and 3' "RACE" protocols known in the art. For example, a method similar to 5 'RACE can be used to produce the missing 5' end of the desired full-length transcript (Frommont-Racine et al., Nucleic Acids Res., 21 (7): 1683-1684). (1993)).

  Briefly, a specific RNA oligonucleotide is ligated to the 5 'end of a population of RNA, possibly containing a full-length gene RNA transcript. PCR amplification is performed on the 5 'portion of the desired full-length gene using a primer set comprising a primer specific for the ligated RNA oligonucleotide and a primer specific for the known sequence of the gene of interest. This amplified product may then be sequenced and used to produce the full-length gene.

  This further method starts with total RNA isolated from the desired source, although poly-A + RNA can be used. The RNA modulator can then be treated with phosphatase, if necessary, to remove 5 'phosphate groups on degraded or damaged RNA that may interfere with subsequent RNA ligase steps. The phosphatase is then treated with tobacco acid pyrophosphatase to inactivate and remove the cap structure present at the 5 'end of the messenger RNA. This reaction leaves a 5 'phosphate group at the 5' end of the cap-cleaved RNA and can be ligated to the RNA oligonucleotide using T4 RNA ligase.

This modified RNA regulator is used as a template for first strand cDNA synthesis using gene specific oligonucleotides. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5 ′ end, using primers specific for the ligated RNA oligonucleotide and primers specific for the known sequence of the gene of interest. The resulting product is then sequenced and analyzed to confirm that the 5 ′ end sequence belongs to the desired gene.

Example 11: Multiple fusions

  An albumin fusion protein (including, for example, a therapeutic protein (or fragment or variant thereof) fused to albumin (or fragment or variant thereof) can be further combined with other proteins to create a “multiple fusion protein”. May be merged. These multiple fusion proteins can be used for various applications. For example, fusion of an albumin fusion protein of the present invention to His-tag, HA-tag, protein A, IgG domain, and maltose binding protein facilitates purification (eg, European Patent No. EP A 394,827, Traunecker et al., Nature 331: 84-86 (1988)). A nuclear localization signal fused to a polypeptide of the present invention can target a protein to a specific intracellular location, while a shared heterodimer or homodimer can increase or decrease the activity of an albumin fusion protein. is there. Furthermore, the fusion of additional protein sequences to the albumin fusion protein may further increase the solubility and / or stability of the fusion protein. Modifying the fusion protein described above using techniques known in the art or routinely and / or modifying the following protocol outlining the fusion of a polypeptide to an IgG molecule Can be produced.

  Briefly, the human Fc site of an IgG molecule can be PCR amplified using primers that extend to the 5 'and 3' ends of the sequence described below. These primers should also have convenient restriction enzyme sites that facilitate cloning into an expression vector, preferably a mammalian or yeast expression vector.

  For example, when using pC4 (ATCC accession number 209646), the human Fc site can be ligated into the BamHI cloning site. Note that the 3'BamHI site should be destroyed. The vector containing the human Fc site is then re-restricted with BamHI, the vector is linearized and encodes the albumin fusion protein of the invention (produced and isolated using techniques known in the art). The existing polynucleotide is ligated into this BamHI site. Note that the polynucleotide encoding the fusion protein of the invention is cloned without a stop codon or otherwise no Fc-containing fusion protein is produced.

  If a naturally occurring signal sequence is used to produce the albumin fusion protein of the invention, pC4 does not require a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence (see, eg, International Patent Specification No. WO 96/34891).

Human IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCT CAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC CTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ ID NO: 52)

Example 12: Production of antibodies from albumin fusion proteins
Hybridoma method

  Antibodies that bind to an albumin fusion protein of the invention, or a portion of a fusion protein of the invention (eg, a therapeutic protein portion or albumin portion of a fusion protein) can be prepared by various methods (Current Protocols, Chapter 2 See As one example of such a method, a preparation of an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention, is prepared in order to be essentially free of natural contaminants, Purify. Such a preparation is then introduced into the animal to produce a polyclonal antibody with a higher specific activity.

  Monoclonal antibodies specific for an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention, are prepared using hybridoma technology (Kohler et al., Nature 256: 495 (1975); Kohler et al. Eur.J. Immunol.6: 511 (1976); Kohler et al., Eur.J. Immunol.6: 292 (1976); N. Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention. Such mouse spleen cells are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be utilized in accordance with the present invention, however, it is preferred to use the parent myeloma cell line (SP2O) available from ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then Wands et al. (Clone by limiting dilution as described by Gastroenterology 80: 225-232 (1981). Hybridoma cells obtained through such sorting are then subjected to the albumin fusion protein of the invention, or of the invention Assay to identify clones that secrete antibodies capable of binding to a portion of the albumin fusion protein.

  Alternatively, additional antibodies capable of binding to an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention can be produced in a two-step procedure using an anti-idiotype antibody. Such a method takes advantage of the fact that the antibody itself is an antigen and may thus obtain an antibody that binds to the second antibody. According to this method, an animal, preferably a mouse, is immunized using a protein specific antibody. The spleen cells of such animals are then used to produce hybridoma cells, which ability to bind to the albumin fusion protein of the invention (or part of the albumin fusion protein of the invention) -specific antibody. Are selected to identify clones that produce an antibody that can be inhibited by the albumin fusion protein of the invention, or a portion of the albumin fusion protein of the invention. Such antibodies include anti-idiotypic antibodies against the fusion protein of the invention (or part of the albumin fusion protein of the invention) -specific antibodies, and further fusion proteins of the invention (or albumin fusion proteins of the invention). Part) —used to immunize animals to induce the formation of specific antibodies.

  For in vivo use of antibodies in humans, antibodies are “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and discussed herein (for review, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques. 4: 214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567, Taniguchi et al., European Patent 17196; Morrison et al., European Patent 173494; Neuberger et al., International Publication No. WO 8601533; Robinson et al., International Publication No. WO 8702671; Boulianne et al., Nature 312: 643 (1984); Neuberger e t al., Nature 314: 268 (1985)).

Isolation of an antibody fragment directed against an albumin fusion protein of the present invention, or a portion of an albumin fusion protein of the present invention, from a library of scFv. A naturally occurring V-gene isolated from human PBL is isolated from the present invention. Or a portion of an antibody fragment that is responsive to a portion of an albumin fusion protein of the invention and may or may not be exposed to a donor (eg, all See US Pat. No. 5,885,793, incorporated herein by reference).

Library rescue. The scFv library is constructed from human PBL RNA as described in International Patent Specification No. WO 92/01047. To rescue phage display antibody fragments, approximately 10 ⁹ E. coli with phagemids were used to inoculate 50 mlm 2 × TY (2 × TY-AMP-GLU) containing 1% glucose and 100 μg / ml ampicillin, While shaking, a 0.8 O.D. D. Grow until. 5 ml of this culture is used to inoculate 50 ml of 2xTY-AMP-GLU and 2x108 TU of delta gene 3 helper (see M13 delta gene III, International Patent Specification No. WO 92/01047). In addition, the culture is incubated for 45 minutes at 37 ° C. without shaking, and then incubated at 37 ° C. for 45 minutes with shaking. The culture broth was 4000 r. p. m. Centrifuge for 10 minutes at RT and resuspend the pellet in 2 liters of 2 × YT containing 100 μg / ml ampicillin and 50 ug / ml kanamycin and grow overnight. Phages are prepared as described in International Patent Specification No. WO 92/01047.

M13 delta gene III is prepared as follows. Since the M13 delta gene III helper phage does not encode the gene III protein, therefore, the phage displaying the antibody fragment (mid) has a binding activity for larger antibodies. Infectious M13 delta gene III particles are generated by growing helper phage in cells with pUC19 derivatives supplying wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37 ° C. without shaking and then for an additional hour at 37 ° C. with shaking. Cells are centrifuged (IEC-Centra 8,400 rpm for 10 minutes), resuspended in 300 ml of 2 × TY containing both 100 μg ampicillin / ml and 25 μg kanamycin / ml and shaken overnight. Gently grow at 37 ° C. The phage particles were purified, concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS, passed through a 0.45 μm filter (Minisart NML; Sartorius), approximately A final concentration of 10 ¹³ transduction units / ml was obtained (ampicillin-resistant clone).

Library panning. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg / ml or 10 μg / ml of an albumin fusion protein of the invention, or a portion of an albumin fusion protein of the invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37 ° C. and washed 3 times in PBS. Approximately 10 ¹³ TU of phage was applied to the tube and incubated at room temperature for 30 minutes with rotation on or under the turntable, then allowed to stand for 1.5 hours. The tube was washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. The phage is eluted by adding 1 ml of 100 mM triethylamine, rotating for 15 minutes on a turntable, and immediately neutralizing the solution with 0.5 ml of 1.0 M Tris-HCl, pH 7.4. The phage is then used to infect 10 ml of mid-log E. coli TG1 by incubating the eluted phage with bacteria for 30 minutes at 37 ° C. The E. coli is then plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then rescued with Delta gene 3 helper phage as described above to prepare phage for subsequent rounds of selection. This process is then repeated for a total of 4 rounds of affinity purification. For rounds 3 and 4, wash the tube 20 times with PBS, 0.1% Tween-20 and 20 times with pBS.

Bond characterization. Eluted phage from the third and fourth rounds of selection are used to infect E. coli HB2151, and soluble scFv is produced from a single colony for the assay (Marks, et al., 1991). The ELISA is performed on microtiter plates coated with either 10 pg / ml of the albumin fusion protein of the invention, or a portion of the albumin fusion protein of the invention, in 50 mM bicarbonate pH 9.6. ELISA positive clones are further characterized by PCT fingerprinting (see, eg, International Patent Specification No. WO 92/01047) followed by sequencing. These ELISA positive clones are still further known in the art, such as epitope mapping, binding affinity, receptor signaling, ability to block or competitively inhibit antibody / antigen binding, and competitive agonist or antagonist activity. It may be characterized by this technique.

Example 13: [ ³ H] -2-Deoxyglucose uptake assay

Adipose, skeletal muscle and liver are insulin-sensitive tissues. Insulin can stimulate glucose uptake / transport into these tissues. In the case of adipose and skeletal muscle, insulin initiates signaling that ultimately leads to the translocation of the glucose transporter 4 molecule, GLUT4, from the specified intracellular component to the cell surface. On the cell surface, GLUT4 allows glucose uptake / transport.
[ ³ H] -2-deoxyglucose uptake

Numerous adipose and muscle-related cell lines may be used in the absence or presence of any one or more therapeutic drug combinations listed for the treatment of diabetes in glucose uptake / transport. Testable for activity. In particular, 3T3-L1 murine fibroblast cell lines and L6 murine skeletal muscle cells are differentiated into 3T3-L1 adipocytes and myotubes, respectively, and appropriate in vitro for [ ³ H] -2-deoxyglucose uptake assays. Available as a model (Urso et al., J Biol Chem, 274 (43): 30864-73 (1999); Wang et al., J Mol Endocrinol, 19 (3): 241-8 (1997); Haspel et al., J Membr Biol, 169 (1): 45-53 (1999); Tsakiridis et al., Endocrinology, 136 (10): 4315-22 (1995)). Briefly, 2 × 10 ⁵ cells / 100 μL adipocytes or differentiated L6 cells were transferred to 96-well Tissue-Culture, “TC” treated plates coated with 50 mg / mL poly-L-lysine in post-differentiation medium, Incubate overnight at 37 ° C. in 5% CO ₂ . Cells are first washed once in low glucose DMEM medium without serum, then listed in 100 μL / well of the same medium and 100 μL / well of buffer for treatment of any one or more diabetics. In combination with increasing concentrations of therapeutic agents of the subject invention, such as 1 nM, 10 nM and 100 nM of the subject invention (eg, a specific fusion disclosed as SEQ ID NO: Y and fragments and variants thereof) In the presence, deplete at 37 ° C. for 16 hours. The plate is washed 3 times with 100 μL / well HEPES buffered saline. Insulin is obtained at 37 ° C. in the presence of 10 μM labeled [ ³ H] -2-deoxyglucose (Amersham, # TRK672) and 10 μM unlabeled 2-deoxyglucose (Sigma, D-3179). Add 1 nM in HEPES buffered saline for 30 minutes. As a control, the same conditions are carried out except in the absence of insulin. A final concentration of 10 μM cytochalasin B (SIGMA, C6762) is added at 100 μL / well in another well and non-specific uptake is measured. Cells are washed 3 times with HEPES buffered saline. Labeling, ie 10 μM [ ³ H] -2-deoxyglucose, and unlabeled, ie 10 μM 2-deoxyglucose, are added for 10 minutes at room temperature. Cells are washed three times with cold phosphate buffered saline “PBS”. Cells are lysed upon addition of 150 μL / well 0.2 N NaOH, followed by incubation for 20 minutes at room temperature with shaking. The sample is then transferred to a sonication vial and 5 mL of sonication solution is added. Vials are counted in a beta-scintillation counter. Uptake under dual conditions, difference in the absence or presence of insulin, the following equation [(Insulin counts per minute “cpm” −nonspecific cpm) / (no insulin cpm−nonspecific cpm) ] To determine. Average responses were within limits of about 5 and 3 times that of controls for adipocytes and myotubes, respectively.
Cell differentiation

Cells were allowed to become confluent in T-75 cm ² flasks. Remove media and replace with 25 mL of predifferentiation media for 48 hours. Cells are incubated at 37 ° C. in 5% CO ₂ , 85% humidity. After 48 hours, the pre-differentiation medium is removed and replaced with 25 mL differentiation medium for 48 hours. Cells are again incubated at 37 ° C. in 5% CO ₂ , 85% humidity. After 48 hours, the medium is removed and replaced with 30 mL post-differentiation medium. Post-differentiation media is maintained for 14-20 days or until full differentiation is achieved. The medium is changed every 2-3 days. Human adipocytes can be purchased from Zen-Bio, INC (# SA-1096).
Example 14

Into the pancreatic cell line [ ³ H] -Thymidine incorporation in vitro assay

GLP-1 induces differentiation of rat pancreatic ductal epithelial cell line ARIP in a time-and / or-times-dependent manner in association with increases in Islet Dual Homeobox-1 (IDX-1) and insulin mRNA levels Has recently been shown (Hui et al., 2001, Diabetes, 50 (4): 785-96). IDX-1 purely increases GLP-1 receptor mRNA levels.
Cell type tested

RIN-M cells : These cells are available from the American Type Tissue Culture Collection (ATCC Cell Line Number CRL-2057). The RIN-M cell line was derived from a radiation-induced transplantable rat islet cell tumor. Strains were established from tumor nude mouse xenografts. Cells produce and secrete islet polypeptide hormones and produce L-dopa decarboxylase (amine precursor uptake and decarboxylation, or APUD, a marker for cells with activity).

ARIP cells : There are epithelial forms of exocrine pancreatic cells available from the American Type Tissue Culture Collection (ATCC Cell Line Number CRL-1674). See also reference Jessop, N .; W. and Hay, R.A. J. et al. , “Characteristics of two pancreatic exocrine cell lines, derived from transplantable tumours”, In M. Vitro, 16: Mig. et al. , “Identification of a cell-specific DNA with binding intensive activity in the acinar pancreas, and identification of cell-specific DNA-binding activity. pancreas) ", Mol. Cell. Biol. 9: 2464-2476, (1989); Roux, E .; , Et al. “The cell-specific transcription factor RTF1 contains two different subunits that interact with DNA (The cell-specific transcription factor PTF1 contentious subunits that interact with the DNA)”. 3: 1613-1624 (1989); and Hui, H .; , Et al. , “Glucagon-like peptide 1 induces the differentiation of islet duodenal homeobox-1-positive pancreatic duct cells into insulin-secreting cells (Glucagon-like peptide 1 See also cells into insulin-secreting cells), Diabetes 50: 785-796 (2001).
Cell preparation

  The RIN-M cell line is grown in RPMI 1640 medium (Hyclone, # SH300027.01) containing 10% fetal calf serum (Hyclone, # SH30088.03) and 1: 3-1 : Subculture every 6-8 days at a ratio of 6 Change medium every 3-4 days.

ARIP (ATCC # CRL-1674) cell line was prepared to contain 1.5 g / L sodium bicarbonate and 10% fetal calf serum, Ham's F12K medium containing 2 mM L-glutamine (ATCC, # 30-2004). ) In. ARIP cell lines are subcultured twice a week at a ratio of 1: 3 to 1: 6. Change medium every 3-4 days.
Assay protocol

Cells are seeded at 4000 cells / well in 96-well plates and cultured for 48-72 hours to 50% confluence. Change cells to medium without serum at 100 mL / well. After incubation for 48-72 hours, serum and / or therapeutic agents of the subject invention (eg, albumin fusion proteins of the invention, and fragments and variants thereof) are added to the wells. Incubation is continued for an additional 36 hours. [ ³ H] -thymidine (5-20 ci / mmol) (Amersham Pharmacia, # TRK120) is diluted to 1 microcurie / 5 microliter. After 36 hours of incubation, 5 microliters are added per well for an additional 24 hours. The reaction is terminated by gently washing the cells once with cold phosphate buffered saline “PBS”. The cells are then fixed with 100 microliters of 10% ice-cold TCA for 15 minutes at 4 ° C. Remove the PBS and add 200 microliters of 0.2N NaOH. The plate is incubated for 1 hour at room temperature with shaking. Transfer the solution to a scintillation vial and add a scintillation liquid compatible with 5 mL of aqueous solution and mix vigorously. Virus is counted in a beta scintillation counter. As a negative control, only buffer is used. Fetal bovine serum is used as a positive control.

Example 15: Diabetes Assay

Diabetes (ie excess sugar in the urine) can be easily assayed to provide an indication of the disease state of diabetes. Compared to normal patient samples, excess urine in patient samples is a sign of IDDM and NIDDM. The effect of treatment of such patients with IDDM and NIDDM is suggested by the resulting reduction in the amount of excess glucose in the urine. In a preferred embodiment for IDDM and NIDDM monitoring, urine samples from patients are assayed for the presence of glucose using techniques known in the art. Diabetes in humans is defined by urinary glucose concentrations in excess of 100 mg per 100 ml. Excess sugar levels in patients exhibiting diabetes can be measured more accurately by obtaining blood samples and assaying serum glucose.

Example 16: Assay to detect stimulation or inhibition of B cell proliferation and differentiation

  The development of a functional humoral immune response requires soluble and similar signaling between B-lineage cells and their microenvironment. The signal may provide a positive stimulus that allows the B lineage cell to allow its programmed development, or a negative stimulus that directs the cell to stop its current developmental pathway. To date, a number of stimulatory and inhibitory signals have been responsive to B cells including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. It was found to affect the. Interestingly, these signals are themselves weak effectors, but in combination with various costimulatory proteins, induce activation, proliferation, differentiation, homing, resistance and death within the B cell population Is possible.

  One of the most studied B-cell costimulatory proteins is the TNF-superfamily. Within this family, it has been discovered that CD40, CD27 and CD30, along with their respective ligands CD154, CD70, and CD153, regulate various immune responses. Assays that allow the detection and / or observation of the proliferation and differentiation of these B cell populations and their precursors are in determining the possible effects of various proteins in these B-cell populations on proliferation and differentiation. It is a valuable tool. Listed below are two assays designed to allow detection of differentiation, proliferation or inhibition of B cell populations and their precursors.

  In vitro assays—Activation, proliferation of albumin fusion proteins of the invention (including therapeutic protein fragments or variants, and / or albumin or albumin fragments or variants) in B cell populations and their precursors Can be assessed for its ability to induce differentiation or inhibition and / or death. The activity of the albumin fusion protein of the present invention in purified human tonsil B cells, measured qualitatively in the range of 0.1 to 10,000 ng / mL / time, using purified tonsil B cells as a priming reagent, formalin Assess in a standard B-lymphocyte costimulation assay, cultured in the presence of fixed Streplococcus aureus Cowan1 (SAC) or immobilized anti-human IgM antibody. Second signals such as IL-2 and IL-15 synergize with SAC and IgM cross-linking and induce B cell proliferation as measured by tritiated-thymidine incorporation. New synergistic drugs can be easily identified using this assay. The assay involves isolating human tonsil cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by CD45R (B220) expression.

Various dilutions of each sample are placed in individual wells of a 96-well plate and in culture medium (10% FBS, 5 × 10 ⁻⁵ M 2ME, 100 U / ml penicillin, 10 ug / ml streptomycin in a total volume of 150 ul. , And 10 ⁵ B-cells suspended in RPMI 1640 containing 10 ⁻⁵ dilution of SAC). Growth or inhibition is quantified by a 20 hour pulse (1 uCi / well) with 3H-thymidine (6.7 Ci / mM) starting 72 hours after factor addition. Positive and negative controls are IL2 and medium, respectively.

  In vivo assay-BALB / c mice contain buffer only or 2 mg / kg (including therapeutic protein fragments or variants, and / or fusion proteins comprising albumin or albumin fragments or variants) The albumin fusion protein is injected (ip) twice a day. Mice were given this treatment for 4 consecutive days, at which time they were sacrificed and various tissues and sera were collected for analysis. Comparison of H & E sections from normal spleen and spleens treated with the albumin fusion protein of the present invention shows a significant increase in nucleic acid in the peripheral artery lymphatic sheath and / or nucleated cytoplasm in the red pulp region The result of the activity of the fusion protein in the cells may be suggested and activation of differentiation and proliferation of the B-cell population may be suggested. Using immunohistochemical studies with the B cell marker, anti-CD45R (B220), any physiological changes to spleen cells, such as spleen destruction, loosely define that infiltrate established T cell regions Determine whether it is due to an increase in B-cell expression in the treated B-cell zone.

  Flow cytometric analysis of spleens from mice treated with albumin fusion protein showed that albumin fusion protein specifically identified a population of ThB +, CD45R (B220) dull B cells, compared to that observed in control mice. Used to suggest whether to increase.

Similarly, the expected result of increased mature B cell expression in vivo is a relative increase in serum Ig titer. Therefore, serum IgM and IgA levels are compared between buffer and fusion protein treated mice.

Example 17: T cell proliferation assay

CD3-induced proliferation assay is performed on PBMC and measured by ³ H-thymidine incorporation. This assay is performed as follows. A 96 well plate was coated overnight at 4 ° C. with a mAb against 100 ml / well CD3 (HIT3a, Pharmingen), or an isotype matched control mAb (B33.1) (0.05 M bicarbonate buffer, pH 9). 1 mg / ml in 5), then washed 3 times with PBS. PBMCs are isolated from human peripheral blood by F / H gradient centrifugation and include various concentrations (including therapeutic protein fragments or variants and / or fusion proteins comprising albumin or albumin fragments or variants) (5 × 10 ⁴ / well) in RPMI containing 10% FCS and P / S (5 × 10 ⁴ / well) (total volume 200 ul). Only relevant protein buffers and media are controls. After 48 hours of incubation at 37 ° C., the plate is spun for 2 minutes at 1000 rpm, 100 ml of supernatant is removed, and the effect on proliferation is observed at −20 ° C. at IL-2 (or other cytokines) ) Save for measurement. Wells are filled with 100 ul medium containing 0.5 uCi ³ H-thymidine and incubated at 37 ° C. for 18-24 hours. Wells are collected and ³ H-thymidine incorporation is used as a measure of proliferation. Only anti-CD3 is a positive control for proliferation. IL-2 (100 U / ml) is also used as a control to enhance proliferation. A control antibody that does not induce T cell proliferation is used as a negative control for the effect of the fusion protein of the invention.

Example 18: Effect of the fusion protein of the invention on monocyte and monocyte-derived human dendritic cell MHC class II expression, costimulatory and adhesion molecules, and cell differentiation

  Dendritic cells are produced by the expression of proliferative progenitors found in peripheral blood. Adherent PBMC or washed-out monocyte fractions are cultured with GM-CSF (50 ng / ml) and IL-4 (20 ng / ml) for 7-10 days. These dendritic cells have a characteristic phenotype of immature cells (CD1, CD80, CD86, CD40 and type II MHC antigens). Treatment with an activator such as TNF-a causes rapid changes in the surface phenotype (type I and type MHC, co-stimulation and increased expression of adhesion molecules, FCγRII down-regulation, CD83 up-regulation) . These changes correlate with increased antigen presentation capacity and functional maturation of dendritic cells.

  FACS analysis of surface antigen is performed as follows. Cells are treated with increasing concentrations of the albumin fusion protein of the invention, or LPS (positive control) for 1-3 days, washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then the appropriate FITC- Alternatively, incubate for 30 minutes at 4 ° C. with a 1:20 dilution of PE-labeled monoclonal antibody. After further washing, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect of cytokine production Cytokines produced by dendritic cells, especially IL-12, are important in the initiation of T cell-dependent immune responses. IL-12 strongly influences the development of ThI helper T-cell immune responses and induces cytotoxic T and NK cell functions. An ELISA is used to measure IL-12 release as follows. Dendritic cells (10 ⁶ / ml) are treated for 24 hours with increasing concentrations of the albumin fusion protein of the invention. LPS (100 ng / ml) is added to the cell culture as a positive control. The supernatant from the cell culture is then collected and analyzed for IL-12 content using a commercially available ELISA kit (eg, R & D Systems (Minneapolis, Minn.)). Use the standard protocol specified.

Effects on type II MHC, costimulatory and adhesion molecule expression Three major families of cell surface antigens, adhesion molecules, molecules involved in antigen presentation, and Fc receptors can be identified as monocytes. Modulating the expression of type II MHC antigens or other costimulatory molecules such as B7 and ICAM-1 results in a change in monocyte antigen-presenting capacity and a change in the ability to induce T cell activation. sell. Increased Fc receptor expression may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

  Using FACS analysis, surface antigens are tested as follows. Monocytes are treated with increasing amounts of the albumin fusion protein of the invention or LPS (positive control) for 1-5 days, washed with PBS containing 1% BSA and 0.02 mM sodium azide, then appropriate FITC -Or incubate for 30 minutes at 4 ° C with a 1:20 dilution of PE-labeled monoclonal antibody. After further washing, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Increased monocyte activation and / or survival Assays for molecules that activate (or inactivate) monocytes and / or increase monocyte survival (or decrease monocyte survival) are described in the art. It is known in the art and is applied routinely to determine whether the molecules of the invention function as monocyte inhibitors or activators. The albumin fusion proteins of the invention can be screened using the three assays described below. For each of these assays, peripheral blood mononuclear cells (PBMC) were isolated from a single donor leucopack (American Red Cross) by centrifugation through a Histopaque gradient (Sigma). ), Baltimore, MD) Monocytes are isolated from PBMC by counterflow centrifugal elution.

Monocyte Survival Assay Human peripheral blood monocytes gradually lose viability when cultured in the absence of serum or other stimuli. These deaths are due to internal control processes (apoptosis). The addition of an activator such as TNF-alpha to the culture dramatically improves cell survival and prevents DNA fragmentation. Using propidium iodide (PI) staining, apoptosis is measured as follows. Monocytes are 48 hours in polypropylene tubes in the presence of 100 ng / ml TNF-alpha (negative control), in serum-free medium (positive control), and in the presence of various concentrations of fusion protein to be tested. Incubate for hours. Cells are suspended at a final concentration of 5 μg / ml at a concentration of 2 × 10 ⁶ / ml in PBS containing PI and then incubated at room temperature for 5 minutes prior to FACScan analysis. PI uptake has been shown to correlate with DNA fragmentation in this experimental paradigm.

Effect of cytokine release An important function of monocytes / macrophages is their regulatory activity in other cell populations of the immune system via post-stimulation cytokine release. To measure cytokine release, an ELISA is performed as follows. Human monocytes are incubated with increasing amounts of the albumin fusion protein of the invention and in the same conditions but in the absence of the fusion protein at a density of 5 × 10 ⁵ cells / ml. For IL-12 production, cells are primed overnight with IFN (100 U / ml) in the presence of the fusion protein. LPS (10 ng / ml) is then added. Conditioned media is collected after 24 hours and frozen until use. The measurement of TNF-alpha, IL-10, MCP-1 and IL-8 was then performed using a commercially available ELISA kit (eg, R & D Systems (Minneapolis, Minn.)) And a standard protocol provided with the kit. It is implemented by applying.

Oxidative disruption purified monocytes are seeded in 96-w plates at 2-1 × 10 ⁵ cells / well. Increasing concentrations of the albumin fusion protein of the invention are added to wells in a total volume of 0.2 ml culture medium (RPMI1640 + 10% FSC, glutamine and antibiotics). After 3 days of incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayer, 0.2 ml / well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U / ml HRPO) was added to the stimulant ( 200 nM PMA). The plate is incubated for 2 hours at 37 ° C. and the reaction is stopped by adding 20 μl 1N NaOH / well. Read absorbance at 610 nm. In order to calculate the amount of H ₂ O ₂ produced by macrophages, a standard curve of known molar amounts of H ₂ O ₂ solution is performed in each experiment.

Example 19: Effect of albumin fusion protein of the present invention on proliferation of vascular endothelial cells

On the first day, human umbilical cord blood endothelial cells (HUVEC) were collected from 4% fetal bovine serum (FBS), 16 units / ml heparin, and 50 units / ml endothelial cell growth supernatant (ECGS, Biotechnique, Inc. .)) In M199 medium containing 2-5 × 10 ⁴ cells / 35 mm dish concentration. On the second day, the medium is changed to M199 containing 10% FBS, 8 units / ml heparin. Albumin fusion proteins of the invention and positive controls such as VEGF and basic FGF (bFGF) are added at various concentrations. On day 4 and 6, change medium. On the 8th day, the number of cells is measured with a Coulter Counter.

An increase in the number of HUVEC cells suggests that the fusion protein can proliferate vascular endothelial cells, and a decrease in the number of HUVEC cells suggests that the fusion protein inhibits vascular endothelial cells.

Example 20: Rat corneal wound healing model

This animal model shows the effect of the albumin fusion protein of the invention on neovascularization. The experimental protocol includes
Making a 1-1.5 mm long incision from the center of the cornea into the stroma,
Inserting a spatula under the incision lip facing the outer corner of the eye,
Creating a pocket (its base is 1-1.5 mm from the edge of the eye),
Placing a pellet containing 50 ng to 5 ug of the albumin fusion protein of the invention in the pocket is included. Treatment with albumin fusion proteins of the present invention can also be applied topically to corneal wounds at a dose range of 20 mg to 500 mg (5 days daily administration) / dose.

Example 21: Diabetic mouse and glucocorticoid injury wound healing model
Diabetes db + / db + mouse model

  In order to demonstrate that the albumin fusion protein of the present invention promotes the healing process, a genetic diabetes mouse model of wound healing is used. The full thickness wound healing model in db + / db + mice is well characterized and is a clinically relevant and reproducible model of wound wound healing. Diabetic wound healing depends on granule tissue formation and re-epithelialization rather than contraction (Gartner, MH et al., J. Surg. Res. 52: 389 (1992); Greenhalgh, DG). Et al., Am. J. Pathol. 136: 1235 (1990)).

  Diabetic animals have many characteristic properties that are observed in type II diabetes. Homozygous (db + / db +) mice are obese compared to their normal heterozygous (db + / + m) siblings. Mutant diabetic (db + / db +) mice have a single autosomal recessive mutation on chromosome 4 (db +) (Coleman et al. Proc. Natl. Acad. Sci. USA 77: 283-293 (1982)). . Animals show bulimia, polyposis and polyuria. Mutant diabetic mice (db + / db +) have elevated blood glucose, increased insulin levels or are normal, and cell-mediated immunity is suppressed (Mandel et al., J. Immunol. 120: 1375 (1978)). Deray-Sachs, M. et al., Clin. Exp. Immunol. 51 (1): 1-7 (1983); Leiter et al., Am. J. of Pathol. 114: 46-55 (1985)). . Peripheral neuropathy, myocardial complications, and microvascular injury, basement membrane hypertrophy and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83 (2): 221- Robertson et al., Diabetes 29 (1): 60-67 (1980); Giacomelli et al., Lab Invest. 40 (4): 460-473 (1979); Coleman, D.L. Diabetes 31 (Suppl): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is similar to insulin resistance to human type II diabetes (Mandel et al., J. Immunol. 120: 1375-1377 (1978)).

  The characteristics observed in these animals suggest that healing in this model may be similar to that observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136). : 1235-1246 (1990)).

  Genetically diabetic female C57BL / KsJ (db + / db +) mice and their non-diabetic (db + / + m) heterozygous siblings are used in this study (Jackson Laboratories) Animals are purchased at 6 weeks of age. Start the study at 8 weeks of age. ) In accordance with the rules and guidelines of the Institutional Animal Care and Use Committee and the Guidelines for the Care of Use of Laboratory Animals Carry out.

  The wound protocol is performed according to the previously repeated method (Tsuboi, R. and Rifkin, DB, J. Exp. Med. 172: 245-251 (1990)). Briefly, on the day of wounding, the animals were intraperitoneally injected with avertin (0.01 mg / mL), 2,2,2-tribromomethanol and 2-methyl-2-butanol dissolved in deionized water. Anesthetize with injection. The back of the animal is shaved and the skin is washed with 70% ethanol ions and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full thickness wound is then created using a Keyes tissue punch. Immediately after wounding, the surrounding skin is gently stretched to remove wound spread. The wound is left open during the experiment. The treatment is applied topically for the first 5 consecutive days on the day of the wound. Prior to treatment, the wound is gently cleaned with saline and gauze sponge.

  Wounds are visually examined and photographed at a fixed distance on the day of surgery, and at intervals of 2 days thereafter. Wound suture is measured by daily measurements on days 1-5 and 8th. Wounds are measured horizontally and vertically using balanced Jameson calipers. A wound is considered healed when granular tissue is no longer seen and the wound is covered by a continuous epithelium.

  The albumin fusion proteins of the invention are administered in excipients using different ranges / times of 4 mg to 500 mg / wound per day for 8 days. The vehicle control group is given 50 mL of vehicle solution.

  The animals are anesthetized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then harvested for histology and immunohistochemistry. Tissue sections are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

  Three groups of 10 animals each (5 diabetics, 5 day diabetic controls) are evaluated. 1) vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area in the vertical and horizontal axes to obtain a square area of the wound. Shrinkage is then estimated by establishing the difference between the initial wound area (Day 0) and after treatment (Day 8). The wound area on the first day is 64 mm ² , corresponding to the size of the skin punch. The calculation is performed using the following formula.
a. [Opening area on the 8th day]-[Opening area on the 1st day] / [Opening area on the 1st day]

  Specimens are fixed with 10% buffered formalin and paraffin-embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using Reichert-Jung slices. Routine hematoxylin-eosin (H & E) staining is performed on the bisected wound section. A wound histological examination is used to assess whether the healing process and the morphological appearance of the repaired skin have been altered by treatment with the albumin fusion protein of the present invention. This assessment included the presence of cell accumulation, confirmation of inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturation (Greenhalgh, DG et al., Am. J. Pathol. 136: 1235 (1990)). A calibrated lens micrometer is used by blind inspectors.

  Tissue sections are also immunohistochemically stained with polyclonal rabbit anti-human keratin antibodies using the ABC Elite detection system. Human skin is used as a positive tissue control and non-immune IgG is used as a negative control. Keratinocyte proliferation is determined by assessing the extent of re-epithelialization using a calibrated lens micrometer.

  Proliferating cell nuclear antigen / cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) in an ABC Elite detection system. Human colon cancer is used as a positive tissue target, and human brain tissue is used as a negative tissue target. Each specimen contained sections where the first antibody was omitted and replaced with non-immune mouse IgG. The ranking of these sections is based on the degree of proliferation on a scale of 0-8, with a low ranking reflecting slight proliferation and a high ranking reflecting intense proliferation.

Experimental data is analyzed using unpaired t-test. A p value of <0.05 is considered significant.
Steroid injury rat model

  Inhibition of wound healing by steroids has been well documented in the in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical 8 1988). Wahlet al., J. Immunol.115: 476-481 (1975); Werb et al., J. Exp. Med.147: 1684-1694 (1978)). Inhibition of angiogenesis, delayed healing of wounds caused by glucocorticoids due to reduced vascular permeability (Ebert et al., An. Inter. Med. 37: 701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)), and producing a transient decrease in cyclic monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammation. Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). Systemic administration of steroids for impaired wound healing is a well-established decrease in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiflammatory Steroid Action: Basic and Clinical Aspects, ace. Natl.Acad.Sci.USA 86: 2229-223 (1989)).

  To demonstrate that the albumin fusion protein of the present invention can accelerate the healing process, the effect of multiple topical application of the fusion protein in full-thickness cutaneous wounds in rats where healing does not function normally by systemic administration of methylprednisolone Assess.

  Young adult male Sprague Dawley rats (Charles River Laboratories) weighing 250-300 g are used in this example. Animals are purchased at 8 weeks of age and the study begins at 9 weeks of age. Rat healing response is impaired by methylprednisolone (17 mg / kg / rat intramuscular). Animals are individually fed and given food and water. All manipulations are performed using aseptic techniques. The experiments are conducted according to the Human Genome Sciences, Inc. Institute of Animal Care and Use Committee and the Guides for the Care of Use of Laboratories and implementation guidelines.

  The wound protocol is performed according to what is described above. On the day of wounding, the animals are anesthetized with an intramuscular injection of ketamine (50 mg / kg) and xylazine (5 mg / kg). The back of the animal is shaved and the skin is washed with 70% ethanol ions and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full thickness wound is then created using a Keyes tissue punch. The wound is left open during the experiment. Application of the test substance is carried out locally for the first time on the wound day, once a day for 7 consecutive days, followed by administration of methylprednisolone. Prior to treatment, the wound is gently cleaned with saline and gauze sponge.

  The wound is visually examined and photographed at a fixed distance on the day of surgery and the last day of treatment. Wound suture is measured by daily measurements on days 1-5 and 8th. Wounds are measured horizontally and vertically using balanced Jameson calipers. A wound is considered healed when granular tissue is no longer seen and the wound is covered by a continuous epithelium.

  The fusion proteins of the invention are administered in excipients using different ranges / times of 4 mg to 500 mg / wound per day for 8 days. The vehicle control group is given 50 mL of vehicle solution.

  The animals are anesthetized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then harvested for histology and immunohistochemistry. Tissue sections are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

  Three groups of 10 animals each (5 methylprednisolone doses, 5 days without glucocorticoid) are evaluated. 1) vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area in the vertical and horizontal axes to obtain a square area of the wound. Shrinkage is then estimated by establishing the difference between the initial wound area (Day 0) and after treatment (Day 8). The wound area on the first day is 64 mm ² , corresponding to the size of the skin punch. The calculation is performed using the following formula.
a. [Opening area on the 8th day]-[Opening area on the 1st day] / [Opening area on the 1st day]

  Specimens are fixed with 10% buffered formalin and paraffin-embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using Olympus slices. Routine hematoxylin-eosin (H & E) staining is performed on the bisected wound section. A wound histological test is used to assess whether the healing process and the morphological appearance of the repaired skin were improved by treatment with the albumin fusion protein of the present invention. A calibrated lens micrometer is used by the blind inspector to determine the distance of the wound gap.

Experimental data is analyzed using unpaired t-test. A p value of <0.05 is considered significant.

Example 22: Lymphedema animal model

  The purpose of this experimental approach is to create an appropriate and consistent model of lymphedema to test the therapeutic effect of the albumin fusion protein of the present invention on lymphogenesis in the rat hind paws and reconstruction of the lymphatic circulation That is. The effect is measured by an inflated dose of the affected paw, quantification of the amount of lymphatic vasculature, total plasma protein and histology. Acute lymphedema is observed for 7-10 days. Perhaps more important is the chronic progression of edema that lasts up to 3-4 weeks.

  Before starting surgery, blood samples are withdrawn for protein concentration analysis. And pentobarbital is administered to male rats weighing ˜350 g. Next, the right foot is shaved from the knee to the buttocks. Wipe the scraped area with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Peripheral and volumetric measurements are performed after making two measurement levels and before injecting the dye into the leg (0.5 cm above the heel, mid pt on the back leg). 0.05 ml of 1% Evan's Blue is injected into the intradermal back of both the right and left legs. Peripheral and volumetric measurements are then performed following dye injection into the leg.

  Using the knee joint as a landmark, a mid-leg groin incision is made to allow the femoral blood vessels to be localized around. Using forceps and hemostatic forceps, dissect and separate the flap. After positioning the femoral vessels, the lymph vessels running along the side and bottom of the vessel are positioned. The main lymphatic vessels in this area are then electrocoagulated or tied with sutures.

  Using a microscope, gently dissect the muscles behind the legs (near the semi-tendonoid and adductor muscles). Next, position the popliteal lymph node. The distal vascular supply of the two proximal and two distal lymphatics and popliteal lymph nodes are then tied by suture. Popliteal lymph nodes and any surrounding adipose tissue are then removed by cutting the connective tissue.

  Care is taken to control the mild bleeding as a result of this procedure. After the lymphatics are occluded, the skin flap is sealed by using liquid skin (Vetbond (AJ Buck)). The separated skin edge is sealed to the underlying muscle tissue, leaving a gap of ~ 0.5 cm around the leg. The skin may also be connected by stitching to the underlying muscle if necessary.

  To avoid infection, animals are individually covered with mesh (no mating). Recovered animals are confirmed daily via the optimal edema peak and typically occur by 5-7 days. The plateau edema peak is then observed. To assess the intensity of lymphedema, the circumference and volume of the two designed parts on each leg are measured before surgery and daily for 7 days. Determine the effects of plasma proteins in lymphedema and investigate whether protein analysis is a useful perimeter. The weight of both the subject and edema leg will be assessed at two locations. The analysis is performed in a blind manner.

Perimeter measurement: Measure the circumference of the leg using a cloth tape measure under simple gas anesthesia to prevent leg movement. Measurements are taken by the two different persons at the talus and back leg and the two readings are averaged. Reads are taken from both subject and edema leg.

Volumetric measurement: On the day of surgery, animals are anesthetized with pentobarbital and tested prior to surgery. For daily volume, animals are briefly anesthetized with halothane (rapid fixation and quick recovery), shaved on both legs, and marked equally with waterproof markers on the legs. The leg is first submerged in water and then immersed in the instrument to the respective recorded level and measured by Buxco edema software (Chen / Victor). One person records the data and immerses the leg to the area where the other person recorded.

Blood-plasma protein measurement: Blood is drawn, spun, and serum is separated prior to surgery, followed by total protein measurement and Ca2 ⁺ comparison.

Leg weight comparison: After drawing blood, animals are prepared for tissue collection. The legs are cut with Kiritin and both the experimental and control legs are cut with a ligature and weighed. A second weighing is performed when the ticanal joint is disassembled and the legs are weighed.

Histological adjustment: the transverse muscle located behind the knee (popliteal) area is cut, placed in a metal mold, filled with freezeGal, soaked in cold methylbutane, in a recorded sample bag, until sectioning , Enter at -80EC. Upon sectioning, the muscle is observed for lymphatic vessels under a fluorescence microscope.

Example 23: Suppression of TNF-induced adhesion molecule expression by the albumin fusion protein of the present invention

  The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between the cell surface adhesion molecule (CAM) on lymphocytes and the vascular endothelium. Adhesion processes in both normal and pathological settings include intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecules on endothelial cells (EC) ( E-selectin) follows a multi-stage cascade involving expression. These molecules and other expression on the vascular endothelium determine the effect of leukocytes adhering to local blood vessels and leaching into local cells during the development of the inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

  Tumor necrosis factor alpha (TNF-a), a potent inflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and can be involved in a wide variety of inflammatory responses, often pathological Result.

  It is possible to test the potential of the albumin fusion proteins of the invention that mediate suppression of TNF-a induced CAM expression. A modified ELISA assay using EC as a solid phase adsorbent is used to measure the amount of CAM expression in TNF-a treated ECs when costimulating with members of the FGF protein family.

To perform the experiment, human umbilical cord blood endothelial cell (HUVEC) broth was obtained from a stored umbilical cord collection and contained 10% FCS and 1% penicillin / streptomycin in a 37 ° C. humidified incubator containing 5% CO _2. Maintain in growth medium (EGM-2, Clonetics, San Diego, Calif.). HUVECs are seeded in 96-well plates at a concentration of 1 × 10 ⁴ cells / well in EGM medium at 37 ° C. for 18-24 hours or until confluence. The monolayer was subsequently washed three times with a serum-free solution of RPMI-1640 containing 100 U / ml penicillin and 100 mg / ml streptomycin, and the cytokine and / or growth factor (s) at 37 ° C. for 24 hours. To process. After incubation, the cells are then evaluated for CAM expression.

Human umbilical cord blood endothelial cells (HUVEC) are grown in standard 96-well plates until confluence. Growth medium is removed from the cells and replaced with 90 ul of 199 medium (10% FBS). Samples for testing and positive or negative controls are added to the plates in triplicate (10 ul volume). Plates are incubated at 37 ° C for either 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). Plates are aspirated to remove media and 100 μl 0.1% paraformaldehyde-PBS (containing Ca ⁺⁺ and Mg ⁺⁺ ) is added to each well. The plate is maintained at 4 ° C. for 30 minutes.

  Next, Fixative is removed from the well, and the well is washed with 1 × PBS (Ca, Mg) + 0.5% BSA and drained. Do not allow the wells to dry. Add 10 μl of diluted primary antibody to test and control wells. Anti-ICAM-1-biotin, anti-VCAM-1-biotin and anti-E-selectin-biotin are used at a concentration of 10 μg / ml (1:10 dilution of 0.1 mg / ml stock antibody). Cells are incubated for 30 minutes at 37 ° C. in a humid environment. The wells are washed with x3, PBS (+ Ca, Mg) + 0.5% BSA.

Then 20 μl of diluted ExtrAvidin-Alkaline Phosphate (1: 5,000 dilution) is added to each well and incubated at 37 ° C. for 30 minutes. The wells are washed with x3, PBS (+ Ca, Mg) + 0.5% BSA. Dissolve 1 tablet of p-nitrophenol phosphate pNPP in 5 ml glycine buffer (pH 10.4). 100 μl pNPP substrate in glycine buffer is added to each test well. Triplicate, standard wells are prepared from working dilutions of ExtrAvidin-Alkaline Phosphate in glycine buffer. 1: 5,000 (10 ⁰ )> 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 . 5 μl of each dilution is added to the triple well, and the AP content obtained in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each standard well. Plates must be incubated for 4 hours at 37 ° C. A volume of 50 μl of 3M NaOH is added to all wells. Results are quantified on a plate reader at 405 nm. The background deduction option is used on blank wells filled with glycine buffer only. A template is set to indicate the concentration of AP-conjugate in each standard well [5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng]. Results are shown as examples of bound AP-conjugate in each sample.

Example 24: Construction of GAS reporter construct

  One signaling pathway involved in cell differentiation and proliferation is called the Jaks-STATs pathway. Activating proteins in the Jaks-STATs pathway bind to gamma active site “GAS” elements, or interferon-sensitive response elements (“ISRE”), which are located within the promoters of many genes. Protein binding to these elements alters the expression of related genes.

  GAS and ISRE elements are recognized by a class of transcription factors called signal transducers and activators of transcription ("Signal Transducers and Activators of Transcribion") or "STATs". There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, such as Stat2 (and the response to IFN-alpha is widespread. Stat4 is more restricted and not present in many cell types, but type 1 Found in cells after treatment with T helper, IL-12, Stat5 was originally called mammalian growth factor, but has been found at higher concentrations in other cells, including thyroid cells. It can be activated in tissue culture cells by many cytokines.

  STATs are activated and translocated from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represents a different family of soluble tyrosine kinases and includes Tyk2, Jak1, Jak2, and Jak3. These kinases show significant sequence similarity and are generally catalytically inactive in the rest of the cell.

  Jaks is activated by a wide range of receptors summarized in the table below (adapted from a review by Schidler and Darnell, Ann. Rev. Biochem. 64: 621-51 (1995)). The cytokine receptor family capable of activating Jaks is divided into two groups. (A) Class 1 includes IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, Receptors for G-CSF, GM-CSF, LIF, CNTF and thrombopoietin are included. And (b) Class 2 includes IFN-a, IFN-g, and IL-10. Class 1 receptors have a conserved cysteine motif (a set of four conserved cysteines and a tryptophan) and a WSXWS motif (membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO: 53)). Share.

  Thus, upon binding of the ligand to the receptor, Jaks are activated, followed by activation of STATs, which then translocate and bind to the GAS element. This entire process is included in the Jaks-STATs signaling pathway. Thus, activation of the Jaks-STATs pathway, reflected by the binding of GAS or ISRE elements, can be used to suggest proteins involved in cell proliferation and differentiation. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway (see Table 5 below). Thus, by using a GAS element linked to a reporter molecule, an activator of the Jaks-STATs pathway can be identified.

  To construct the synthetic GAS-containing promoter elements used in the biological assays described in Examples 27-29, a PCR-based strategy is used to produce the GAS-SV40 promoter sequence. Although other GAS or ISRE elements can be used instead, the 5 ′ primer is found in the IRF1 promoter and previously (Rothman et al., Immunity 1: 457-468 (1994)) a wide range of cytokines It contains four tandem copies of the GSA binding site that have been shown to bind to STATs upon induction with. The 5 'primer also contains a sequence 18 bp complementary to the SV40 early promoter sequence and flank the XhoI site. The sequence of the 5 'primer is 5': GCCGCCTCGAGATTTCCCGAAATCTAGATTTCCCGAGAAATGATTTCCCCCAAAATGATTCCCCGAAAATTCTGCCATCTCAATTTAG: 3 '(SEQ ID NO: 54).

  The downstream primer is complementary to the SV40 promoter and is adjacent to the HindIII site. 5 ': GCGGCAAGCTTTTTGCAAAGCCTAGGC: 3' (SEQ ID NO: 55).

PCR amplification is performed using the SV40 promoter template present in the B-gal: promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI / HindIII and subcloned into BLSK2- (Stratagene.). Sequencing with the forward and reverse primers confirms that the insert contains the following sequence: 5 ': CTCGAG ATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA AAGCTT: 3 ' ( SEQ ID NO: 56)

  At this GAS promoter element linked to the SV40 promoter, the GAS: SEAP2 reporter construct is then modified. Here, the reporter molecule is secreted alkaline phosphatase, or “SEAP”. However, clearly in this example or any other example, any reporter molecule may be substituted for SEAP. Well known reporter molecules that can be used in place of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescein protein (GFP), or detectable by antibodies Any protein is included.

  With the above sequence, the synthetic GAS-SV40 promoter element is effectively replaced with the proliferating GAS: SV40 promoter element to create a GAS-SEAP vector, and using Clonetech with HindIII and XhoI Was subcloned into the pSEAP-Promoter vector obtained from However, this vector does not contain a neomycin resistance gene and is therefore not preferred for mammalian expression systems.

  Thus, to produce a mammalian stable cell line expressing the GAS-SEAP reporter, the GAS-SEAP cassette was removed from the GAS-SEAP vector using SalI and NotI and these within the multiple cloning site. Using a restriction site, it inserts into a backbone vector containing a neomycin resistance gene, such as pGFP-1 (Clontech), to produce a GAS-SEAP / Neo vector. Once this vector has been transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding, as described in Examples 27-29.

Other constructs can be made using the above description and replacing the GAS with a different promoter sequence. For example, reporter molecule constructs containing EGR and NF-KB promoter sequences are described in Examples 27-31. However, many other promoters can be replaced using the protocols described in these examples. For example, the SRE, IL-2, NFAT, or osteocalcin promoter, alone or in combination (GAS / NF-KB / EGR, GAS / NF-KB, Il-2 / NFAT, or NF-KB / GAS), It can be replaced. Similarly, other cell lines can be used, such as HELA (epithelium), HUVEC (endothelium), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aorta), or cardiomyocytes, Reporter construct activity can be tested.

Example 25: Assay for SEAP activity

  As a reporter molecule for the assays described in the Examples disclosed herein, SEAP activity is assayed using Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure . The Tropix Phospho-light Kit supplies dilutions, assays, and reaction buffers using:

  A dispenser is prepared with 2.5 × Dilution Buffer and 15 ul of 2.5 × dilution buffer is dispensed into an Optiplate containing a solution containing 35 ul of the albumin fusion protein of the present invention. The plate is sealed with a plastic sealer and incubated at 65 ° C. for 30 minutes. Separate the Optiplate to avoid irregular heating.

  Cool the sample to room temperature for 15 minutes. Empty the dispenser and prepare the assay buffer. Add 50 ml assay buffer and incubate at room temperature for 5 minutes. Empty the dispenser and prepare the reaction buffer (see table below). Add 50 ul reaction buffer and incubate for 20 minutes at room temperature. Since the chemiluminescent signal intensity is time dependent, it takes about 10 minutes to read 5 plates on the luminometer, thus processing 5 plates at each time point and starting the second set after 10 minutes.

  Read the relative light unit in the luminometer. Set H12 to blank and print the result. Increased chemiluminescence suggests reporter activity.

Example 26: Assay to identify neuronal activity

  When cells are differentiated and proliferating, a group of genes is activated through many different signaling pathways. One of these genes, EGR1 (Early Proliferation Response Gene 1), is induced in various tissues and cell types upon activation. The EGR1 promoter is responsive to such significant pathways. The EGR1 promoter linked to a reporter molecule can be used to assess the ability of the fusion proteins of the invention to activate cells.

  In particular, the following protocol is used to assess neuronal activity in the PC12 cell line. PC12 cells (rat pheochromocytoma cells) proliferate upon activation with a number of mitogenic factors such as TPA (tetradecanoylphorbol acetate), NGF (nerve growth factor) and EGF (epidermal growth factor). And / or is known to differentiate. EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to a SEAP reporter, activation of PC12 cells by the albumin fusion protein of the present invention can be assessed.

The EGR / SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (−633 to +1) (Sakamoto K et al., Oncogene 6: 867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers.
First primer: 5 ′ GCGCTCGAGGGGATGACAGCGATAGAACCCCGG-3 ′ (SEQ ID NO: 57)
Second primer: 5 ′ GCGAAGCTTCGCGCACTCCCCGGATCCGCTCTC-3 ′ (SEQ ID NO: 58)

  Using the GAS: SEAP / Neo vector produced in Example 24, the EGR1 amplification product can then be inserted into this vector. Linearization of GAS: SEAP / Neo vector with restriction enzymes XhoI / HindIII, removal of GAS / SV40 stuffer. EGR1 amplification products are restricted with these same enzymes. Ligate the vector and the EGR1 promoter.

  To prepare a 96-well plate for cell culture, 2 ml of coating solution (1:30 dilution of collagen type I in 30% ethanol (Upstate Biotech Inc.) catalog number 08- 115) (filter sterilization)) is added per 10 cm plate or per well of a 96-well plate and allowed to air dry for 2 hours.

  PC12 cells were pre-coated on 10 cm tissue culture dishes with 10% horse serum (JRH BIOSCIENCES, catalog number 12449-78P), 5% heat-inactivated fetal bovine serum (100 RH / ml penicillin and 100 ug / ml streptomycin) Growing daily in RPMI-1640 medium (Bio Whitaker) with FBS). Perform 1-4 splits every 3-4 days. Cells are removed from the plate by scraping and resuspended by pipetting for more than 15 minutes.

  The EGR / SEAP / Neo construct is transfected into PC12 using techniques known in the art. EGR-SEAP / PC12 stable cells are obtained by growing the cells in 300 ug / ml G418. Medium without G418 should be used for daily growth, but every 1-2 months, and cells should be regrown in 300 ug / ml G418 for passage.

  To assay for neuronal activity, 10 cm plates containing 70-80% confluent cells are screened by removing old media. Cells are washed once with PBS (phosphate buffered saline). It is then craved overnight in RPMI-1640 containing low serum medium (1% horse serum with antibiotics and 0.5% FBS).

The next morning, the medium is removed and the cells are washed with PBS. Scrape the cells from the plate and suspend the cell wells in 2 ml low serum medium. Count the number of cells and add additional low serum medium until a final cell density of 5 × 10 ⁵ cells / ml is reached.

200 ul of cell suspension is added to each well of a 96-well plate (equivalent to 1 × 10 ⁵ cells / well). A series of different concentrations of albumin protein of the invention are added at 37 ° C. for 48-72 hours. As a positive control, growth factors known to activate PC12 cells via EGR, such as 50 ng / ul neuronal growth factor (NGF), can be used. More than 50-fold induction of SEAP is typically seen in positive control wells. SEAP assays may be routinely performed using techniques known in the art and / or as described in Example 25.

Example 27: Assay for T-cell activity

  The following protocol is used to assess T cell activity by identifying factors and determining whether the albumin fusion proteins of the invention proliferate and / or differentiate T cells. T cell activity is assessed using the GAS / SEAP / Neo construct provided in Example 24. Thus, agents that increase SEAP activity exhibit the ability to activate the Jaks-STATS signaling pathway. The T cells used in this assay are Jurkat T cells (ATCC accession number TIB-152), although Molt-4 cells (ATCC accession number CRL-1582) can also be used.

  Jurkat T-cells are lymphoblastic CD4 + Th1 helper cells. To produce a stable cell line, approximately 2 million Jurkat cells are transfected with GAS / SEAP / Neo vectors using DMRIE-C (Life Technologies) (transfection procedure described below) ). Transfected cells are seeded to a density of approximately 20,000 cells / well, and transfectants resistant to 1 mg / ml gentisin are selected. Resistant colonies are grown and then tested for their response to increasing concentrations of interferon gamma. The volume response of the selected clone is shown.

  In particular, the following protocol produces sufficient cells for 75 wells containing 200 ul of cells. Thus, or scale up or do a large number to produce enough cells for a large number of 96 well plates. Jurkat cells are maintained in 10% serum containing RPMI + 1% Pen-Strep. In a T25 flask, 2.5 ml of OPTI-MEM (Life Technologies) is mixed with 10 ug of plasmid DNA. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 minutes.

During the incubation period, the cell concentration is counted and the required number of cells (10 ⁷ / transfection) is spun down and resuspended in OPTI-MEM to a final concentration of 10 ⁷ cells / ml. Then 1 ml of 1 × 10 ⁷ cells in OPTI-MEM is added to the T25 flask and incubated at 37 ° C. for 6 hours. After incubation, add 10 ml RPMI + 15% serum.

  The Jurkat: GAS-SEAP stable reporter strain is maintained in RPMI + 10% serum, 1 mg / ml gentisin, and 1% Pen-Strep. These cells are treated with various concentrations of one or more fusion proteins of the invention.

  On the day of treatment with the fusion protein, cells are washed and resuspended in fresh RPMI + 10% serum to a density of 500,000 cells / ml. The actual number of cells required depends on the number of fusion proteins and the number of different concentrations of fusion protein screened. Approximately 10 million cells are needed for a 96 well plate (eg, 10 million cells for 10 plates).

  Well dishes containing Jurkat cells treated with the fusion protein are placed in an incubator for 48 hours (Note: this time varies between 48-72 hours). A 35 ul sample from each well is then transferred to an opaque 96 well plate using a 12 channel pipette. Cover the opaque plate (using a cellophene cover) and store at −20 ° C. until the SEAP assay is performed according to Example 25. Plates containing the remaining cells to be tested are placed at 4 ° C. and used as a source of material to repeat the assay in specific wells if desired.

  As a positive control, 100 units / ml interferon gamma can be used and is known to activate Jurkat T cells. Over 30-fold induction is typically observed in positive control wells.

The above protocol may be used in the production of both transient as well as stably transfected cells and will be understood by those skilled in the art.

Example 28: Assay for T-cell activity

  NF-KB (nuclear factor KB) is produced by a wide variety of drugs, including inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and certain viruses A transcription factor that is activated by the expression of a gene product. As a transcription factor, NF-KB activates immune cells, regulates apoptosis (NF-KB appears to protect cells from apoptosis), B and T cell development, antiviral and antimicrobial responses, and multiple stresses Controls the expression of genes involved in the response.

  In unstimulated conditions, NF-KB remains in the cytoplasm with I-KB (inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, and NF-KB reciprocates into the nucleus, thereby activating transcription of the target gene. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

  Due to its central role and ability to respond to a wide range of stimuli, reporter structures using the NF-KB promoter element are used to screen fusion proteins. An activator or inhibitor of NF-KB is useful in treating, preventing and / or diagnosing a disease. For example, inhibitors of NF-KB can be used to treat diseases associated with acute or chronic activation of NF-KB, such as rheumatoid arthritis.

A PCR-based strategy is used to construct a vector containing the NF-KB promoter element. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCCC) (SEQ ID NO: 59), an 18 bp sequence complementary to the 5 ′ end of the SV40 early promoter sequence, flanked by XhoI sites.
5 ': GCGGCCTCGAGGGGACTTTCCCCGGGGACTTTCCGGGGACTTTCCGGACTTCCATCCTGCCATCTCAATTTAG: 3' (SEQ ID NO: 60)

  The downstream primer is complementary to the 3 'end of the SV40 promoter and is flanked by a HindIII site. 5 ': GCGGCAAGCTTTTTGCAAAGCCTAGGC: 3' (SEQ ID NO: 55)

PCR amplification is performed using the SV40 promoter template present in the pB-gal: promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI and HindIII and subcloned into BLSK2- (Stratagene). Sequencing with T7 and T3 primers confirms that the insert contains the following sequence:
5 ': CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT: 3' (SEQ ID NO: 61)

  Next, the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) is replaced with the NF-KB / SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene and is therefore not preferred for mammalian cell lines.

  To produce a stable mammalian cell line, the NF-KB / SV40 / SEAP cassette is removed from the NF-KB / SEAP vector using restriction enzymes SalI and NotI and inserted into a vector containing neomycin resistance. . In particular, the NF-KB / SV40 / SEAP cassette was inserted into pGFP-1 (Clontech) by replacing pGFP-1 with SalI and NotI and then replacing the GFP gene.

Once the NF-KB / SV40 / SEAP / Neo vector is generated, it is generated and maintained according to the stable Jurkat T-cell, protocol described in Example 25. Similarly, a method for assaying fusion proteins in these stable Jurkat T-cells is also described in Example 25. As a positive control, exogenous TNF alpha (0.1, 1, 10 mg) is added to wells H9, H10, and H11, and 5-10 fold activation is typically observed.

Example 29: Assay to identify bone marrow activity

  The following protocol is used to assess the bone marrow activity of an albumin fusion protein of the present invention by determining whether the fusion protein will grow and / or differentiate bone marrow cells. Bone marrow cell activity is assessed using the GAS / SEAP / Neo construct produced in Example 24. Thus, agents that increase SEAP activity exhibit the ability to activate the Jaks-STATS signaling pathway. The bone marrow cells used in this assay are the pre-monocytic cell line U937, although TF-1, HL60 or KG1 can also be used.

To transiently transfect U937 cells with the GAS / SEAP / Neo construct produced in Example 24, the DEAE-Dextran method (Kharbanda et. Al., 1994, Cell Growth & Differentiation, 5: 259-). 265). First, 2 × 10 ⁷ U937 cells are collected and washed with PBS. U937 cells are usually grown in RPMI 1640 medium containing 10% heat inactivated fetal bovine serum (FBS) containing 100 units / ml penicillin and 100 mg / ml streptomycin.

Next, the cells contain 1 ml of 0.5 mg / ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na ₂ HPO ₄ · 7H ₂ O, 1 mM MgCl ₂ , and 675 uM CaCl ₂ . Suspend in 20 mM Tris-HC1 (pH 7.4) buffer. Incubate at 37 ° C for 45 minutes.

  Cells are washed with RPMI 1640 containing 10% FBS, resuspended in 10 ml complete medium and incubated at 37 ° C. for 36 hours.

  GAS-SEAP / U937 stable cells are obtained by growing the cells in 400 ug / ml G418. Medium without G418 is used for daily growth, but is used every 1-2 months, and cells are re-grown for several passages in 400 ug / ml G418.

These cells are tested by harvesting 1 × 10 ⁸ cells (which is sufficient for 10 96-well plate assays), washing with PBS. Cells are suspended in 200 ml of the above growth medium at a final concentration of 5 × 10 ⁵ cells / ml. Plate 200 ul cells / well in 96-well plates (or 1 × 10 ⁵ cells / well).

Add different concentrations of fusion protein. Incubate at 37 ° C. for 48-72 hours. As a positive control, 100 units / ml interferon gamma can be used and is known to activate U937 cells. Over 30-fold induction is typically observed in positive treated wells. SEAP assay the supernatant according to methods known in the art and / or the protocol described in Example 25.

Example 30: Assay to identify changes in small molecule concentration and membrane permeability

  Binding of ligands to receptors is known to alter intracellular levels of small molecules such as calcium, potassium, sodium, and pH, as well as membrane potential. These changes can be measured in assays to identify fusion proteins that bind to specific cellular receptors. Although the following protocol is described in terms of calcium, this protocol can be easily used to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule that can be detected by a fluorescent probe. It can be modified.

  The following assay uses a Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules that bind to small molecules (Molecular Probes). Obviously, any fluorescent molecule that detects small molecules can be used in place of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc., catalog number F-14202) as used herein. It is.

For adherent cells, seed cells at 10,000-20,000 cells / well in a clear-bottomed Co-star black 96-well plate. The plate is incubated for 20 hours in a CO ₂ incubator. Adherent cells are washed twice with 200 ul HBSS (Hank Balance Salt Solution) in a Biotek washer, leaving 100 ul buffer after the final wash.

A stock solution of 1 mg / ml fluo-4 is made in 10% pluronic acid DMSO. To load cells at fluo-4, add 50 ul of 12 ug / ml fluo-4 to each well. Incubate the plate at 37 ° C. in a CO ₂ incubator for 60 minutes. The plate is washed 4 times with HBSS in a Biotek washer leaving 100 ul of buffer.

For non-adherent cells, the cells are spun down from the culture medium. Cells are resuspended with HBSS in a 50 ml conical tube to 2-5 × 10 ⁶ cells / ml. Add 4 ul of 1 mg / ml fluo-4 solution in 10% pluronic acid DMSO to each ml of cell suspension. The tube is then placed in a 37 ° C. water bath for 30-60 minutes. Cells are washed twice with HBSS, resuspended to 1 × 10 ⁶ cells / ml and dispensed into microplates at 100 ul / well. Centrifuge the plate at 1000 rpm for 5 minutes. The plate is then washed once with 200 ul in a Denley Cell Wash followed by aspiration to a final volume of 100 ul.

  For non-cell based assays, each well contains a fluorescent molecule such as fluo-4. The fusion protein of the invention is added to the wells and changes in fluorescence are detected.

In order to measure the fluorescence of intracellular calcium, FLIPR is set with the following parameters. (1) The system gain is 300 to 800 mVV. (2) The exposure time is 0.4 seconds. (3) The camera F / stop is F / 2. (4) Excitation is at 488 nm. (5) The radiation is 530 nm. And (6) Sample addition is 50ul. An increase in radiation at 530 nm suggests an extracellular signaling event triggered by the albumin fusion protein of the invention, or a molecule induced by the albumin fusion protein of the invention, resulting in an increase in intracellular Ca ⁺⁺ concentration. It becomes.

Example 31: Assay to identify tyrosine kinase activity

  Protein tyrosine kinases (PTKs) represent a diverse group of transmembrane and cytoplasmic kinases. The receptor protein tyrosine kinase (RPTK) group includes receptors for a wide range of mitogens and metabolic growth factors, including PDGF, FGF, EGF, NGF, HGF and the insulin receptor subfamily. Furthermore, there is a large family of RPTKs whose corresponding ligands are unknown. Ligands for RPTKs include mainly secreted small proteins, but also include membrane bound and extracellular matrix proteins.

  Activation of RPTK by a ligand includes ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunit and activation of cytoplasmic tyrosine kinase. Cytoplasmic tyrosine kinases include receptor-associated tyrosine kinases of the src-family (eg src, yes, lck, lyn, fyn), non-receptor binding and cytosolic protein tyrosine kinases, such as the receptor cytokine super, such as the Jak family. Included are members that mediate signaling triggered by the family (eg, interleukins, interferons, GM-CSF, and leptin).

  Because of the wide range of known factors that can stimulate tyrosine kinase activity, is the albumin fusion protein of the invention or molecules induced by the albumin fusion protein of the invention capable of activating the tyrosine kinase signaling pathway? Whether it is possible to identify whether or not is an object of interest. Thus, the following protocol is designed to identify such molecules capable of activating the tyrosine kinase signaling pathway.

  Target cells (eg primary keratinocytes) are plated at a concentration of approximately 25,000 cells per well in 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, IL). Plates are sterilized twice with 100% ethanol for 30 minutes, rinsed with water, and dried overnight. Plates are 100 ml cell culture grade type I collagen (50 mg / ml), gelatin (2%) or polylysine (50 mg / ml), all commercially available from Sigma Chemicals (St. Louis, MO) for 2 hours. Alternatively, coat with 10% matrigel, available from Becton Dickinson (Bedford, Mass.), Or bovine serum, rinse with PBS, and store at 4 ° C. Cells grown on these plates were seeded at 5,000 cells / well in growth culture and 48 hours as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, Calif.). Later, using alamarBlue, the number of cells is assayed indirectly. Falcon plate cover # 3071 from Becton Dickinson (Bedford, Mass.) Is used to cover Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in proliferation experiments.

To prepare the extract, A431 cells are seeded (20,000 / 200 ml / well) on nylon membranes on Loprodyne plates and cultured overnight in complete medium. Cells are quiesced by incubation in basal medium without serum for 24 hours. After 5-20 minutes treatment with EGF (60 ng / ml) or different concentrations of the albumin fusion protein of the invention, the medium was removed and 100 ml extraction buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton). Protease inhibitor cocktail (# 1836170)) from X-100, 0.1% SDS, 2 mM Na ₃ VO ₄ , 2 mM Na ₄ P ₂ O ₇ and Boehringer Mannheim (Indianapolis, Ind.) In addition to each well, the plate is shaken on a rotating shaker for 5 minutes at 4 ° C. The plate is then placed in a suction delivery manifold and the extract is filtered through a 0.45 mm membrane bottom in each well using house suction. The extract is collected in a 96-well capture / assay plate at the bottom of the aspiration manifold and immediately placed on ice. To obtain an extract purified by centrifugation, solubilizing the surfactant for 5 minutes, the components in each well are removed and centrifuged at 16,000 × g at 4 ° C. for 15 minutes.

  The filtered extract is tested for the level of tyrosine kinase activity. Although many methods for detecting tyrosine kinase activity are known, one method is described herein.

  In general, the tyrosine kinase activity of an albumin fusion protein of the invention is assessed by determining its ability to phosphorylate tyrosine residues on a particular substrate (biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a wide range of tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the following components in order. First, 10 ul of 5 uM biotinylated peptide, then 10 ul ATP / Mg ²⁺ (5 mM ATP / 50 mM MgCl ₂ ), then 10 ul 5 × Assay Buffer (40 mM imidazole hydrochloride, pH 7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM _{MgCl 2, 5 mM MnCl 2,} 0.5 mg / ml BSA), then van dating sodium 5ul (1 mM), then added water 5ul. The reaction is initiated by slowly mixing the components and adding 10 ul of control enzyme or filtered supernatant that preincubates the reaction mixture at 30 ° C. for 2 minutes.

  The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120 mm EDTA and the reaction is placed on ice.

  Tyrosine kinase activity is determined by transferring a 50 ul aliquot of the reaction mixture to a microtiter plate (MTP) module and incubating at 37 ° C. for 20 minutes. This allows the streptavidin coated 96 well plate to bind to the biotinylated peptide. Wash MTP module 4 times with 300 ul / well PBS. Next, 75 ul of anti-phosphotyrosine antibody conjugated to horseradish peroxidase (anti-P-Tyr-POD (0.5 u / ml)) is added to each well and incubated at 37 ° C. for 1 hour. Wash wells as above.

Next, add 100 ul peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 minutes (up to 30 minutes) Measure sample absorbance at 405 mm by using an ELISA reader. The level of bound peroxidase activity is quantified using an ELISA reader and reflects the level of tyrosine kinase activity.

Example 32: Assay to identify phosphorylation activity

  As a possible alternative and / or complement of the protein tyrosine kinase activity assay described in Example 31, an assay that detects activation (phosphorylation) of key intracellular signaling intermediates can also be used. It is. For example, as described below, one particular assay can detect tyrosine phosphorylation of Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules such as Raf, JNK, p38 MAP, Map Kinase Kinase (MEK), MEK Kinase, Src, Muscle Specific Kinase (MuSK), IRAK, Tec, and Janus, As well as any other formoserine, phosphotyrosine or phosphothreonine molecules can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assays.

  In particular, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1 ml protein G (1 ug / ml) for 2 hours at room temperature (RT). The plate is then rinsed with PBS and blocked with 3% BSA / PBS for 1 hour at RT. Protein G plates are then treated with two commercially available monoclonal antibodies against Erk-1 and Erk-2 (100 ng / well) (1 hour at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can be easily modified by substituting monoclonal antibodies that detect any of the molecules described above.) After 3-5 minutes in PBS, the plates are stored at 4 ° C. until use.

  A431 cells are seeded at 20,000 / well in 96-well Loprodyne filter plates and cultured overnight in growth medium. The cells are then depleted in basal medium (DMEM) for 48 hours and then treated with EGF (6 ng / well) or various concentrations of the fusion protein of the invention for 5-20 minutes. The cells are then solubilized and the extract is filtered directly into the assay plate.

After incubation with the extract for 1 hour at RT, the wells are rinsed again. As a positive control, a commercially available MAP kinase modulator (10 ng / well) is used in place of the A431 extract. The plates are then treated with a commercial polyclonal (rabbit) antibody (1 ug / ml) that specifically recognizes phosphorylated epitopes of Erk-1 and Erk-2 kinases (1 hour at RT). This antibody is biotinylated by standard procedures. Bound polyclonal antibody is then quantified by serial incubation with Europium-Streptavidin and Europium fluorescence enhancing reagents in a Wallac DELFIA instrument (time-resolved fluorescence). An increase in the fluorescent signal on the background indicates phosphorylation by the fusion protein of the invention or a molecule induced by the albumin fusion protein of the invention.

Example 33: Phosphorylation assay

To assay for the phosphorylation activity of the albumin fusion protein of the invention, a phosphorylation assay as disclosed in US Pat. No. 5,958,405 (incorporated herein by reference) is used. . Briefly, phosphorylation activity may be measured by phosphorylation of protein substrates using gamma-labeled ³² P-ATP and quantification of incorporated radioactivity using a gamma radioisotope counter. The fusion protein of the invention is incubated with protein substrate, ³² P-ATP, and kinase buffer. ³² P incorporated into the substrate is then separated from free ³² P-ATP by electrophoresis, and the incorporated ³² P is counted and compared to a negative control. A radioactivity count over the negative control is an indicator of the phosphorylation activity of the fusion protein.

Example 34: Detection of phosphorylation activity (activation) of the albumin fusion protein of the present invention in the presence of a polypeptide ligand

The phosphorylation activity of the albumin fusion protein of the invention is measured using methods known in the art or described herein. A preferred method of measuring phosphorylation activity is by use of a tyrosine phosphorylation assay as described in US Pat. No. 5,817,471 (incorporated herein by reference).

Example 35: Assay for stimulation of bone marrow CD34 + cell proliferation

  This assay is based on the ability of human CD34 + to proliferate in the presence of hematopoietic growth factors and evaluates the ability of the fusion proteins of the invention to stimulate the proliferation of CD34 + cells.

  It has been previously shown that most mature precursors respond only to a single signal. More immature precursors require at least two signals to respond. Therefore, to test the effect of the fusion protein of the invention on the hematopoietic activity of a wide range of progenitor cells, the assay includes the fusion protein of the invention in the presence or absence of hematopoietic growth factors. included. The isolated cells are cultured for 5 days in the presence of stem cell factor (SCF) in combination with the tested sample. Only SCF has a very limited effect on bone marrow (BM) growth, and in such a condition only serves as a “survival” factor. However, in combination with any factor that exhibits a stimulatory effect in these cells (eg, IL-3), SCF can cause a synergistic effect. Thus, if the tested fusion protein has a stimulatory effect on hematopoietic progeny, such activity can be easily detected. Since normal BM cells have low levels of cycling cells, any inhibitory effect of the fusion protein may not be detected. Thus, assays for inhibitory effects in progeny are preferably tested in cells that are first subjected to in vitro stimulation with SCF + IL + 3 and then contacted with a compound being evaluated for inhibition of such induced proliferation.

Briefly, CD34 + cells are isolated using methods known in the art. Cells are thawed and placed in medium (QBSF 60 serum-free medium with 1% L-glutamine (500 ml), Quality Biological, Inc., Gaithersburg, MD catalog number 160-204-101). Resuspend. After several gentle centrifugation steps at 200 × g, the cells are rested for 1 hour. The cell number is adjusted to 2.5 × 10 ⁵ cells / ml. During this time, 100 μl of sterile water is added to the end well of a 96-well plate. In this assay, the cytokines that can be tested with the albumin fusion proteins of the present invention are rhSCF and rhIL-3 (R & D Systems, Minneapolis, Minn., Catalog number 203-) alone and at 30 ng / ml. ML) at 50 ng / ml, rhSCF (R & D Systems, Minneapolis, MN, catalog number 255-SC). After 1 hour, 10 μl of prepared cytokine, various concentrations of the albumin fusion protein of the invention, and 20 μl of diluted cells are added into the medium already present in the wells to allow a final total volume of 100 μl. . The plate is then placed in a 37 ° C / 5% CO ₂ incubator for 5 days.

  Collect 18 hours before the assay and add 0.5 μCi / well of [3H] thymidine in a 10 μl volume to each well to determine the growth rate. The experiment is terminated by collecting cells from each 96-well plate into a filter mat using a Tomtec Harvester 96. After collection, the filter mat is dried, tidy and placed in an OmniFilter assembly consisting of one OmniFilter plate and one OmniFilter tray. 60 μl Microscint is added to each well and the plate is sealed with TopSeal-A press-on sealing film. A barcode 15 sticker is added to the first plate for counting. The sealed plate is then loaded and the level of radioactivity is determined via the Packard Top Count and the print data collected for analysis. The level of radioactivity reflects the amount of cell proliferation.

The study described in this example tests the activity of the fusion protein that stimulates bone marrow CD34 + cell proliferation. One skilled in the art can readily modify the exemplified studies to test the activity of the fusion proteins and polynucleotides of the invention (eg, gene therapy), and their agonists and antagonists. The ability of the albumin fusion protein of the present invention to stimulate bone marrow CD34 + proliferation is useful for diagnosis and treatment of diseases in which the albumin fusion protein and / or polynucleotide corresponding to the fusion protein affects the immune system and hematopoiesis It is suggested that. Exemplary uses are described in “Immune Activity” and “Infectious Diseases” above, and elsewhere herein.

Example 36: Assay for Extracellular Matrix Enhanced Cell Response (EMCR)

  The purpose of the extracellular matrix enhanced cell response (EMCR) assay is to assess the ability of the fusion proteins of the invention to work on hematopoietic stem cells with respect to the extracellular matrix (ECM) induced signal.

Cells respond to regulatory factors with respect to signals received from the surrounding microenvironment. For example, fibroblasts, endothelium and epithelial stem cells cannot replicate without signal from the ECM. Hematopoietic stem cells can undergo self-renewal in the bone marrow but not in an in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro depends on their interaction with stromal cells and the ECM protein fibronectin (fn). Adhesion to fn cells is mediated by the α ₅ · β ₁ and α ₄ · β ₁ integrin receptor expressed by human and mouse hematopoietic stem cells. The factor (s) involved in being able to integrate into the ECM environment and stimulate stem cell self-renewal has not yet been identified. The discovery of such factors should be of great interest in gene therapy and bone marrow transplantation indications.

Briefly, polystyrene, non-tissue culture treatment, 96-well plates are coated with fn fragments at a coating concentration of 0.2 μg / cm ² . Mouse bone marrow cells are plated (1,000 cells / well) in 0.2 ml serum free medium. Cells cultured in the presence of IL-3 (5 ng / ml) + SCF (50 ng / ml) are used as a positive control, and there is almost no self-renewal of stem cells, but conditions under which differentiation is determined are predicted. The albumin fusion protein of the present invention is tested in an appropriate negative control in the presence or absence of SCF (5.0 ng / ml), where the volume of administered composition comprising the albumin fusion protein of the present invention is: Represents 10% of total assay volume. The plated cells then hypoxic environment _{(5% CO 2, 7%} O 2, and 88% _{N 2)} are grown by incubating for 7 days in a tissue culture incubator. The number of proliferating cells in the well is then quantified by measuring thymidine incorporation into the cellular DNA. Confirmation of positive hits in the assay requires phenotypic characterization of the cells and can be achieved by scaling up the culture system and using appropriate antibody reagents against cell surface antigens and FACScan.

  Where it is clear that a particular fusion protein of the invention is a stimulant of hematopoietic progeny, the diagnosis of a disease in which the fusion protein and the polynucleotide corresponding to the fusion protein are affecting, for example, the immune system and hematopoiesis And may be useful in treatment. Exemplary uses are described in “Immune Activity” and “Infectious Diseases” above, and elsewhere herein. Fusion proteins may also be useful in expanding committed progeny of stem cells and various blood systems, and also in the differentiation and / or proliferation of various cell types.

  Alternatively, albumin fusion proteins of the invention, and polynucleotides encoding albumin fusion proteins of the invention may also be used to inhibit hematopoietic cell proliferation and differentiation, and thus during chemotherapies It may be used to protect bone marrow stem cells from therapeutic agents. This anti-proliferative effect may allow for the administration of larger / dose chemotherapeutic agents and thus allow for more effective chemotherapeutic treatment.

Furthermore, since stromal cells are important in the production of hematopoietic cells, the fusion protein of the present invention and the polynucleotide encoding the albumin fusion protein of the present invention are anemic, pancytopenia, leukopenia. May be useful for the treatment and diagnosis of hematopoietic related diseases such as thrombosis, thrombocytopenia or leukemia. Applications include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, neoplasia radiotherapy or chemotherapy.

Example 37: Human skin fibroblasts and aortic smooth muscle cell proliferation

  The albumin fusion protein of the invention is added to a culture of normal human skin fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC), and two co-assays are performed on each sample. The first assay tests the effect of the fusion protein on the proliferation of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC). Abnormal proliferation of fibroblasts or smooth muscle cells is part of various pathological processes, including fibrosis and restenosis. The second assay tests IL6 production by both NHDF and SMC. IL6 production is an indicator of functional activation. In activated cells, the production of numerous cytokines and other factors increases, which may result in inflammatory or immunomodulation. The assay is performed with or without co-TNFa stimulation to confirm for costimulatory or inhibitory activity.

  Briefly, on day 1, 96-well black plates are prepared at 1000 cells / well (NHDF) or 2000 cells / well (AoSMC) in 100 μl culture medium. NHDF culture medium contains Clonetics FB basal medium, 1 mg / ml hFGF, 5 mg / ml insulin, 50 mg / ml gentamicin, 2% FBS, while AoSMC culture medium is Clonetics SM basal medium, 0.5 μg / ml hEGF, 5 mg / Ml insulin, 1 mg / ml hFGF, 50 mg / ml gentamicin, 50 μg / ml amphotericin B, 5% FBS. After incubation at 37 ° C. for at least 4-5 hours, the culture medium is aspirated and replaced with growth stop medium. Growth stop medium for NHDF includes fibroblast basal medium, 50 mg / ml gentamicin, 2% FBS, and growth stop medium for AoSMC includes SM basal medium, 50 mg / ml gentamicin, 50 μg / ml Amphotericin B, 0.4% FBS is included. Incubate at 37 ° C until day 2.

On the second day, serial dilutions and templates of the albumin fusion proteins of the invention are designed to always include media controls and known protein controls. The protein is diluted in growth arrest medium for both stimulation and inhibition experiments. For inhibition experiments, TNFa is added to a final concentration of 2 ng / ml (NHDF) or 5 ng / ml (AoSMC). Add 1/3 volume medium containing control or albumin fusion protein of the invention and incubate at 37 ° C./5% CO ₂ until day 5.

  Transfer 60 μl from each well to another labeled 96-well plate, cover with plate-sealer and store at 4 ° C. until day 6 (for IL6 ELISA). Alamar Blue is aseptically added to the remaining 100 μl in the cell culture plate in an amount equal to 10% culture volume (10 μl). Return plate to incubator for 3-4 hours. The CytoFluor is then used to measure fluorescence with excitation at 530 nm and emission at 590 nm. This produces growth stimulation / inhibition data.

  On the fifth day, an IL6 ELISA was performed by coating 96-well plates with 50-100 ul / well anti-human IL6 monoclonal antibody diluted in PBS, pH 7.4, and ON at room temperature. Incubate.

  On day 6, the plates are emptied into the sink and blotted onto a paper towel. Prepare an assay buffer containing PBS containing 4% BSA. Plates are blocked with 200 ml / well of Pierce Super Block blocking buffer in PBS for 1-2 hours and then washed with wash buffer (PBS, 0.05% Tween-20). Blot plates on paper towels. Then 50 μl / well of diluted anti-human IL-6 monoclonal, biotin-labeled antibody is added at 0.50 mg / ml. Make dilutions of IL-6 stock in medium (30, 10, 3, 1, 0.3, 0 ng / ml). Duplicate samples are added to the first row of the plate. Cover plate and incubate on shaker at RT for 2 hours.

  The plate is washed with wash buffer and blotted on a paper towel. Dilute EU-labeled streptavidin 1: 1000 in assay buffer and add 100 μl / well. Cover the plate and incubate at RT for 1 hour. The plate is again washed with wash buffer and blotted onto a paper towel.

  Add 100 μl / ml enhancement solution. Shake for 5 minutes. Read the plate on a Wallac DELFIA Fluorometer. Readings from triplicate samples in each assay were tabulated and averaged.

A positive result in this assay suggests AoSMC cell proliferation, suggesting that the albumin fusion protein may be involved in dermal fibroblast proliferation and / or smooth muscle cell proliferation. A positive result also suggests many potential uses of the fusion protein and the polynucleotide encoding the fusion protein. For example, inflammation and immune responses, wound healing, and angiogenesis as detailed throughout the specification. In particular, the fusion protein may be used in promoting wound healing and skin remodeling, and angiogenesis of both blood vessels and lymphatic vessels. Vascular proliferation can be used, for example, in the treatment of cardiovascular disease. In addition, fusion proteins exhibiting antagonist activity in this assay are useful in treating diseases, diseases and / or conditions involving angiogenesis by acting as anti-angiogenic agents (eg, anti-angiogenesis). sell. These diseases, diseases and / or conditions are, for example, malignant, solid tumors, benign tumors such as blood vessel types, acoustic neuromas, neurofibromas, trachoma, and pyogenic granulomas, atheromatous plaques, ocular neovascular diseases, Such as diabetic retinopathies, premature infant retinopathies, macular degeneration, corneal transplant rejection, neovascular glaucoma, posterior lens fibroproliferation, rubeosis, retinoblastoma, ocular uveitis and pterygium (abnormal vascular growth), rheumatism -Like arthritis, psoriasis, delayed wound healing, endometriosis, angiogenesis, granulation, scar hypertrophy (keloid), non-adhesive fracture, scleroderma, trachoma, vascular adhesion, myocardial neovascularization, coronary artery branch, brain Side branch, arteriovenous malformation, ischemic limb angiogenesis, Osler-Webber syndrome, plaque new blood Known in the art, such as tube formation, telangiectasia, hemophilia joint, hemangiofibroma, fibromuscular dysplasia, wound granulation, Crohn's disease and atherosclerosis, and / or As described herein. Furthermore, albumin fusion proteins that act as antagonists in this assay may be useful in treating anti-hyperproliferative diseases and / or anti-inflammatory known in the art and / or described herein. .

Example 38: Cell adhesion molecule (CAM) expression on endothelial cells

  The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAM) on lymphocytes and the vascular endothelium. Adhesion processes in both normal and pathological settings include intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecules on endothelial cells (EC) ( E-selectin) follows a multi-stage cascade involving expression. These molecules and other expression on the vascular endothelium determine the effect of leukocytes adhering to local blood vessels and leaching into local cells during the development of the inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

Briefly, endothelial cells (eg, Human Umbilical Vein Endothelial cells (HUVEC)) are grown to confluence in standard 96 well plates, the growth medium is removed from the cells, and 100 μl of 199 medium ( 10% fetal bovine serum (FBS)) The sample for testing (including the albumin fusion protein of the invention) and the positive or negative control are added to the plate in triplicate (in a 10 μl volume). Incubate for either 5 hours (selection and integrin secretion) or 24 hours (integrin expression only) at 37 ° C. The plates are aspirated to remove the media and 100 μl 0.1% paraformaldehyde-PBS (Ca Wipe ⁺⁺ and Mg ⁺⁺ The plate is maintained for 30 minutes at 4 ° C. The fixative is removed from the wells, and the wells are washed with 1 × PBS (+ Ca, Mg) + 0.5% BSA and drained. 10 μl of diluted primary antibody is added to test and control wells, anti-ICAM-1-biotin, anti-VCAM-1-biotin and anti-E-selectin-biotin are used at a concentration of 10 μg / ml (0 (1:10 dilution of 1 mg / ml stock antibody) Cells are incubated in a humid environment for 30 minutes at 37 ° C. Wells are washed three times with PBS (+ Ca, Mg) + 0.5% BSA. 20 μl of diluted ExtrAvidin-Alkaline Phosphate (1: 5,000 dilution) is added to each well and incubated for 30 minutes at 37 ° C. Wash with PBS (+ Ca, Mg) + 0.5% BSA Dissolve 1 tablet of p-nitrophenol phosphate pNPP in 5 ml glycine buffer (pH 10.4) 100 μl pNPP in glycine buffer Substrate is added to each test well, triplicate, standard wells are prepared from working dilutions of ExtrAvidin-Alkaline Phosphate in glycine buffer: 1: 5,000 (10 ⁰ )> 10 ^−0.5 > 10 ^{− 1} > 10 ^{−1.5 5} μl of each dilution is added to a triple well and the AP content obtained in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to each standard well, and the plate is incubated at 37 ° C. for 4 hours. A volume of 50 μl of 3M NaOH is added to all wells. Plates are read on a plate reader at 405 nm using the background subtraction option on blank wells filled with glycine buffer only. In addition, a template is set to indicate the concentration of AP-conjugate in each standard well [5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng]. Results are shown as examples of bound AP-conjugate in each sample.

Example 39: Alamar Blue Endothelial Cell Proliferation Assay This assay was performed using bovine lymphatic endothelial cells (Bovine Lymphatic Endothelial Cells (LEC)), bovine aortic endothelial cells (Bovine Aortic Endothelial Cells (BAEC)), or human microvascular uterine muscle cells (BAEC). It is used to quantitatively determine protein-mediated inhibition of bFGF-induced proliferation of Human Microvascular Uterine Myometric Cells (UTMEC)). This assay incorporates a fluorescence growth index based on detection of metabolic activity. A standard Alamar Blue proliferation assay is prepared in EGM-2MV containing 10 ng / ml bFGF added as a source of endothelial cell stimulation. This assay may be used on various endothelial cells with slight changes in growth media and cell concentrations. Dilute the dilutions of the protein batch to be tested as appropriate. Serum free medium without FGF (GIBCO SFM) is used as an unstimulated control and angiostatin or TSP-1 is included as a known inhibition control.

  Briefly, LEC, BAEC or UTMEC are seeded in growth medium at a density of 5000-2000 cells / well in 96 well plates and left at 37 ° C. overnight. After overnight incubation of the cells, the growth medium is removed and replaced with GIBCO EC-SFM. Cells are treated in triple wells with appropriate dilutions of the albumin fusion protein of the invention (prepared in SFM) or control protein sample (s) and further treated with bFGF to a concentration of 10 ng / ml. Once the cells have been treated with the sample, the plate (s) are returned to the 37 ° C. incubator for 3 days. Three days later, 10 ml of stock Alamar blue (Biosource catalog number DAL1100) is added to each well and the plate (s) is returned to the 37 ° C. incubator for 4 hours. The plate (s) are then read using a CytoFluor fluorescence reader at 530 nm excitation and 590 nm emission. Direct output is recorded in the relative fluorescence unit.

Alamar blue is an oxidation-reduction indicator of both fluorescence and color change in response to chemical reduction of the growth medium as a result of cell growth. As cell growth in culture, the intrinsic metabolic activity is an immediate chemical reduction of the surrounding environment. Reduction on growth changes the indicator from an oxidized (non-fluorescent blue) form to a reduced (fluorescent red) form (ie, stimulated growth produces a stronger signal and suppressed growth produces a weaker signal) Produced and the total signal is proportional to the total number of cells as well as their metabolic activity). A background level of activity is observed only with starvation medium. This is compared to the output observed from the positive control sample (bFGF in growth medium) and protein dilutions.

Example 40: Detection of inhibition of mixed lymphocyte reaction

  This assay can be used to detect and assess inhibition of mixed lymphocyte reaction (MLR) by the fusion proteins of the present invention. Inhibition of MLR may be by direct effects on cell proliferation and viability, regulation of costimulatory molecules in interacting cells, regulation of lymphocyte and accessory cell tube adhesion, or regulation of cytokine production by accessory cells. A large number of cells can be targeted by albumin fusion proteins that inhibit MLR because the peripheral blood mononuclear segments used in this assay include T, B and natural killer lymphocytes, as well as monocytes and dendritic cells. .

  The albumin fusion proteins of the invention found to inhibit MLR are applied in diseases associated with lymphocyte and monocyte activation or proliferation. These include, but are not limited to, asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, atopic dermatitis, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, Crohn's disease, ulcerative It includes colitis, arteriosclerosis, cirrhosis, graft-versus-host disease, host body graft disease, hepatitis, leukemia and lymphoma.

Briefly, PBMCs from human donors were lysed using Lymphocyte Separation Medium (LSM®, density .0770 g / ml, Organon Technika Corporation, West Chester, PA). Purify by density gradient centrifugation Prepare PBMC from 2 donors at 2 × 10 ⁶ cells / ml in RPMI-1640 (Life Technologies, Grand Island, NY) with 10% FCS and 2 mM glutamine. . PBMC from third donor, prepared to 2x10 ⁵ cells / ml. the PBMC of 50 microliters from each donor, a 96 well round bottom Add dilutions of fusion protein test substance (50 μl) to the microtiter plate in triplicate, add test samples (of the protein of interest) at a final dilution of 1: 4, and add rhuIL- 2 (R & D Systems), Minneapolis, MN, catalog number 202-IL) was added at a final concentration of 1 mg / ml, and anti-CD4 mAb (R & D Systems, clone 34930.11, catalog number MAB379) was added. Add to a final concentration of 10 μg / ml Cells are cultured for 7-8 days at 37 ° C. in 5% CO ₂ and 1 μC [ ³ H] thymidine is added to the wells for the last 16 hours of culture. Collect and thymidine uptake by Packard TopCount It shows a. Data measured had a mean and standard deviation of triplicates.

Samples of the fusion protein of interest are screened in a separate experiment to inhibit lymphocyte proliferation, negative control treatment, anti-CD4 mAb, and positive control treatment to enhance lymphocyte proliferation, IL-2 (set Compared to either replacement material or supernatant).

Example 41: Assay for protease activity

  The following assay may be used to assess the protease activity of the albumin fusion protein of the invention.

  Gelatin and casein zymography are performed essentially as described (Heusen et al., Anal. Biochem., 102: 196-202 (1980); Wilson et al., Journal of Urology, 149: 653-. 658 (1993)). Samples were run on a 10% polyacrylamide / 0.1% SDS gel containing 1% xelin orcacein, 1 hour at room temperature, 5% to 16 hours at 37 ° C. in 2.5% Triton, 0.1 M glycine Soak in pH 8.3. After staining with the amide black area of proteolysis, a transparent area appears against the blue-black background. Trypsin (Sigma T8642) is used as a positive control.

Protease activity is also measured by monitoring the cleavage of na-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B-4500). The reaction is set up at pH 7.5 (25 mM NaPO ₄ , 1 mM EDTA, and 1 mM BAEE). Samples are added and changes in absorbance at 260 nm are monitored on a Beckman DU-6 spectrophotometer in a time driven fashion. Trypsin is used as a positive control.

Additional assays based on the release of acid-soluble peptides from casein or hemoglobin measured as absorbance at 280 nm or using the Folin method colorimetrically are described by Bergmeyer, et al. , Methods of Enzymatic Analysis, 5 (1984). Other assays include solubilization of chromogenic substrates (Ward, Applied Science, 251-317 (1983)).

Example 42: Identification of serine protease substrate specificity

The substrate specificity of an albumin fusion protein of the invention having serine protease activity may be measured using methods known in the art or described herein. A preferred method for determining substrate specificity is by the use of a positional scanning synthetic combinatorial library, as described in British Patent GB 2 324 529, all of which are incorporated herein.

Example 43: Ligand binding assay

  The following assay may be used to assess the ligand binding activity of the albumin fusion protein of the invention.

Ligand binding assays provide a direct way to elucidate receptor pharmacology and can be adapted to high throughput formats. Purified ligands for the albumin fusion proteins of the invention are radiolabeled to high characteristic activity (50-2000 Ci / mmol) for binding studies. The measurement is then performed such that the radiolabeling step does not reduce the activity of the ligand to the fusion protein. Assay conditions for other regulators such as buffers, ions, pH and nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell polypeptide sources. For these assays, a specific polypeptide binding is defined as the total associated radioactivity—the radioactivity measured in the presence of excess unlabeled competing ligand. Where possible, one or more competing ligands are used to define residual non-specific binding.

Example 44: Functional assay in Xenopus oocytes

Cap RNA transcripts from linearized plasmid templates encoding albumin fusion proteins of the invention are synthesized in vitro with RNA polymerase according to standard procedures. The in vitro transcript is suspended in water at a final concentration of 0.2 mg / mi. An ovarian lobe is taken from an adult female toad to obtain stage V vesicular oocytes and an RNA transcript (10 ng / oocyte) is injected in a 50 nl bolus using a microinjection device. Two electrode voltage clamps are used to measure the current from individual Xenopus oocytes in response to fusion protein and polypeptide agonist exposure. Recording was performed at room temperature in Barth medium without Ca ²⁺ . The Xenopus system can be used to screen for known ligands and tissue / cell extracts to activate the ligands.
Example 45: Microphysical measurement assay

Activation of a wide variety of second messenger systems results in the excretion of small amounts of acid from the cells. The acid formed is primarily as a result of the increased metabolic activity required to stimulate intracellular signaling processes. Changes in the pH of the medium around the cells are very small but can be detected by a CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, Calif). CYTOSENSOR can therefore measure the ability of the albumin fusion proteins of the present invention to activate second messengers linked to energy using intracellular signaling pathways.

Example 46: Extraction / cell supernatant screening

A number of mammalian receptors exist without any co-activating ligands (agonists). Therefore, active ligands for these receptors may not be included in the ligand bank as identified to date. Thus, the albumin fusion proteins of the present invention can also be functionally screened against tissue extracts (using functional screens such as calcium, cAMP, microphysiometer, oocyte electrophysiology, etc.) Natural ligands for the therapeutic and / or albumin protein portions of the albumin fusion proteins can be identified. Extracts that produce a positive functional response can be successively subdivided until the active ligand is isolated and identified.

Example 47: ATP-binding assay

  The following assay may be used to assess the ATP-binding assay of the fusion proteins of the invention.

The ATP-binding activity of the albumin fusion protein of the present invention is determined using the ATP-binding assay described in US Pat. No. 5,858,719, all of which are incorporated herein by reference. It may be detected. Briefly, ATP-binding to the albumin fusion protein of the invention is measured via optical affinity labeling with 8-azido-ATP in a competition assay. Reaction mixtures containing 1 mg / ml ABC transport protein are incubated with various concentrations of ATP, or non-hydrolyzable ATP analog adenyl-5′-imidodiphosphate, at 4 ° C. for 10 minutes. 8-Azido-ATP (Sigma Chem. Corp., St. Louis, MO.) + 8-Azido-ATP ( ³² P-ATP) (5 mCi / μmol, ICN, Irvine CA.) Is added at a final concentration of 100 μM and 0.5 ml aliquots are placed in the wells of a magnetic spot plate on ice. The plate is irradiated using a short wave 254 nm UV lamp at a distance of 2.5 cm from the plate at two 1 minute intervals with a 1 minute cooling period in between. The reaction is stopped by the addition of a final concentration of 2 mM dithiothreitol. Incubations are subjected to SDS-PAGE electrophoresis, dried and autoradiographed. A protein band corresponding to the albumin fusion protein of the present invention is cut out and the radioactivity is quantified. The decrease in radioactivity associated with an increase in ATP or adenyl-5′-imidodiphosphate provides a measure of ATP affinity to the fusion protein.

Example 48: Identification of signal transduction proteins that interact with albumin fusion proteins of the invention

The albumin fusion proteins of the present invention may be utilized as research tools for the identification, characterization and purification of signal transduction pathway proteins or receptor proteins. Briefly, the labeled fusion protein of the present invention is useful as a reagent for purification of interacting molecules. In one embodiment of affinity purification, the albumin fusion protein of the invention is conjugated to a chromatography column. An extract containing no cells derived from putative target cells such as cancer tissue is passed over the column and molecules with appropriate affinity bind to the albumin fusion protein. The protein complex is recovered from the column, separated, and the recovered molecule is subjected to N-terminal protein sequencing. This amino acid sequence is then used to design degenerate oligonucleotide probes to identify capture molecules or to clone the corresponding gene from an appropriate cDNA library.
Example 49: IL-6 bioassay

Various assays are known in the art for testing the proliferative effects of the albumin fusion proteins of the present invention. For example, one such assay is described in Marz et al. IL-6 Bioassay as described by (Proc. Natl. Acad. Sci., USA, 95: 3251-56 (1998), incorporated herein by reference). . After 68 hours at 37 ° C., the number of viable cells is determined by adding the tetrazolium salt thiazolyl blue (MTT) and incubating for an additional 4 hours at 37 ° C. B9 cells are lysed by SDS and the optical density is measured at 570 nm. Controls containing IL-6 (positive) and no cytokine (negative) are used. Briefly, IL-6 dependent B9 murine cells are washed three times in IL-6 free medium, plated at 50 μl at 5,000 cells / well, and 50 μl of the fusion protein of the invention is added. Increased proliferation in test sample (s) (including albumin fusion protein of the invention) relative to a negative control is an indication of the proliferation effect mediated by the fusion protein.

Example 50: Supporting chick embryo neuron survival

To test whether sympathetic cell survival is supported by the albumin fusion protein of the present invention, Senaldi et al may be utilised (Proc. Natl. Acad. Sci., USA, 96: 11458). -63 (1998), incorporated herein by reference) may be used. Briefly, motor and sympathetic neurons were isolated from chicken embryos, each containing L15 medium (containing 10% FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine and insulin, Life Technologies). , Rockville, MD.) And Dulbecco's Modified Eagle Medium [10% FCS, Glutamine, Penicillin, and 25 mM Hepes Buffer (pH 7.2), Life Technologies, Rockville, MD. And incubate in 5% CO ₂ at 37 ° C. in the presence of different concentrations of the purified fusion protein of the invention, as well as a negative control lacking any cytokines. After 3 days, neuronal survival is measured through assessment of cell morphology and use of a colorimetric assay of Mosmann (Mosmann, T., J. Immunol. Methods, 65: 55-63 (1983)). Enhanced neuronal cell survival compared to controls lacking cytokines is an indication of the ability of the albumin fusion protein to enhance neuronal cell survival.

Example 51: Assay for phosphate activity

  The following assay may be used to assess the serine / threonine phosphatase (PTPase) activity of the albumin fusion proteins of the invention.

Assays well known to those skilled in the art can be used to assay for serine / threonine phosphatase (PTPase). For example, the serine / threonine phosphatase (PSPase) activity of the albumin fusion protein of the present invention may be measured using a PSPase assay kit from New England Biolabs, Inc. Myelin basic protein (MyBP), a substrate for PSPase, is phosphorylated on serine and threonine with cAMP-dependent protein kinase in the presence of [ ³² P] ATP. The protein serine / threonine phosphatase activity is then determined by measuring the release of inorganic phosphate from 32P-labeled MyBP.

Example 52: Interaction of serine / threonine phosphatase with other proteins

A fusion protein of the invention with serine / threonine phosphatase activity (eg, as determined in Example 51) can be used, for example, to identify, characterize and purify additional interacting or receptor proteins, or other signaling pathway proteins. Useful as a research tool for. Briefly, the labeled fusion protein of the present invention is useful as a reagent for purification of interacting molecules. In one embodiment of affinity purification, the albumin fusion protein of the invention is covalently bound to a chromatography column. A cell-free extract, such as a nerve or liver cell, derived from a putative target cell is passed over the column and a molecule with the appropriate affinity binds to the fusion protein. The fusion protein-complex is recovered from the column, separated, and the recovered molecule is subjected to N-terminal protein sequencing. This amino acid sequence is then used to design a degenerate oligonucleotide probe for cloning the corresponding gene from an appropriate cDNA library.

Example 53: Assay for heparanase activity

There are a number of assays known in the art that may be used to assay for the heparanase activity of an albumin fusion protein of the invention. In one example, the heparanase activity of an albumin fusion protein of the present invention is measured according to Vlodavsky et al. (Vlodavsky et al., Nat. Med., 5: 793-802 (1999)). Briefly, cell lysate, conditioned medium, intact cells (1 × 10 ⁶ cells / 35-mm dish), cell culture supernatant, or purified fusion protein are brought to 37 ° C., pH 6.2-6.6. Incubate with ³⁵ S-labeled ECM or soluble ECM-derived peak I proteoglycans for 18 hours. The incubation medium is centrifuged and the supernatant is analyzed by gel filtration on a Sepharose CL-6B column (0.9 x 30 cm). Fractions are eluted with PBS and their radioactivity is measured. The heparan sulfate side chain fragment is eluted from Sepharose 6B with 0.5 <K _av <0.8 (peak II) and each experiment is performed at least three times. Vlodavsky et al. As described by, the degradation fragment corresponding to “Peak II” is an indicator of the activity of the albumin fusion protein of the present invention in the cleavage of heparan sulfate.

Example 54: Immobilization of biomolecules

This example is a non-host cell lipid bilayer construct (eg, incorporated herein by reference in its entirety) that is adaptable to study the fusion proteins of the invention in the various functional assays described above. (See Bieri et al., Nature Biotech 17: 1105-1108 (1999)), for the immobilization of albumin fusion proteins of the present invention. Briefly, a sugar specific chemistry for biotinylation is used to anchor the biotin tag to the albumin fusion protein of the present invention, allowing a uniform direction in immobilization. A 50 uM solution of the albumin fusion protein of the invention in a wash membrane is incubated with 20 mM NaIO ₄ and 1.5 mg / ml (4 mM) BACH or 2 mg / ml (7.5 mM) biotin-hydrazine for 1 hour at room temperature ( Reaction volume, 150 ul). The sample was then dialyzed for 5 hours at 4 ° C. for the first time ((Pierce Sliderizer Cassett, 10 kDa cutoff; Pierce Chemical Co., Rockford IL)), changing the buffer every 2 hours, and finally Dialyze against 500 ml buffer R (0.15 M NaCl, 1 mM MgCl ₂ , 10 mM sodium phosphate, pH 7) for 12 hours, immediately before adding to the cuvette, the sample contains buffer ROG50 (containing 50 mM octyl glycoside) Dilute 1: 5 with buffer R).

Example 55: Assay for metalloproteinase activity

Metalloproteinases are peptide hydrases that use metal ions such as Zn ²⁺ as a catalytic mechanism. The metalloproteinase activity of the albumin fusion protein of the invention can be assayed according to methods known in the art. The following exemplary methods are provided.
Proteolysis of alpha-2-macroglobulin

In order to confirm protease activity, the purified fusion protein of the present invention was added to 1 × assay buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl ₂ , 25 mM ZnCl ₂ and 0.05% Brij-35. ) With substrate alpha-2-megaglobulin (0.2 units / ml, Boehringer Mannheim, Germany) and incubate at 37 ° C. for 1-5 days. Trypsin is used as a positive control. The negative control is alpha-2-megaglobulin in assay buffer. Samples are collected and boiled in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for 5 minutes and then loaded onto an 8% SDS-polyacrylamide gel. After electrophoresis, the protein is visualized by silver staining. Proteolysis is manifested by the expression of lower molecular weight bands compared to the negative control.
Inhibitors of alpha-2-macroglobulin proteolysis by inhibitors of metalloproteinases

Known metalloproteinase inhibitors (metal chelators (EDTA, EGTA, and HgCl ₂ ), peptide metalloproteinases (TIMP-1 and TIMP-2) and commercially available small molecule MMP inhibitors) are also used in the albumin fusion of the present invention. It may be used to characterize the proteolytic activity of a protein. These synthetic MMP inhibitors that may be used are MMP inhibitor I, [IC ₅₀ = 1.0 μM for MMP-1 and MMP-8, IC ₅₀ = 30 μM for MMP-9, MMP-3. IC ₅₀ = 150 μM], MMP-3 (Stromelysin-1) inhibitor I [IC ₅₀ = 5 μM for MMP-3] and MMP-3 inhibitor II [K _i = 130 nM for MMP-3 ], Inhibitors available via Calbiochem, catalog numbers 444250, 444218, and 444225, respectively. Briefly, different concentrations of small molecule MMP inhibitors were added to 22.9 μl of 1 × HEPES buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl ₂ , 25 mM ZnCl ₂ and 0.05% In Brij-35), mixed with the purified fusion protein of the invention (50 μg / ml), incubated at room temperature (24 ° C.) for 2 hours, then 7.1 μl of substrate alpha-2-megaglobulin (0.2 Units / ml) and incubate at 37 ° C. for 20 hours. The reaction is stopped by adding 4 × sample buffer and immediately boiled for 5 minutes. After SDS-PAGE, protein bands are visualized by silver staining.
Synthetic fluorescent peptide substrate cleavage assay

Substrate characteristics for the fusion protein of the present invention having the indicated metalloproteinase activity were determined using synthetic fluorescent peptide substrates (purchased from BACHEM Bioscience Inc) using techniques known in the art. May be measured. Test substrates include M-1985, M-2225, M-2105, M-2110, and M-2255. The first four are MMP substrates and the remaining one is a substrate for tumor necrosis factor-α (TNF-α) converting enzyme (TACE). These substrates are preferably prepared in 1: 1 dimethyl sulfoxide (DMSO) and water. The stock solution is 50-500 μM. The fluorescence assay is performed by using a Perkin Elmer LS 50B emission spectrometer equipped with a constant temperature water bath. The excitation λ is 328 nm and the emission λ is 393 nm. Briefly, the assay was performed using 176 μl 1 × HEPES buffer (0.2 M NaCl, 10 mM CaCl ₂ , 0.05% Brij-35 and 50 mM HEPES, pH 7.5) for 15 minutes at 25 ° C., 4 μl Incubation with a substrate solution (50 μM) followed by addition of 20 μl of the purified fusion protein of the invention to the assay cuvette. The final concentration of substrate is 1 mM. The initial hydrolysis rate is monitored for 30 minutes.

Example 56: Development of diabetes in NOD mice

  Although the disease is more pronounced in females than in male NOD, female NOD (non-obese diabetic) mice are characterized by showing IDDM with a course similar to that seen in humans. Hereinafter, unless stated otherwise, the phrase “NOD mouse” means a female NOD mouse. NOD mice have a progressive destruction of beta cells caused by chronic autoimmune diseases. Therefore, NOD mice begin life with euglycemia or begin with normal blood glucose levels. However, by about 15-16 weeks of age, NOD mice begin to become hyperglycemic, suggesting the destruction of their major pancreatic beta cells and the inability of the pancreas to produce correspondingly sufficient insulin. . Thus, both the cause and progression of the disease is similar to human IDDM patients.

  An in vivo assay of the effect of the immune regimen can be assessed in female NOD / LtJ mice (commercially available from The Jackson Laboratory, Bar Harbor, Me.). It has been reported in the literature that 80% of female mice develop diabetes by 24 weeks of age, and the onset of islet inflammation begins between 6 and 8 weeks of age. NOD mice are mated and are very responsive to various immunoregulatory strategies. Adult NOD mice (6-8 weeks old) have an average body weight of 20-25 g.

  These mice are not treated (control), treated with the inventive therapy (eg, the inventive albumin fusion protein and fragments and variants thereof) alone or in combination with other therapeutic compounds referred to above. Also good. The effect of these various treatments on the progression of diabetes can be measured as follows.

At 14 weeks of age, female NOD mice can be phenotypically determined according to glucose tolerance. Glucose tolerance can be measured by an intraperitoneal glucose tolerance test (IPGTT). Briefly, blood was drawn from paraorbital network at 0 and 60 minutes after intraperitoneal injection of glucose (1 g / kg body weight). Normal tolerance is defined as plasma glucose at 0 minutes below 144 mg% and 60 minutes below 160 mg%. Blood glucose levels are measured with a Glucometer Elite instrument.

  Based on this phenotypic decision analysis, animals can be assigned to different experimental groups. In particular, animals with higher blood glucose levels can be assigned to impaired glucose tolerance groups. Mice can optionally be fed and given oxidized water (pH 2.3).

  Glucose and glucose intolerant mice can be further divided into controls, albumin fusion proteins of the invention, and albumin fusion protein / therapeutic compound combinations. A control group of mice may receive intraperitoneal injections of vehicle only 6 times a week daily. The albumin fusion group can be given an intraperitoneal injection of the therapeutic of the invention (eg, the albumin fusion protein of the invention and fragments and variants thereof) daily, six times a week. Mice in the albumin fusion protein / therapeutic compound combination group can be given both the albumin fusion protein and the therapeutic compound combinations described above.

  Urinary glucose levels in NOD mice can be measured on a biweekly basis using Labstix (Bayer Diagnostics, Hampshire, England). Body weight and fluid intake can also be measured on a biweekly basis. The onset of diabetes is defined after the onset of diabetes on two consecutive measurements. Ten weeks after treatment, additional IPGTT is performed and animals are sacrificed the following day.

  Over the 10-week course of treatment, control animals in both glucose tolerance and intolerance groups develop diabetes at a rate of 60% and 86%, respectively (see US Pat. No. 5,866,546, Gross et al.). ) Thus, a high rate of diabetes occurs in NOD mice that are initially glucose tolerant when not treated.

  Results can be confirmed by measurement of blood glucose levels in NOD mice before and after treatment. Blood glucose levels are measured as described above in both glucose and intolerant mice in all groups described.

In other embodiments, the therapeutics of the invention (eg, the specific fusion disclosed as SEQ ID NO: Y, and fragments and variants thereof) can be quantified using spectroscopic analysis, and the appropriate protein amount determined by Resuspend in 50 μl phosphate buffered saline (PBS) per dose prior to injection. Two injections per week can be administered subcutaneously under the dorsal skin of each mouse. Monitoring can be performed on two separate occasions prior to immunization, can be performed weekly throughout the procedure, and can continue thereafter. Urine can be tested weekly for glucose (Keto-Diastix.RTM .; Miles Inc., Kankake, Ill.) And diabetic mice can be checked for serum glucose (ExacTech.RTM., Medisense). (MediSense, Inc.), Waltham, Mass.). Diabetes is diagnosed when fasting blood glucose is 2.5 g / L or more.

Example 57: Histological experiment of NOD mice

  Histological experiments of tissue samples from NOD mice increase the relative concentration of beta cells in the pancreas of the compounds of the invention and / or combinations of compounds of the invention with other therapeutic drugs for diabetes Capability can be demonstrated. The experimental method is as follows.

  Mice from Example 56 can be sacrificed at the end of the treatment period and tissue samples can be taken from the pancreas. Samples are fixed in 0.9% saline in 10% formalin and embedded in wax. Two sets of 5 consecutive 5 μm sections can be cut for immunolabeling with a 150 μm cut interval. Sections were immunostained for insulin (guinea pig anti-insulin antiserum dilution 1: 1000, ICN Themes UK) and glucose (rabbit anti-pancreatic glucagon antiserum dilution 1: 2000) and peroxidase conjugated anti-guinea pig (Dako, High Wycombe). , UK) or peroxidase-conjugated anti-guinea pig (Dako, High Wycombe, UK) or peroxidase-conjugated anti-rabbit antiserum (dilution 1:50, Dako).

The compositions of the present invention may or may not have a strength effect similar to that in the clinical signs of diabetes in glucose tolerance and intolerant glucose actives in the visible mass of beta cells.

Example 58: NIDDM in vivo mouse model

  Male C57BL / 6J mice from Jackson Laboratories (Jackson Laboratory (Bar Harbor, ME)) were obtained at 3 weeks of age, conventional diet, or fat (35.5% wt / wt; Bioserv. Frenchtown, NJ) or fructose (60% wt / wt; Harlan Teklad, Madison, Wis.) Any of these may be fed. A normal bait consists of 4.5% wt / wt fat, 23% wt / wt protein, 31.9% wt / wt starch, 3.7% wt / wt fructose, and 5.3% wt / wt fiber. The high fat diet is 35.5% wt / wt fat, 20% wt / wt protein, 36.4% wt / wt starch, 0.0% wt / wt fructose, and 0.1% wt / wt Made of fiber. High fructose diet consists of 5% wt / wt fat, 20% wt / wt protein, 0.0% wt / wt starch, 60% wt / wt fructose, and 9.4% wt / wt fiber. Mice are housed at no more than 5 animals per cage in a 12 hour light (6 am-6pm) / dark cycle at a temperature of 22 ° C ± 3 ° C and a 50% ± 20% humidity control room ( Luo et al., 1998, Metabolism 47 (6): 663-8, “Nongenetic mouse models of non-insulin-dependent dibesets melitus”, Larsen et al .; (11): 2530-9 (2001), “Systemic administration of long-acting GLP-1 derivative NN2211 induces long-term and reversible weight loss in normal and obese rats (Systemic administration o) the long-acting GLP-1 derivative NN2211 induces lasting and reversible weight loss in both normal and obese rats) "). After 3 weeks exposure to each diet, mice were treated with streptozotocin “STZ” (Sigma, St. Louis, MO) or vehicle (0.05 mol / L citrate) at 100 mg / kg body weight. , PH 4.5) may be injected intraperitoneally and maintained on the same diet for the next 4 weeks. Under non-fasting conditions, blood is obtained at 1, 2, and 4 weeks after STZ by cutting the distal site of the tail. Samples are used to measure non-fasting plasma glucose and insulin concentrations. Body weight and food intake are recorded weekly.

To directly measure the effect of high fat diet on insulin's ability to stimulate glucose removal, the experiment was conducted at the end of the 7 weeks described above with vehicle-injected fat-fed, food-fed mice, STZ. Can be started in three groups of fat-fed mice injected with. Mice can be fed for 4 hours before the experiment. In the first series of experiments, mice can be anesthetized with methoxyfuran (Pitman-Moor, Mundelein, IL) inhalation. Insulin (Sigma) is usually injected intravenously via the tail vein. ([IV] 0.1 U / kg body weight), blood can be collected from different tail veins at 3, 6, 9, 12 and 15 minutes after injection Plasma glucose concentrations are measured on these samples Yes, the half-life of glucose elimination from plasma (t ^1/2 ) was calculated using the pharmacokinetic / pharmacodynamic software program WinNonlin (Scientific Consulting, Apex, NC) Is possible.

  In a second series of experiments, mice can be anesthetized with intraperitoneal pentobarbital sodium (Sigma). The abdominal cavity is opened and the main abdominal vein is exposed and catheterized with a 24-gauge IV catheter (Johnson-Johnson Medical, Arlington, TX). The catheter is fixed to the muscular tissue adjacent to the abdominal vein, cut on the bottom of the syringe connection, connected to a previously filled PE50 plastic tube, and then connected to the syringe containing the infusion solution. The abdominal cavity is then sutured closed. With this approach, there is no backflow occlusion of blood from the lower part of the body. Mice can be continuously infused with glucose (24.1 mg / kg / min) and insulin (10 mU / kg / min) at an infusion volume of 10 μL / min. Retroorbital blood samples (70 μL each) may be taken 90, 105, 120 and 135 minutes after the start of infusion for measurement of plasma glucose and insulin concentrations. The average of these four samples is used to estimate steady state plasma glucose (SSPG) and insulin (SSPI) concentrations for each animal.

  Finally, the ability to reduce plasma glucose of albumin fusion proteins, therapeutic compositions herein, alone or in combination with one or more therapeutic drugs listed for the treatment of diabetes Experiments can be performed on the following two groups of “NIDDM” mouse models that are STZ-injected: (1) fat feeding C57BL / 6J, and (2) fructose feeding C57BL / 6J. Mouse plasma glucose concentrations for these studies can range from 255 to 555 mg / dL. Mice are randomized with any of the albumin fusion therapeutics of the present invention, either vehicle, alone or in combination with one or more of any therapeutic drugs listed for the treatment of diabetes. Assign randomly to treatment. A total of 3 / dose can be administered. Tail vein blood samples can be taken for measurement of plasma glucose concentration before the first dose and 3 hours after the last dose.

Plasma glucose concentration can be measured using an enzyme colorimetric assay, the Glucose Diagnostic Kit from Sigma (Sigma No. 315). Plasma insulin levels can be measured using a Rat Insulin RIA Kit (# RI-13K; St. Charles, MO) from Linco Research.

Example 59: In vitro H4IIe-SEAP reporter assay establishing improvement in insulin activity
Various H4IIe reporters

H4IIe / rMEP-SEAP: The malate enzyme promoter (rMEP) isolated from rats contains a PPAR-gamma element that is in the insulin pathway. This reporter construct is stably transfected into the liver H4IIe cell line.

H4IIe / SREBP-SEAP: Sterol regulatory element binding protein (SREBP-1c) works on the promoters of many insulin responsive genes, such as fatty acid synthase (FAS), and fatty acids in fibroblasts, adipocytes, hepatocytes A transcription factor that regulates the expression of a key gene in metabolism. SREBP-1c, also known as adipocyte determination and differentiation factor 1 (ADD-1), is considered as a major mediator of insulin effects in gene expression in adipocytes. Its activity is regulated by insulin, sterol and glucose levels. This reporter construct is stably transfected into the liver H4IIe cell line.

The H4IIe / FAS-SEAP: fatty acid synthase reporter construct contains a minimal SREBP-responsive FAS promoter. This reporter construct is stably transfected into the liver H4IIe cell line.

H4IIe / PEPCK-SEAP: The phosphoenolpyruvate carboxykinase (PEPCK) promoter is a major site of humoral regulation of PEPCK gene transcription that regulates PEPCK activity. PEPCK catalyzes the associated rate limiting step in hepatic gluconeogenesis and therefore must be carefully controlled to maintain blood glucose levels within normal limits. This reporter construct is stably transfected into the liver H4IIe cell line.

These reporter constructs can also be stably transfected into 3T3-L1 fibroblasts and L6 myoblasts. These stable cell lines then differentiate into 3T3-L1 adipocytes and L6 myotubes as previously described in Example 13. Differentiated cell lines can then be used in the SEAP assay described below.
Growth and assay media

  Growth medium is 10% fetal bovine serum (FBS), 10% bovine serum, 1% NEAA, 1 × penicillin / streptomycin, and 0.75 mg / mL G418 (for H4IIe / rFAS-SEAP and H4IIe / SREBP-SEAP) Or 0.50 mg / mL G418 (for H4IIe / rMEP-SEAP). For H4IIe / PEPCK-SEAP, the growth medium consists of 10% FBS, 1% penicillin / streptomycin, 15 mM HEPES buffered saline, and 0.50 mg / mL G418.

The assay medium consists of low glucose DMEM medium (Life Technologies), 1% NEAA, 1 × penicillin / streptomycin for H4IIe / rFAS-SEAP, H4IIe / SREBP-SEAP, H4IIe / rMEP-SEAP reporters. . The assay medium for the H4IIe / PEPCK-SEAP reporter consists of 0.1% FBS, 1% penicillin / streptomycin, and 15 mM HEPES buffered saline.
Method

96-well plates are seeded at 75,000 cells / well in 100 μL / well growth medium until cells in the log growth phase adhere. Cells are craved for 48 hours by replacing growth medium with assay medium, 200 μmL / well (for H4IIe / PEPCK-SEAP cells, assay medium containing 0.5 μM dexamethasone is seeded at 100 μL / well, approximately Incubate for 20 hours). The assay medium is subsequently replaced with 100 mL / well of fresh assay medium and obtained from a transfected cell line expressing the therapeutic of the invention (eg, the albumin fusion protein of the invention and fragments and variants thereof). Add a 50 μL aliquot of clear to wells. Supernatant from the empty vector transfected cell line is used as a negative control. Addition of 10 nM and / or 100 nM insulin to wells is used as a positive control. After 48 hours incubation, the conditioned medium is collected and SEAP activity is measured (Phospha-Light System protocol, Tropix # BP2500). Briefly, samples are diluted 1: 4 in dilution buffer and incubated at 65 ° C. for 30 minutes to inactivate the endogenous non-placenta of SEAP. A 50 μL aliquot of the eluted sample is mixed with 50 μL SEAP assay buffer containing a mixture of inhibitors active against non-placental SEAP isoenzyme and incubated for an additional 5 minutes. A 50 μL aliquot of CSPD chemiluminescent substrate diluted 1:20 in emerald fluorescence enhancer is added to the mixture and incubated for 15-20 minutes. Read plate in Dynax plate luminometer.

Example 60: Transgenic animals

  The albumin fusion protein of the invention can be expressed in transgenic animals. Using any species of animal, including but not limited to mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates such as baboons, monkeys and chimpanzees Transgenic animals may be produced. In certain embodiments, techniques described herein or known in the art are used to express the fusion proteins of the invention in humans as part of a gene therapy protocol.

  Any technique known in the art may be used to introduce a polynucleotide encoding an albumin fusion protein of the invention into an animal to produce the founder strain of a transgenic animal. Such techniques include, but are not limited to, pronuclear microinjection (Patterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263- 1270 (1993); Wright et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., US Pat. No. 4,873,191 (1989)), germline. Retroviral-mediated gene delivery into the cell (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985 ), Blastocysts or embryos, gene targeting in embryonic stem cells (Thompson et al., Cell 56: 313-321 (1989)), electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3). : 1803-1814 (1983)), introduction of a polynucleotide of the present invention using a gene cancer (see, eg, Ulmer et al., Science 259: 1745 (1993)), nucleic acid constructs into embryonic pleural stem cells And delivery of stem cells to blastocysts (Lavitrano et al., Cell 57: 717-723 (1989); etc.), all of which are hereby incorporated by reference for an overview of such techniques. Gordon, “T, incorporated by literature . Ansgenic Animals, "Intl Rev. Cytol 115:. 171-229 (1989) refer to.

  For example, nuclear delivery from cultured embryos, fetuses or adult cells induced to rest into enucleated oocytes (Campell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385). : 810-813 (1997)) using any technique known in the art to produce a transgenic clone containing a polynucleotide encoding an albumin fusion protein of the invention. Also good.

  The invention includes transgenic animals having a polynucleotide encoding an albumin fusion protein of the invention in all their cells, as well as some, but not all, of these cells. Provide animals, ie mosaic animals or chimeras. Transgenes may be integrated as a single transgene or in multiple copies, such as head-to-head or head-to-tail tandems, in a concatamer. Transgenes are also described, for example, in Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992)), which can be selectively introduced into a specific cell type or activated. Good. The regulatory sequences required for such cell type specific activity will depend on the particular cell type of the control and will be apparent to those skilled in the art. Gene targeting is preferred when it is desired that the polynucleotide encoding the fusion protein of the invention integrates into the chromosomal site of the endogenous gene corresponding to the therapeutic protein portion or albumin portion of the fusion protein of the invention. . Briefly, if such a technique is to be used, the vector containing the nucleotide sequence homologous to the endogenous gene is integrated via homologous recombination with the chromosome sequence and the nucleotide sequence of the endogenous gene. Designed for the purpose of disrupting the function of. Transgenes are also described in, for example, Gu et al. (Gu et al., Science 265: 103-106 (1994)) may be selectively introduced into a particular cell type and thus inactivate endogenous genes only in that cell type. To do. The regulatory sequences required for such cell type specific inactivation will depend on the particular cell type of interest and will be apparent to those skilled in the art.

  Once transgenic animals have been produced, recombinant gene expression may be assayed using standard techniques. Initial screening may be performed by Southern blot analysis or PCR techniques, and animal tissue may be analyzed to confirm that integration of the polynucleotide encoding the fusion protein of the invention has been performed. The level of mRNA expression of the polynucleotide encoding the fusion protein of the present invention in the tissue of the transgenic animal is also, but not limited to, Northern blot analysis, in situ hybridization analysis of a tissue sample obtained from the animal, And may be assessed using techniques including reverse transcriptase-PCR (rt-PCR). Fusion protein-expressing tissue samples may also be assessed immunocytochemically or immunohistochemically using antibodies specific for the fusion protein.

Once a founder is produced, it may be bred, inbred, outbred or cross-crossed to produce a particular animal colony. Examples of such mating strategies include, but are not limited to, the effect of cross-breeding of founder animals at one or more integration sites to establish another lineage, the additive expression of each transgene. In order to produce compound transgenics that express transgenes at higher levels, inbred another strain, to increase expression and eliminate the need for animal screening by DNA analysis Inbreds of heterozygous transgenic animals to produce animals that are homozygous for the integration site, crosses of other homozygous lines to produce compound heterozygous or homozygous lines, And a transgene (ie, a polynucleotide encoding an albumin fusion protein of the invention) on a different background that is appropriate for the experimental model of interest That mating, are included. The transgenic animal of the present invention includes, but is not limited to, a fusion protein of the present invention, a change in biological function of a therapeutic protein and / or albumin component of the fusion protein of the present invention, a condition associated with abnormal expression and It has uses including animal model systems that are useful in studying diseases and / or screening for compounds that are effective in alleviating such conditions and / or diseases.

Example 61: Method of treatment using gene therapy-ex vivo

  One method of gene therapy transplants fibroblasts capable of expressing an albumin fusion protein of the present invention on a patient. In general, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in a tissue-culture medium and separated into small portions. A small chunk of tissue is placed on the wet flask of the tissue culture flask and approximately 10 portions are placed in each flask. Shake the flask up and down, close tightly and leave at room temperature overnight. After 24 hours at room temperature, the flask is inverted and the tissue mass remains fixed at the bottom of the flask and fresh medium (eg, Ham F12 medium containing 10% FBS, penicillin and streptomycin) is added. The flask is then incubated for approximately 1 week at 37 ° C.

  At this point, fresh media is added and subsequently changed every few days. After an additional 2 weeks of culture, a monolayer of fibroblasts appears. The monolayer is trypsinized and expanded into a large flask.

  PMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25 (1988)), flanked by Moloney murine sarcoma virus long terminal repeats, was subsequently digested with EcoRI and HindIII, followed by bovine intestine Treat with phosphatase. The linear vector is fractionated on an agarose gel and purified using glass beads.

  A polynucleotide encoding an albumin fusion protein of the invention is produced using techniques known in the art and corresponds to 5 'and 3' terminal sequences, optionally with appropriate restriction sites. Amplify using PCR primers with start / stop codons. Preferably, the 5 'primer comprises an EcoRI site and the 3' primer comprises a HindIII site. Equivalent Moloney murine sarcoma virus linear backbone and amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of the two fragments. The ligation mixture is then used to transduce bacterial HB101 and then plated on agar containing kanamycin for the purpose of confirming that the vector has the gene of interest inserted correctly.

  Amphoteric pA317 or GP + am12 packaging cells are grown in tissue culture medium to a confluent concentration in Dulbecco's modified Eagle medium (DMEM) containing 10% bovine serum (CS), penicillin and streptomycin. The MSB vector containing the gene is then added to the medium and the packaging cells are transduced with the vector. The packaging cell now produces infectious viral particles that contain the gene (the packaging cell is now referred to as the producer cell).

  Fresh medium is added to the transduced producer cells, and then the medium is collected from a 10 cm plate of confluent producer cells. The spent medium containing infectious virus particles is filtered through a Millipore filter to remove the isolated producer cells and this medium is then used to infect fibroblasts. The medium is removed from the fibroblast subconfluence plate and immediately replaced with the medium from the producer cells. Remove this medium and replace with fresh medium. If the virus titer is high, virtually all fibroblasts are infected and do not require sorting. If the titer is very low, it is necessary to use a retroviral vector with a selectable marker, such as neo or his. Once the fibroblasts are effectively infected, the fibroblasts are analyzed to determine the mobilization in which albumin protein was produced.

The modified fibroblasts are then transplanted onto the host, either alone or after growing to confluence on cytodex 3 microcarrier beads.

Example 62: Method of treatment using gene therapy-in vivo

  Another aspect of the invention is to use in vivo gene therapy methods to treat diseases, illnesses and conditions. Gene therapy methods relate to the introduction of naked nucleic acid (DNA, RNA and antisense DNA or RNA) sequences encoding the albumin fusion protein of the invention into animals. A polynucleotide encoding an albumin fusion protein of the invention may be operably linked (ie, linked) to a promoter or any other genetic element necessary for expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, for example, International Patent Nos. WO 90/11092, WO 98/11779, US Pat. Nos. 5,693,622, 5,705,151, 5580859; Tabata et al. , Cardiovasc. Res. 35 (3): 470-479 (1997); Chao et al. Pharmacol. Res. 35 (6): 517-522 (1997); Wolff, Neuromuscul. Disorder. 7 (5): 314-318 (1997); Schwartz et al. , Gene Ther. 3 (5): 405-411 (1996); Tsurumi et al. , Circulation 94 (12): 3281-3290 (1996), incorporated herein by reference.

  Any method of delivering a polynucleotide construct to an animal cell, such as an injection into the interstitial space of a tissue (such as heart, muscle, skin, lung, liver, intestine, etc.) May be delivered by. The polynucleotide construct can be delivered in a pharmacologically acceptable liquid or aqueous carrier.

  The phrase “naked” polynucleotide, DNA or RNA is any that serves to assist, enhance or facilitate entry into the cell, such as a viral sequence, viral particle, liposomal formulation, lipofectin or precipitated agent. Means a sequence that does not contain any delivery excipients. However, it can be prepared by methods well known by those skilled in the art (Felgner PL et al. (1995) Ann. NY Acad. Sci. 772: 126-139 and Abdallah B. et al. (1995). ) Polynucleotides encoding albumin fusion proteins of the present invention (such as those taught in Biol. Cell 85 (1): 1-7) may also be delivered in liposome formulations.

  The polynucleotide vector constructs used in gene therapy methods are preferably constructs that do not integrate into the host genome or do not contain sequences that allow replication. Strong promoters known to those skilled in the art can be used to drive the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked tail nucleic acids into target cells is the transient nature of polynucleotide synthesis within the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods up to 6 months.

  Polynucleotide construct, muscle, skin, brain, lung, liver, spleen, bone marrow, thyroid, heart, lymph, blood, bone, joint, pancreas, kidney, bladder, stomach, intestine, testis, ovary, uterus, rectum, nerve It can be delivered to the interstitial space of tissues in animals, including systems, eyes, glands and connective tissues. In the interstitial space of tissue, intercellular fluid, mucopolysaccharide matrix of reticulated fiber tube of organ tissue, elastic fiber in the wall of tube or chamber, collagen fiber of fiber tissue, or connective tissue that houses muscle cells in sheath Of matrix, or missing bones. Similarly, the space occupied by circulating plasma and lymph channel lymph. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. It may be delivered conventionally by injection into a tissue containing these cells. Preferably, delivery and expression can be achieved in non-differentiated or not fully differentiated cells, such as blood stem cells or dermal fibroblasts, but delivered to differentiated, persistent, non-dividing cells. To express. In vivo muscle cells are particularly competent in their ability to take up and express their polynucleotides.

  For naked polynucleotide injection, an effective dosage of DNA or RNA is in the range of about 0.05 g / kg body weight to about 50 mg / kg body weight. Preferably the dose is from about 0.005 g / kg to about 20 mg / kg, more preferably from about 0.05 mg / kg to about 5 mg / kg. Of course, as will be appreciated by those skilled in the art, this / time varies according to the tissue site of the injection. The appropriate / effective nucleic acid sequence / time can be readily determined by one skilled in the art and may depend on the condition being treated and the mode of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of the tissue. However, other parenteral routes may also be used, such as inhalation of aerosol formulations, especially for delivery to lung or bronchial tissue, throat or nasal mucosa. Furthermore, naked polynucleotide constructs can be delivered to the artery during angioplasty by the catheter used in the procedure.

  The in vivo effect of the injected polynucleotide / muscle in vivo is measured as follows. Suitable template DNA for production of mRNA encoding the polypeptide of the invention is prepared according to standard recombinant DNA methods. Template DNA, which can be either circular or linear, is used as naked DNA or complexed with liposomes. Subsequently, various amounts of template DNA are injected into the quadriceps of the mouse.

  5-6 week old female and male Balb / C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made in the anterior thigh and the quadriceps muscle is directly visualized. Template DNA is injected in 0.1 ml carrier in a 1 cc syringe through a 27 gauge needle for 1 minute into the knee at a depth of approximately 0.5 cm, approximately 0.2 cm from the distal insertion site of the muscle. Sutures are placed over the injection site for further localization and the skin is closed with stainless steel staples.

After an appropriate incubation time (eg 7 days), a muscle extract is prepared by removing the entire quadriceps. A 15um section of every fifth individual quadriceps is stained histochemically for protein expression. The time course of fusion protein expression may be performed in a similar manner, except that the quadriceps muscles from different mice are collected at different time points. Intramuscular DNA persistence after injection may be measured by Southern blot analysis after preparing total cell DAN and HIRT supernatants from injected and control mice. Using the results of the above experiments in mice, naked DNA can be used to extrapolate appropriate doses and other therapeutic parameters in humans or other animals.

Example 63: Biological effects of the fusion protein of the present invention
Astrocyte and neuron assay

  Inducing the activity of the albumin fusion protein of the present invention in promoting the survival of cortical neurons, neurite outgrowth, or phenotypic differentiation, and proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes Can be tested. The selection of cortical neurons for biopsy is based on the widespread expression of FGF-1 and FGF-2 in the skin structure and the previously reported enhancement of skin nerve survival in the results of FGF-2 treatment ing. For example, a thymidine incorporation assay can be used to elucidate the activity of the albumin fusion protein of the invention in these cells.

Furthermore, previous reports describing the biological effects of FGF-2 (basic FGF) in skin or hippocampal neurons in vitro showed a decrease in both neuronal survival and neurite outgrowth ( All of which are the assays incorporated herein by reference (Wallike et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite outgrowth (profibrous growth factor promoters). survival of dissociated hippocampal neurons and enhances neuroextension.) "Proc. Natl. Acad. Sci. USA 83: 3012-3016 (1986)). However, reports from experiments performed on PC-12 cells show that these two responses need not be synonymous, not only which FGF is being tested, but also the receptor (s) on the target cell. The ability to induce neurite outgrowth of an albumin fusion protein of the present invention using a primary skin nerve culture paradigm, response achieved with FGF-2 using, for example, a thymidine incorporation assay. And can be compared.
Fibroblast and endothelial cell assays

Human lung fibroblasts are obtained from Clontics (Clonetics (San Diego, Calif.)) And maintained in growth media from Clonticus. Skin microvascular endothelial cells were obtained from Cell Applications (San Diego, Calif.). For proliferation assays, human embryonic fibroblasts and skin microvascular endothelial cells can be cultured in growth medium in 96-well plates at 5,000 cells / well for 1 day. The cells are then incubated in 0.1% BSA basal medium for 1 day. After exchanging the medium with fresh 0.1% BSA medium, the cells are incubated with the test fusion protein of the invention for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) Is added to each well to a final concentration of 10%. Incubate cells for 4 hours. Cell survival is measured by reading in a CytoFluor fluorescence reader. For the PGE ₂ assay, human lung fibroblasts are cultured at 5,000 cells / well for 1 day in 96-well plates. After medium change to 0.1% BSA basal medium, cells are incubated with FGF-2 or fusion protein of the invention for 24 hours in the presence or absence of IL-1a. Supernatants are collected and assayed for PGE ₂ by EIA kit (Cayman, Ann Arbor, MI). For the IL-6 assay, human embryo fibroblasts are cultured for 1 day at 5,000 cells / well in 96-well. After medium change to 0.1% BSA basal medium, cells are incubated for 24 hours with FGF-2 or a fusion protein of the invention, with or without IL-1a. Supernatants are collected and assayed for PGE ₂ by ELISA (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured in basal medium for 3 days with FGF-2 or an albumin fusion protein of the present invention prior to the addition of Alamar Blue to assess the effect on fibroblast proliferation. FGF-2 should exhibit stimulation at 10-2500 ng / ml, which can be used to compare with stimulation with the fusion protein of the present invention.
Cell proliferation based on [3H] thymidine incorporation

    The following [3H] thymidine incorporation assay can be used to measure the effect of therapeutic proteins, such as growth factor proteins, on the growth of cells such as fibroblasts, epithelial cells or immature muscle cells.

Sub-confluent cultures are stopped in the G1 phase by an 18 hour incubation in medium without serum. The therapeutic protein is then added for 24 hours, and for the last 4 hours, the culture is labeled with [3H] thymidine at a final concentration of 0.33 μM (25 Ci / mmol, Amersham, Arlington Heights, IL). Incorporated [3H] thymidine is precipitated with ice-cold 10% trichloroacetic acid for 24 hours. Subsequently, the cells are successively rinsed with ice-cold 10% trichloroacetic acid and then with ice water. After dissolution in 0.5M NaOH, store the lysate and PBS rinse (500 ml) and measure the amount of radioactivity.
Parkinson model

The lack of motor function in Parkinson's disease is due to a lack of striatal dopamine, a result of degeneration of nigrostriatal dopaminergic projection neurons. An animal model for Parkinson that has been widely characterized includes the systemic administration of 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken up by astrocytes, catabolized by monoamine oxidase B to 1-methyl-4-phenylpyridine (MPP ⁺ ) and released. MPP ⁺ subsequently accumulates actively in dopaminergic neurons by a high affinity reuptake transporter for dopamine. MPP ⁺ is then concentrated in the mitochondria by an electrochemical gradient, selectively inhibiting nicotide amide adenine diphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transfer, Oxygen radicals are generated in the final body.

  It has been shown in a tissue culture paradigm that FGF-2 (basic FGF) has trophic activity on nigrodopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, the group of Dr. Unsiker et al. Showed that administration of FGF-2 in gel foam implants in the striatum resulted in nearly complete protection of nigrodopaminergic neurons from toxicity associated with MPTP exposure. (Otto and Unsicker, J. Neuroscience, 1990).

To determine whether the albumin fusion protein of the present invention has activity similar to that of FGF-2 in enhancing dopaminergic neuron survival in vitro based on data on FGF-2 And can be tested in vivo for the protection of dopaminergic neurons in the striatum from disorders associated with MPTP treatment. Cultures are prepared by dissecting the midbrain floor plate from the 14th day of pregnancy Wistar rat lung. Tissues were separated with trypsin, at a concentration of 200,000 cells / cm ^2, Poriruchinin - plated on laminin coated glass coverslips. Cells are maintained in Dulbecco's modified Eagle's medium and F12 medium containing humoral supplement (N1). Cultures are fixed in paraformaldehyde after 8 days in vitro and processed for tyrosine hydroxylase immunohistochemical staining, a specific marker for dopaminergic neurons. Dissociated cell culture medium is prepared from fetal rats. The culture medium is changed every 3 days and factors are also added at that time.

Since dopaminergic neurons were isolated from animals on day 14 of gestation, the growth time of the stage where dopaminergic progenitor cells proliferate, the number of tyrosine hydroxylase immunopositive neurons, dopaminergic surviving in vitro Represents an increase in the number of sex neurons. Thus, it is suggested that the fusion protein may be involved in Parkinson's disease when the therapeutic protein of the invention works to prolong dopaminergic neuron survival.

Example 64: Pancreatic beta-cell transplant combination therapy

  Transplantation is a common form of treatment of autoimmune diseases, especially when the target self tissue is severely damaged. For example, without limitation, pancreatic transplantation and islet cell transplantation are common treatment options for IDDM (eg, Stewart et al., Journal of Clinical Endocrinology & Metabolism 86 (3): 984-988. Brunicardi, Transplant. Proc. 28: 2138-40 (1996); Kendall & Robertson, Diabetes Metab. 22: 157-163 (1996): Hamano et al., Kobe J. 93. -104 (1996); Larsen & Stratta, Diabetes Metab. 22: 139-14 ... (1996); and Kinkhabwala, et al, Am J. Surg 171: 516-520 (1996)). As with any transplantation method, transplantation therapy for patients with autoimmune disease includes treatment to minimize the risk of host rejection of the transplanted tissue. However, autoimmune diseases involve an additional, independent risk that the preexisting autoimmune response that damages the original self tissue exerts a similar damaging effect in the transplanted tissue. Accordingly, the present invention provides a method for the treatment of autoimmune pancreatic disease using an albumin fusion protein of the present invention in combination with an immunomodulator / immunosuppressant in an individual receiving autoimmune disease transplant therapy. And a composition.

  In accordance with the present invention, compositions and formulations based on albumin fusion as described above can be used to prevent damage to transplanted organs, tissues or cells that are the result of the host individual's autoimmune response that originally targeted the native self tissue. Administer for prevention and treatment. Administration may be performed 2-4 times per week, both before and after transplantation.

Without limitation, AI-401, CDP-571 (anti-TNF monoclonal antibody), CG-1088, Diamyd (diabetes vaccine), ICM3 (anti-ICAM-3 monoclonal antibody), linomide (Roquinimex),
NBI-6024 (modified peptide ligand), TM-27, VX-740 (HMR-3480), caspase-8 protease inhibitor, thalidomide, hOKT3gamma1 (Ala-ala) (anti-CD3 monoclonal antibody), Oral Interferon-Alpha, oral The following immunomodulators / immunosuppressors, including Lactobacillus, and LymphoStat-B ™ can be used with the albumin fusion proteins of the present invention in islet cells or pancreas transplants.

Example 65: Identification and cloning of VH and VL domains

  One method for identifying and cloning VH and VL domains from a cell line expressing a particular antibody is to perform PCR with VH and VL specific primers on cDAN generated from the antibody expressing cell line. Is to implement. Briefly, RNA is isolated from cell lines and used as a template for RT-PCR designed to amplify the VH and VL domains of antibodies expressed by EBV cell lines. Cells may be lysed in TRIzol® reagent (Life Technologies, Rockville. MD) and extracted with 1/5 volume of chloroform. Following the addition of chloroform, the solution is incubated for 10 minutes at room temperature and centrifuged in a tabletop centrifuge at 14,000 rpm for 15 minutes at 4 ° C. The supernatant is collected and RNA is precipitated using an equal volume of isopropanol. The precipitated RNA is pelleted by centrifugation in a tabletop centrifuge at 14,000 rpm for 15 minutes at 4 ° C. Following centrifugation, the supernatant is rinsed and washed with 75% ethanol. Following washing, the RNA is again centrifuged at 800 rpm for 5 minutes at 4 ° C. Discard the supernatant and allow the pellet to air dry. RNA is dissolved in DEPC water and heated to 60 ° C. for 10 minutes. The amount of RNA is determined using optical density measurements.

  cDNA may be synthesized from 1.5 to 2.5 micrograms of RNA using reverse transcriptase and random hexamer primers according to methods well known in the art. The cDNA is then used as a template for PCR amplification of VH and VL domains. The primers used to amplify the VH and VL genes are shown in Table 7. Typically, a PCR reaction utilizes a single 5 'primer and a single 3' primer. Often, groups of 5 'and / or 3' primers may be used when the amount of available RNA is limited or for higher efficiency. For example, often all five VH-5 'primers and all JH3' primers are used in a single PCR reaction. The PCR reaction was performed in a 50 microliter volume containing 1 × PCR buffer, 2 mM of each dNTP, 0.7 units of High Fidelity Taq polymer, 5 ′ primer mix, 3 ′ primer mix, and 7.5 microliter cDNA. carry out. Both VH and VL 5 'and 3' primer mixes can be made by storing 22 pmol and 28 pmol of each individual primer together, respectively. PCR conditions are 96 ° C., 5 minutes, followed by 94 ° C., 1 minute, 50 ° C., 1 minute and 72 ° C., 1 minute 25 cycles, followed by 72 ° C., 10 minutes extension cycle. After the reaction is complete, the sample tube is stored at 4 ° C.

ＰＣＲ試料をついで、１．３％アガロースゲル上で電気泳動する。予想したサイズ（ＶＨドメインに関して〜５０６塩基対、ＶＬドメインに関して３４４塩基対）のＤＮＡバンドをゲルから切り取り、本技術分野でよく知られている方法を用いて精製する。精製したＰＣＲ産物を、ＰＣＲクローニングベクター（インビトロジェン社Ｉｎｖｉｔｒｏｇｅｎ Ｉｎｃ．）、Ｃａｒｌｓｂａｄ， ＣＡからのＴＡベクター）内にライゲート可能である。個々のクローン化ＰＣＲ産物を、大腸菌のトランスフェクションおよび青／白色選別の後に単離可能である。クローン化したＰＣＲ産物をついで、本技術分野で一般的に公知の方法を用いて配列決定してもよい。

The PCR sample is then electrophoresed on a 1.3% agarose gel. A DNA band of the expected size (˜506 base pairs for the VH domain, 344 base pairs for the VL domain) is excised from the gel and purified using methods well known in the art. The purified PCR product can be ligated into a PCR cloning vector (Invitrogen Inc., TA vector from Carlsbad, Calif.). Individual cloned PCR products can be isolated after transfection of E. coli and blue / white sorting. The cloned PCR product may then be sequenced using methods generally known in the art.

A PCR band containing a VH domain and a VL domain can also be used to create a full-length Ig expression vector. Heavy (eg, human IgG1 or human IgG4) or light chain (human kappa or human lambda) so that complete heavy or light chain molecules can be expressed from these vectors when the VH and VL domains are transfected into an appropriate host cell. It can be cloned into a vector containing the nucleotide sequence of the constant region. In addition, if the cloned heavy and light chains are both expressed in one cell line (from either one or two vectors) they are contained within a fully functional antibody molecule that is secreted into the cell culture medium. It can be assembled. Methods for using polynucleotides encoding VH and VL antibody domains to produce expression vectors encoding complete antibody molecules are well known in the art.

Example 66: Preparation of HA-cytokine or HA-growth factor fusion proteins such as NGF, BFNFa, BDNFb and BDNFc

Exclusive cDNAs for the cytokine or growth factor of interest, such as NGF, all from standard cDNA methods, from cDNA libraries, by RT-PCR, and by PCR with a series of overlapping synthetic oligonucleotide primers Although not critical, it can be isolated by a variety of methods. The nucleotide sequences of all these proteins are known and available. For cloning of the cDNA into a vector containing the cDNA for HA, the cDNA can be tailored at the 5 ′ and 3 ′ ends so that an oligonucleotide linker can be used to create a restriction enzyme. This can be at the N or C-terminus with or without the use of a spacer sequence. NGF (or other cytokine) cDNA is removed from pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or the complete expression cassette and inserted into plasmid pSAC35 to allow expression of albumin fusion proteins in yeast pC4: HSA In a vector such as The albumin fusion protein secreted from yeast can then be recovered from the culture medium and purified and tested for its biological activity. For expression in mammalian cell lines, the same procedure is adapted except that the expression cassette used utilizes a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a suitable plasmid for transfection of mammalian cell lines.

Example 67: Preparation of a HA-IFN fusion protein such as IFNa

Includes, but not exclusively, cDNA of interest interferon, such as IFNa, from cDNA libraries, using standard methods, by RT-PCR, and by PCR using a series of overlapping synthetic oligonucleotide primers. It can be isolated by various methods. Nucleotide sequences for interferons such as IFNα are known, for example, in US Pat. Is available. For cloning of the cDNA into a vector containing the cDNA for HA, the cDNA can be tailored at the 5 ′ and 3 ′ ends so that an oligonucleotide linker can be used to create a restriction enzyme. This can be at the N or C-terminus with or without the use of a spacer sequence. IFNα (or other interferon) cDNA is removed from pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or the complete expression cassette and inserted into plasmid pSAC35 to allow expression of albumin fusion proteins in yeast: Clone into a vector such as HSA. The albumin fusion protein secreted from yeast can then be recovered from the culture medium and purified and tested for its biological activity. For expression in mammalian cell lines, the same procedure is adapted except that the expression cassette used utilizes a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a suitable plasmid for transfection of mammalian cell lines.
Maximum protein recovery from vials

The albumin fusion protein of the present invention has a high degree of stability even when packaged at low concentrations. Furthermore, despite the low protein concentration, good fusion-protein recovery is observed even when the aqueous solution does not add other proteins to minimize binding to the vial wall. The recovery of the vial-storage HA-IFN solution is compared with the storage solution. 6 or 30 μg / ml HA-IFN solution is placed in a vial and stored at 4 ° C. After 48 or 72 hours, a volume essentially equal to a 10 ng sample was taken and measured in an IFN sandwich ELISA. The estimated value was compared with that of a high concentration stock solution. As indicated, there is essentially no sample loss in these vials, suggesting that the addition of exogenous material such as albumin is not necessary to prevent sample loss to the vial wall. is doing.
In vivo stability and bioavailability of HA-α-IFN fusion

  To determine the in vivo stability and bioavailability of HA-α-IFN fusion molecules, purified fusion molecules (from yeast) were administered to monkeys. Pharmacological compositions formulated from HA-α-INFN fusions are counted for increased serum half-life and bioavailability. Thus, a pharmacological composition may be formulated to contain a lower dose of alpha-interferon activity compared to a negative alpha-interferon molecule.

  A pharmacological composition comprising a HA-α-IFN fusion may be used to treat or prevent a disease in any disease or disease patient that can be modulated by administration of α-IFN. Such diseases include, but are not limited to, hairy cell leukemia, Kaposi's sarcoma, genital and anal warts, chronic hepatitis B, chronic non-A non-B hepatitis, especially hepatitis C, hepatitis D, chronic myelogenous leukemia , Renal cell carcinoma, bladder cancer, ovarian and cervical cancer, skin cancer, recurrent respiratory papillomatosis, non-Hodgkins and cutaneous T-cell lymphoma, melanoma, multiple myeloma, AIDS, multiple sclerosis, Brain tumors and the like are included (see Alpha, In: AHFS Drug Information, 1997).

Accordingly, the present invention includes a pharmacological composition comprising an HA-α-IFN fusion protein, a polypeptide or peptide formulated at a dosage suitable for human administration. The invention also includes a method of treating a patient in need of such therapy comprising at least the step of administering a pharmacological composition comprising at least one HA-α-IFN fusion protein, polypeptide or peptide. .
Bifunctional HA-α-IFN fusion

  The HA-α-IFN expression vector may be modified to include an insert for expression of the bifunctional HA-α-IFN fusion protein. For example, the cDNA for the second protein of interest may be inserted in-frame downstream of the “rHA-IFN” sequence after removing the double stop codon or being shifted downstream of the coding sequence.

In one version of the bifunctional HA-α-IFN fusion protein, an antibody or fragment to a B-lymphocyte stimulating protein (GenBank Acc 4455139) or polypeptide can be fused to one end of the HA component of the fusion molecule. Good. This bifunctional protein is useful for modulating any immune response produced by the α-IFN component of the fusion.

Example 68: Preparation of HA-hormone fusion protein

CDNA for the hormone of interest is obtained by a variety of methods including, but not exclusively, using standard methods, from cDNA libraries, by RT-PCR, and by PCR using a series of overlapping synthetic oligonucleotide primers. It can be isolated. Nucleotide sequences for all these proteins are known and available in public databases such as GenBank. For cloning of the cDNA into a vector containing the cDNA for HA, the cDNA can be tailored at the 5 ′ and 3 ′ ends so that an oligonucleotide linker can be used to create a restriction enzyme. This can be at the N or C-terminus with or without the use of a spacer sequence. Hormone cDNA is removed in a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or then the complete expression cassette and inserted into plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. To clone. The albumin fusion protein secreted from yeast can then be recovered from the culture medium and purified and tested for its biological activity. For expression in mammalian cell lines, the same procedure is adapted except that the expression cassette used utilizes a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a suitable plasmid for transfection of mammalian cell lines.

Example 69: Preparation of HA-soluble receptor or HA-binding protein fusion protein

All, but not exclusively, cDNA for the soluble receptor or binding protein of interest is included, using standard methods, from a cDNA library, by RT-PCR, and by PCR using a series of overlapping synthetic oligonucleotide primers. It can be isolated by various methods. Nucleotide sequences for all these proteins are known and available in public databases such as GenBank. For cloning of the cDNA into a vector containing the cDNA for HA, the cDNA can be tailored at the 5 ′ and 3 ′ ends so that an oligonucleotide linker can be used to create a restriction enzyme. This can be at the N or C-terminus with or without the use of a spacer sequence. The receptor cDNA can be taken in a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or the complete expression cassette and then inserted into plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. To clone. The albumin fusion protein secreted from yeast can then be recovered from the culture medium and purified and tested for its biological activity. For expression in mammalian cell lines, the same procedure is adapted except that the expression cassette used utilizes a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a suitable plasmid for transfection of mammalian cell lines.

Example 70: Preparation of HA-growth factor

Various methods including, but not exclusively, cDNA for a growth factor of interest from a cDNA library, by RT-PCR, and by PCR using a series of overlapping synthetic oligonucleotide primers, all using standard methods Can be isolated by (See GenBank Acc. No. NP — 600009) Tailoring the cDNA at the 5 ′ and 3 ′ ends so that an oligonucleotide linker can be used for cloning the cDNA into a vector containing the cDNA for HA. Thus, a restriction enzyme can be produced. This can be at the N or C-terminus with or without the use of a spacer sequence. Hormone cDNA is removed in a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or then the complete expression cassette and inserted into plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. To clone. The albumin fusion protein secreted from yeast can then be recovered from the culture medium and purified and tested for its biological activity. For expression in mammalian cell lines, the same procedure is adapted except that the expression cassette used utilizes a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a suitable plasmid for transfection of mammalian cell lines.

Example 71: Preparation of HA-single chain antibody fusion protein

  Single chain antibodies, including but not limited to, selection from phage libraries, cloning of antibody cDNAs, using flanking constant regions as primers for cloning variable regions, or of any particular antibody It is produced by a variety of methods including cloning of the variable region of a particular antibody by synthesizing oligonucleotides corresponding to the variable region. For cloning of the cDNA into a vector containing the cDNA for HA, the cDNA can be tailored at the 5 'and 3' ends to make a restriction enzyme, so that an oligonucleotide linker can be used. This can be at the N or C-terminus with or without the use of a spacer sequence. Hormone cDNA is removed in a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or the complete expression cassette and then inserted into plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. To clone.

In the fusion molecule of the present invention, V _H and V _L can be linked by one of the following methods or a combination thereof. Peptide linker between the N- terminus of the C- terminal and _{V L} of V _H, is cleaved upon secretion of two, then to self-bond, Kex2p protease cleavage site between the _{V H} and _{V L,} and _{V H} and V Cysteine residues arranged so that _L can be linked together to form a disulfide bond between them. Another option is to place V _H at the N-terminus of the HA or HA domain fragment and _VL at the C-terminus of the HA or HA domain fragment.

The albumin fusion protein secreted from yeast can then be recovered from the medium, purified and tested for its activity. For expression in mammalian cell lines, the same procedure is adapted except that the expression cassette used utilizes a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a suitable plasmid for transfection of mammalian cell lines. Antibodies produced in this manner can be purified from the culture medium and tested for their binding to the antigen using standard immunochemical methods.

Example 72: Preparation of HA-cell adhesion molecule fusion protein

The cDNAs for the cell adhesion molecules of the present invention are not exclusive of all, using standard methods, from cDNA libraries, by RT-PCR, and by PCR using a series of overlapping synthetic oligonucleotide primers, It can be isolated by a variety of methods including: Nucleotide sequences for all these proteins are known and available in public databases such as GenBank. For cloning of the cDNA into a vector containing the cDNA for HA, the cDNA can be tailored at the 5 ′ and 3 ′ ends so that an oligonucleotide linker can be used to create a restriction enzyme. This can be at the N or C-terminus with or without the use of a spacer sequence. Cell adhesion molecule cDNA, such as pCPC0005 (FIG. 2), pScCHSA, pScNHSA, or the complete expression cassette is then removed and inserted into plasmid pSAC35 to allow expression of albumin fusion proteins in yeast. Clone into vector. The albumin fusion protein secreted from yeast can then be recovered from the culture medium and purified and tested for its biological activity. For expression in mammalian cell lines, the same procedure is adapted except that the expression cassette used utilizes a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a suitable plasmid for transfection of mammalian cell lines.

Example 73: Preparation of inhibitors and peptides as HA fusion proteins such as HA-antiviruses, HA-antibiotics, HA-enzyme inhibitors and HA-anti-allergenic proteins

CDNA for the peptide of interest, such as an antibiotic peptide, is not exclusive, using standard methods, from a cDNA library, by RT-PCR, and by PCR using a series of overlapping synthetic oligonucleotide primers. , And can be isolated by various methods. Nucleotide sequences for all these proteins are known and available in public databases such as GenBank. For cloning of the cDNA into a vector containing the cDNA for HA, the cDNA can be tailored at the 5 ′ and 3 ′ ends so that an oligonucleotide linker can be used to create a restriction enzyme. This can be at the N or C-terminus with or without the use of a spacer sequence. Peptide cDNA can be taken in a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or the complete expression cassette and then inserted into plasmid pSAC35 to allow expression of the albumin fusion protein in yeast. To clone. The albumin fusion protein secreted from yeast can then be recovered from the culture medium and purified and tested for its biological activity. For expression in mammalian cell lines, the same procedure is adapted except that the expression cassette used utilizes a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a suitable plasmid for transfection of mammalian cell lines.

Example 74: Preparation of targeted HA fusion protein

The cDNA for the protein of interest can be isolated from a cDNA library or can be made synthetically using a variety of overlapping oligonucleotides using standard molecular biology methods. Appropriate nucleotides can be modified in the cDNA to form convenient restriction sites and to allow adhesion of the protein cDNA to the albumin cDNA. A targeting protein or peptide cDNA, such as a single chain antibody or peptide, such as a nuclear localization signal that can direct the protein inside the cell, can be fused to the other end of albumin. The protein of interest and targeting peptide are cloned into a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA or pC4: HSA that allows fusion with albumin cDNA. In this manner, both the N- and C-termini of albumin are fused to other proteins. The fusion cDNA is then excised from pPPC0005 and inserted into a plasmid such as pSAC35 to allow expression of the albumin fusion protein in yeast. All the above procedures can be performed using standard methods in molecular biology. Albumin fusion protein secreted from yeast can be recovered and purified from the culture medium and tested for its biological activity and its targeting activity using appropriate biochemical and biological tests.

Example 75: Preparation of HA-enzyme fusion

CDNA for the enzyme of interest can be obtained by a variety of methods including, but not exclusively, using standard methods, from cDNA libraries, by RT-PCR, and by PCR using a series of overlapping synthetic oligonucleotide primers. It can be isolated. For cloning of the cDNA into a vector containing the cDNA for HA, the cDNA can be tailored at the 5 ′ and 3 ′ ends so that an oligonucleotide linker can be used to create a restriction enzyme. This can be at the N or C-terminus with or without the use of a spacer sequence. Enzymatic cDNA is removed in a vector such as pPPC0005 (FIG. 2), pScCHSA, pScNHSA, or then the complete expression cassette and inserted into plasmid pSAC35 to allow expression of albumin fusion proteins in yeast. To clone. The albumin fusion protein secreted from yeast can then be recovered from the culture medium and purified and tested for its biological activity. For expression in mammalian cell lines, the same procedure is adapted except that the expression cassette used utilizes a mammalian promoter, leader sequence and terminator (see Example 1). This expression cassette is then excised and inserted into a suitable plasmid for transfection of mammalian cell lines.

Example 76: Construct ID 2249, IFNa2-HSA, production

Construct ID 2249, pSAC35: IFNa2. HSA is a DNA encoding an IFNa2 albumin fusion protein with an HSA chimeric leader sequence, followed by C.I. The mature form of IFNa2 protein fused to the amino terminus of the mature form of HSA in cerevisiae expression vector pSAC35, ie C1-E165.
Cloning of IFNa2 cDNA

  A polynucleotide encoding IFNa2 was PCR amplified using primers IFNa2-1 and IFNa2-2, described below. PCR amplification is cut with Sal I / Cla I and ligated into Xho I / Cla I cut pScCHSA. Construct ID # 2249 encodes an albumin fusion protein comprising a chimeric leader sequence of HSA, a mature form of IFNa2, followed by a mature HSA protein.

Two oligonucleotides suitable for PCR amplification of polynucleotides encoding mature forms of IFNa2, IFNa2-1 and IFNa2-2 are synthesized.
IFNa2-1: 5′-CGCGGCGC GTCGAC AAAAGATGTGATCTGCCTCAAACCCACA-3 ′ (SEQ ID NO: 348)
IFNa2-2: 5′-GCGCGGC ATCGAT GAGCAACCCTCACTCTTGTGTGCATCTTCCTTACTTCTTAAACTTTCT-3 ′ (SEQ ID NO: 349)

  The IFNa2-1 primer encodes the SalI cloning site (shown underlined), the nucleotide encoding the last 3 amino acid residues of the chimeric HSA leader, and the first 7 amino acid residues of the mature form of IFNa2. Contains 22 nucleotides (shown in bold). In IFNa2-2, the CalI site (indicated by underlining) followed by DNA is the reverse complement of DNA encoding the first 10 amino acids of the mature HSA protein, and the last 22 nucleotides (shown in bold) Is the reverse complement of the reverse complement of DNA encoding the last 7 amino acid residues of IFNa2 (see Example 2). A PCR amplification of IFNa2-HSA was produced using these primers, purified, digested with Sal I and Cla I restriction enzymes, and cloned into the Xho I and Cla I sites of the pScCHSA vector. After confirming the sequence, the expression cassette encoding this IFNa2 albumin fusion protein was subcloned into Not I designed pSAC35.

  Furthermore, the presence of the expected IFNa2 sequence can be confirmed by analysis of the N-terminus of the expressed albumin fusion protein by amino acid sequencing (see below).

  Other IFNa2 albumin fusion proteins using different leader sequences were constructed by methods known in the art (see Example 2). Examples of various leader sequences include, but are not limited to, invertase “INV” (constructs 2343 and 2410) and mating alpha factor “MAF” (construct 2366). These IFNa2 albumin fusion proteins can be subcloned into mammalian expression vectors such as pC4 (construct 2383) and pEE12.1 as previously described (see Example 5). An IFNa2 albumin fusion protein with a therapeutic protein fused to the C-terminus of HSA can also be constructed (construct 2381).

The IFNa2 albumin fusion protein of the invention is preferably a mature form of HSA fused to either the N- or C-terminus of the mature form of IFNa2, ie, Cys-1 to Glu-165, ie Asp-25 to Leu- 609. In one embodiment of the invention, the INFa2 albumin fusion protein of the invention further comprises a signal sequence directed to the developmental fusion polypeptide in the host secretory pathway used for expression. In a further preferred embodiment, the signal peptide encoded by the signal sequence is removed and the mature IFNa2 albumin fusion protein is secreted directly into the culture medium. The IFNa2 albumin fusion protein of the present invention includes, but is not limited to, MAF, INV, Ig, fibrin B, clusterin, insulin-like growth factor binding protein 4, but not limited to a variant HSA comprising a chimeric HSA / MAF leader sequence Heterologous signal sequences including leader sequences or other heterologous signal sequences known in the art may be included. In a preferred embodiment, the IFNa2 albumin fusion protein of the invention comprises native IFNa2. In a further preferred embodiment, the IFNa2 albumin fusion protein of the invention includes an N-terminal methionine residue. Polynucleotides encoding these polypeptides, including fragments and / or variants, are also encompassed by the present invention.
Expression and purification of construct ID 2249 Expression in cerevisiae

The yeast 2 of construct 2249. Transduction into S. cerevisiae strain BXP10 was performed by methods known in the art (see Example 3). Cells can be harvested in stationary phase after 72 hours of growth. The supernatant is collected by clarification of the cells at 3000 g for 10 minutes. Expression levels are tested by immunoblot detection with anti-HSA serum (Kent Laboratories) or as a primary antibody. An IFNa2 albumin fusion protein with a molecular weight of approximately 88.5 kDa can be obtained.
Yeast S. Purification from cerevisiae cell supernatant

Yeast S. Cell supernatants containing IFNa2 albumin fusion protein expressed from construct ID # 2249 in cerevisiae cells can be obtained on a Dyax peptide affinity column on a small scale (see Example 4) or in the following five steps: dialysis, DEAE On a large scale by anion exchange chromatography using Sepharose Fast Flow column, hydrophobic interaction chromatography (HIC) using Butyl 650S column, cation exchange chromatography using SP-Sepharose Fast Flow column or Blue-Sepharose chromatography on a large scale ( (See Example 4) Purification is possible. IFNa2 albumin fusion protein is obtained from DEAE-Sepharose Fast Flow column with 100-250 mM NaCl, from SP-Sepharose Fast Flow column at 150-250 mM NaCl, and from Q-Sepharose High Performance column at 5-7.5 mS / cm. It may be eluted. N-terminal sequencing produces the sequence CDLPQ (SEQ ID NO: 98), which corresponds to the mature form of IFNa2.
INFa2 activity can be assayed using an in vitro ISRE-SEAP assay

The IFNa2 albumin fusion protein encoded by construct ID # 2249 can be tested for activity in an ISRE-SEAP assay as previously described in Example 76. Briefly, conditioned yeast supernatants were tested at a 1: 1000 dilution for their ability to direct ISRE signaling on the ISRE-SEAP / 293F reporter cell line. ISRE-SEAP / 293F reporter cells were plated one day prior to treatment at 3 × 10 ⁴ cells / well in 96-well poly-D-lysine coated plates. The reporter cells were then incubated for 18-24 hours and 40 μL was taken for use in the SEAP Reporter Gene Chemiluminescent Assay (Roche catalog # 1779842). Recombinant human interferon beta “rhIFNb” (Biogen) was used as a positive subject.
result

IFNa2-HSA purification modulators at concentrations ranging from 10 ⁻¹ to 10 ¹ ng / mL (see FIG. 4) or 10 ⁻¹⁰ to 10 ⁻⁸ ng / mL (see FIG. 5). The ISRE-SEAP assay showed a relatively linear increase.
In vivo induction of OAS by interferon alpha fusion encoded by construct ID 2249

The OAS enzyme, 2 ′, 5′-oligoadenylate synthase is activated at the transcriptional level by interferon in response to antiviral infection. The effects of interferon constructs can be measured by obtaining blood samples from treated monkeys and analyzing these samples for transcriptional activation of the two OAS mRNAs, p41 and p69. A 0.5 mL volume of whole blood was collected per group at 7 different time points, 0, 1, 2, 4, 8, 10, and 14 per animal. Obtained from 4 animals. The various groups included vehicle control, intravenous injection of 30 μg / kg HSA-IFN on day 1, subcutaneous injection of 30 μg / kg HSA-IFN on day 1, 300 μg / kg HSA-IFN on day 1. And subcutaneous injections of 40 μg / kg interferon alpha (Schering-Plough) on days 1, 3 and 5 as a positive control. The levels of p41 and p69 mRNA transcripts were measured by real-time quantitative PCR (Taqman) using probes specific for p41-OAS and p69-OAS. OAS mRNA levels were quantified compared to the 18S ribosomal RNA endogenous control. Albumin fusion encoded by construct 2240 can be subjected to similar experiments.
result

Significant increases in mRNA transcript levels for both p41 and p69 OAS were observed in HSA-interferon treated monkeys versus IFNa treated monkeys (see FIG. 6 for p44 data). The effect was maintained for approximately 10 days.

Example 77: Indication for IFNa2 albumin fusion protein

  IFN alpha albumin fusion proteins (including but not limited to those encoded by constructs 2249, 2343, 2410, 2366, 2382, and 2381) to treat, prevent, reduce and / or detect multiple sclerosis Can be used. Other indications include, but are not limited to, severe acute respiratory syndrome (SARS) and other coronavirus infections including but not limited to Ebola virus and Marburg virus, Arena virus including Lhasa virus, Junin virus, Macpo virus, Guanarito virus, and lymphocytic choriomeningitis virus (LCMV), including but not limited to Puntatrovirus, Crimean-Congo bleeding Fever virus, sand fever fever virus, rift valley fever virus, bunya virus, including but not limited to yellow fever, banzi virus, West Nai West Nile virus, Dengue virus, Japan Encephalitis virus, Tick-borne encephalitis, Omsk Hemorrhagic Fever and Kasenur forest disease (Kyaseur forest disease) Including, but not limited to flavivirus, Venezuela, eastern and western equine encephalitis virus, Ross River virus and Rubella virus, Togavirus, Vaccinia, Cowpox, Smallpox And orthopocs including, but not limited to, monkeypox , Herpes virus, FluA / B, Respiratory Sincytial virus (RSV), paraflu, measles, rhinovirus, adenovirus, Semliki Forest virus, Viral Hemorrhagic fevers, abd virus R Paramyxoviruses, including Nipah and Hendra viruses, and other viral reagents identified by the Centers for Disease Control and Prevention as the most important diseases (ie, Category A, B and C reagents such as Moran , Emerg. Med. Clin. North. Am. 2002; 20 (2): 311-30 and Darling et al. , Emerg. Med. Clin. North Am. 2002; 20 (2): 273-309).

Preferably, the IFNa-albumin fusion protein or IFN hybrid fusion protein is treated naive and in treatment-experienced adults and pediatric patients, in combination with CCR5 antagonists, alone or with HIV-1 infection, HCV or HIV-1 and HCV. For the preparation of a medicament for the treatment of co-infection, it is administered in combination with at least one ribavirin, IL-2, IL-12, pentafuside in combination with an anti-HIV drug therapy, eg HAART.

Example 78: Construct ID # 3691, BNP-HSA, production

Construct ID 3691, pC4: SPCON. BNP1-32 / HSA encodes an albumin fusion protein with an active BNP (amino acids 1-32) fused to the amino-terminus of the mature form of HSA in the mammalian first fragment vector pC4, followed by a secreton. DNA.
Cloning of BNP cDNA for construct 3691

  A polypeptide encoding BNP is PCR amplified using the primers BNP-1 and BNP-2 described below, cut with Bam HI / Cla I, and ligated into Bam HI / Cla I cut pC4: HSA As a result, the structure ID # 3691 is obtained. Construct ID # 3691 encodes the consensus leader sequence (SEQ ID NO: 111) and the albumin fusion protein containing the processed active form of BNP, followed by the mature HSA protein (SEQ ID NO: for construct 3691 in Table 2). 204).

Two oligonucleotides, BNP-1 and BNP-2, were synthesized, suitable for PCR amplification of polynucleotides encoding BNP activity and processed form.
BNP-1: 5′-GAGCGC GGATCC AAGCTTCCCGCCATCATGTGTGGCCGCCTGTGTGGCTGTCTGCTCGCTCGCTGCTGCGTGTGGCCATGGTGTGGGGCCCGCCCAAGCTGTGCAAGG-3 ′
BNP-2: 5′-AGTCCC ATCGAT GAGCAACCTCACTCTTGTGTGCATCATGCCCCCTCAGCACTTTGC-3 ′ (SEQ ID NO: 365)

  BNP-1 is a BamHI cloning site (underlined), a polynucleotide encoding a consensus leader sequence (SEQ ID NO: 111) (italicized), and a polynucleotide encoding the first 7 amino acid sequence of BNP (bold) including. In BNP-2, the underlined sequence is the ClaI site, and the subsequent polynucleotide contains the reverse complement of DNA (bold) encoding the last 6 amino acids of BNP and the first 10 amino acids of the mature HSA protein. Including. Using these two primers, the BNP protein was PCR amplified. Annealing and extension temperatures and times must be determined empirically for each particular primer pair and template.

The PCR product was purified (eg, using the Wizard PCR Preps DNA Purification System (Promega Corp)) and then digested with Bam HI and Cla I. Further purification of the Bam HI-Cla I fragment by gel electrophoresis. The product was later cloned into Bam HI / Cla I digested pC4: HSA to produce construct ID # 3691. The expression construct was sequence verified.
Expression of construct ID3691 and expression in purified 293F cells

Construct ID # 3691, pC4: SPCON. BNP1-32 / HSA was transfected into 293F cells by methods known in the art (see Example 6).
Purification from 293F cell supernatant

Two liters of supernatant were collected 3 days after transfection. Recombinant protein was captured by a 5 ml Blue Sepharose CL-6B column (Amersham Biosciences, Piscataway, NJ, USA) and extracted with 2M NaCl. The material was bound to a HiPrep 16/10 Phenyl FF (High Sub) column and eluted with 20 mM MES, pH 6.7. BNP-HSA was further purified by hydroxyapatite column chromatography at pH 6.8 in a sodium phosphate buffer gradient (0-20 mS / cm in 200 ml). The final product was exchanged into a PBS pH 7.2 into a HiPrep 26/10 desalting column (Amersham Biosciences).
BNP-HSA activity can be assayed using in vitro NPR-A / cGMP assay

Natriuretic peptide receptor-A (NRP-A) is a signal receptor for BNP and is therefore responsible for most of the biological effects of BNP. BNP biological activity is mediated by an NPR-A guanylyl cyclase domain that converts GTP to cGMP via activation. A simple assay for BNP activity is to measure BNP stimulation of a 293F cell line stably overexpressing NPR-A. Following exposure to BNP, intracellular cGMP production can be measured by cGMP ELISA.
Method for screening NPR-A 293F stable clone

The open reading frame of human NPR-A is constructed in a pcDNA3.1 expression vector (Invitrogen). 293F cells were stably transfected with plasmid DNA by the lipofectamine method and sorted by 0.8 μg / ml G418. 293F / NPR-A stable clones were selected for optimal response to recombinant BNP.
Measurement of cGMP activation

CGMP activation by BNP is performed in 293F / NPR-A cells and measured by the catchpoint cyclic-GMP fluorescence assay kit (Molecular Devices, Sunnyvale, CA, USA). Briefly, 50,000 cells / well of 293F / NPR-A cultured in 96-well plates were mixed with 80 μl pre-stimulation buffer (10 mM glucose, pH 7.4, 15 nM sodium bicarbonate, and 0 Washed in Krebs-Ringer Bicarbonate Buffer) containing .75 mM 3-isobutyl-1-methylxatin. BNP-HSA or recombinant BNP in 40 μl pre-stimulation buffer was added to the cells for 10 minutes at 37 ° C. The cells were lysed with 40 μl Lysis Buffer for 10 minutes with shaking. The amount of cGMP in the lysate was quantified according to the instruction manual.
result

The BNP-HSA and recombinant BNP / times responsiveness association was determined (see FIG. 7). The maximum activity of construct ID # 3691 and recombinant BNP was similar with EC50 values of 28.4 ± 1.2 and 0.46 ± 1.1 nM, respectively (1.63 ± 0.016 pair, respectively) 1.80 ± 0.016 pm).
BNP-HSA reduces blood pressure in vivo

BNP reduces blood pressure by direct vasodilation and by suppression of the renin / angiotensin / endothelin / aldosterone system. The ability of BNP-HSA to reduce arterial blood pressure was tested in 3-month-old male essential hypertensive rats purchased from Taconic (Germantown, NY, USA). Essentially hypertensive rats are genetically hypertensive after 3 months of age when hypertension begins. BNP-HSA or recombinant BNP was reconstituted in 0.3 cc PBS / rat. Drug was delivered by tail vein infusion. Systolic and diastolic blood pressures were recorded by tail-cutting using an XBP-1000 System (Kent Scientific, Torrington, CT, USA). For each blood pressure data point, 4-5 continuous recordings were taken and averaged. Mean arterial pressure (MAP) was calculated as 1/3 systolic pressure / 2/3 diastolic pressure. For determination of the response / time, the blood pressure was pC4: SPCON. Measurements were made 20 hours after administration of BNP1-32 / HSA.
result

  The typical systole of essential hypertensive rats was 180-200 mmHg before administration. A single bolus of 6 nmol / kg BNP-HSA delivered via the tail vein reduced both diastolic and systolic pressure and measured a 30 mm Hg mean arterial pressure (MAP) decrease. Decreased blood pressure was stable and lasted for several days, then gradually returned to baseline over several days (see FIG. 8). On the other hand, due to instantaneous clearance, a single 6 nmol / kg bolus of recombinant BNP produced a very transient MAP reduction of approximately ˜15 mmHg.

In addition, a 20 hour dose response after BNP-HSA bolus injection was measured in 4 essential hypertensive rats. 0.5 nmol / kg BNP-HSA has an average of 7 mmHg MAP reduction, while 6 nmol / kg BNP-HSA has an average of 30 mmHg MAP reduction and 18 mmol / kg dose of BNP-HSA is only 6 nmol Only the blood pressure of / kg was lowered.
In vivo induction of plasma cGMP by BNP-HSA Method

Intracellular cGMP activation by BNP results in its release from the cell into the circulation. Plasma cGMP levels correlate with BNP-induced cardiovascular and renal physiology. Plasma cGMP has been used as a biomarker for in vivo BNP activity. To study the induction of plasma cGMP by BNP-HSA in vivo, 11-12 week old male C57 / BL6 was administered to the recombinant BNP or BNP-HSA at a 6 nmol / kg dose via the tail vein. Gave one bolus. Plasma was removed from tail bleeding at time points of 5, 10, 20, 40 and 80 minutes for the recombinant BNP administration group and at further time points of 640, 1440, 2880 and 5760 minutes for the BNP-HSA group. Prepared. Plasma samples from mice treated with PBS as vehicle control were collected at the zero time point. cGMP levels were measured by the catchpoint cyclic-GMP fluorescence assay kit according to the instructions.
result

After a single intravenous bolus of 6 nmol / kg recombinant BNP or BNP-HSA, the peak plasma cGMP levels above baseline increased 3.9- or 5.6-fold, respectively (see FIG. 9) . Furthermore, the monophasic exponential decay half-life after recombinant BNP treatment is 16 minutes (10-42 min, 95% Cl), and the monophasic exponential decay half-life of cGMP after BNP-HSA administration Was 1538 minutes (1017-3153 minutes, 95% Cl).
In vivo pharmacokinetic analysis of BNP albumin fusion encoded by construct ID3691

  The 11-12 week old male C57 / BL6 (obtained from Ace Animals, Boyertown, PA, USA) weighed 25.1 ± 0.12 g at the time of testing. All animals were dosed at a dose of 10 mg / kg body weight. PBS was administered to predose animals. Recombinant BNP was injected in the tail vein or subcutaneously in the middle shoulder region.

  Pharmacokinetic analysis was performed on the following groups:

  Blood was sampled from the inferior vena cava and placed in EDTA-coated microcontainers and stored on ice. Samples were centrifuged in a microcentrifuge at 14,000 rpm (16,000 × g) for 10 minutes at room temperature. The crystals were transferred into a cluster tube and stored at -80 ° C.

  BNP-HSA concentrations in plasma samples were measured using a BNP EIA Kit (Phoenix Pharmaceutical, Belmont, CA, USA). A standard curve was performed at the same time on the same plate containing the test sample. The detection limit is 0.11 ng / mL for recombinant BNP. The assay detected recombinant BNP and did not cross-react with mouse BNP.

Analysis was performed by the septumless method (WinNonlin; version 4.1; Pharsight Corp., Mountain View, CA, USA). The average plasma concentration at each time was used in the analysis. AUC0- _t was calculated using the linear up / log down trapezoidal method. Extrapolation to infinite AUC _0-8 was performed by dividing the last observed concentration by the terminal elimination rate constant. Data were weighted homogeneously for these analyses.
result

  The mean baseline concentration of BNP-HSA in plasma as detected in the pre-dosed sample was approximately 0.081-0.095 μg / ml. After single intravenous or subcutaneous injection, BNP-HSA has a terminal elimination half-life of 11.2 (intravenous administration), or 19.3 hours (subcutaneous administration), and the half-life of recombinant BNP in mice is 3.1 minutes. BNP-HSA compartmental analysis revealed that BNP-HSA has the following characteristics:

Five points in the final phase of the venous profile and four points in the final phase of the subcutaneous profile were selected for the final half-life calculation. The resulting AUC during this final phase was approximately 10% of the total AUC for intravenous and subcutaneous profiles, respectively. This is compared to only 2% and 4% of the total AUC for the intravenous and subcutaneous profiles, respectively, when the last 3 points are selected for the final half-life calculation.

Example 79: Construct ID # 3618, BNP (2x) -HA, production

Construct ID # 3618, pC4: SPCON. BNP1-32 (2x) / HSA is a two-step, active BNP (amino acid) fusion in the mammalian initial vector pC4 fused to the consensus leader sequence, secreton, followed by the amino-terminus of the mature form of HSA. 1-32) which contains DNA encoding an albumin fusion protein.
Cloning of BNP cDNA for construct 3618

  A polypeptide encoding a double BNP motif is first PCR amplified using the four primers BNP-1, BNP-2, BNP-3 and BNP-4 described below, and two fragments A and B was produced. Following amplification, the two purified fragments (A and B) were mixed in equimolar amounts and used as a PCR template and amplified with primers BNP-5 and BNP-6 as described below. The BNP (2 ×) insert was then cut with Bam HI / Cla I and ligated into the pC4: HSA vector previously digested with Bam HI / Cla I, resulting in construct ID # 3618. Construct ID # 3618 encodes an albumin fusion protein containing a consensus leader sequence (SEQ ID NO: 111) and two copies of the processed active form of BNP, followed by mature HSA protein (in Table 2, construct 3618). (See SEQ ID NO: 226 for).

Four oligonucleotides suitable for PCR amplification of polynucleotides encoding two fragments of BNP protein were first synthesized.
BNP-1 5′AGCCCCAAGATGGTGCAAGGGTCTGGCTGCTTTGGGAGGGAAGATGGACCGGATCAGCTCCCTCAGTG GCTGGGCTGCAAAGGTGCTGAGGCGGGCAT-3 ′ (SEQ ID NO: 460)
BNP-2 5′-CCTTGCACCCATCTTGGGGCTATGCCGCTCCAGCACTTTGC-3 ′ (SEQ ID NO: 461)
BNP-3 5′-GCAAAGTGCTGAGGCGGGCATAGCCCCAAGATGGTGCAAGG-3 ′ (SEQ ID NO: 462)
BNP-4 5′-AGTCCCCATCGATGAGCACACCTCACTCTTGTGTGCATCATGCCCTCTCAGCACTTTGC-3 ′ (SEQ ID NO: 463)

Two BNP protein fragments (A and B, respectively) were PCR amplified using primer sets BNP-1 / BNP-2 and BNP-3 / BNP-4. Annealing and extension temperatures and times must be determined empirically for each particular primer pair and template. Fragments A and B are purified (eg, using the Wizard PCR Preps DNA Purification System (using Promega Corp)), mixed in equimolar amounts, and two additional oligonucleotides suitable for PCR amplification, BNP-5 and Use as a template for PCR amplification with BNP-6.
BNP-5: 5'-GAGCGC GGATCC AAGCTTCCCGCCATCATGGTGTGGGCGCCTGTGTGGCTGCTGCTGCTGCTGCTGCTGCTG
BNP-6: 5′-AGTCCC ATCGAT GAGCAACCCTCACTCTTGTGTGCATCATGCCGCCTCAGCACTTTGC-3 ′ (SEQ ID NO: 383)

  BNP-5 is a BamHI cloning site (underlined), a polynucleotide encoding the consensus leader sequence (SEQ ID NO: 111) (italicized), and a polynucleotide encoding the first 7 amino acid sequence of BNP (bold) including. In BNP-6, the underlined sequence is the ClaI site, and the subsequent polynucleotide contains the reverse complement of DNA (bold) encoding the last 6 amino acids of BNP and the first 10 amino acids of the mature HSA protein. Including. Using these two primers, the consensus leader sequence and two tandem copies of the active BNP peptide were PCR amplified. Annealing and extension temperatures and times must be determined empirically for each particular primer pair and template.

The PCR product was purified (eg, using the Wizard PCR Preps DNA Purification System (Promega Corp) and then digested with Bam HI and Cla I. Further purification of the Bam HI-Cla I fragment by gel electrophoresis. The product was later cloned into Bam HI / Cla I digested pC4: HSA to produce construct ID # 3618. The expression construct was sequence verified.
Expression of construct ID3618 and expression in purified 293F cells

Construct ID # 3618, pC4: SPCON. BNP1-32 (2x) / HSA was transfected into 293F cells by methods known in the art (see Example 6).
Purification from 293F cell supernatant

PC4 encoded by construct ID # 3618: SPCON. BNP1-32 (2x) / HSA was purified as described above in Example 78 under the sub-item entitled "Purification from 293F cell supernatant".
BNP (2x) -HSA activity can be assayed using an in vitro NPR-A / cGMP assay

The activity of BNP (2 ×) -HSA encoded by construct ID # 3618 can be assayed using the NPR-A / cGMP assay, “the activity of BNP-HSA can be assayed using the in vitro NPR-A / cGMP assay. It can be assayed as described above in Example 78 under the sub-item named “A”.
Results The BNP (2 ×) -HSA and recombinant BNP / times responsiveness association was determined (see FIG. 7). The maximal activities of BNP (2 ×) and recombinant BNP encoded by construct ID # 3618 were similar with EC50 values of 9.8 ± 1.1 and 0.46 ± 1.1 nM, respectively (respectively 1.68 ± 0.02 vs. 1.80 ± 0.016 pm).

Example 80: In vitro NRP-A / cGMP assay for BNP
Background and method

Natriuretic peptide receptor-A (NRP-A) is a signal receptor for BNP and is therefore responsible for most of the biological effects of BNP. BNP biological activity is mediated by an NPR-A guanylyl cyclase domain that converts GTP to cGMP via activation. A simple assay for BNP activity is to measure BNP stimulation of a 293F cell line stably overexpressing NPR-A. Following exposure to BNP, intracellular cGMP production can be measured by cGMP ELISA.
Method for screening NPR-A 293F stable clone

The open reading frame of human NPR-A is constructed in a pcDNA3.1 expression vector (Invitrogen). 293F cells were stably transfected with plasmid DNA by the lipofectamine method and sorted by 0.8 μg / ml G418. 293F / NPR-A stable clones were selected for optimal response to recombinant BNP.
Measurement of cGMP activation

CGMP activation by BNP is performed in 293F / NPR-A cells and measured by the catchpoint cyclic-GMP fluorescence assay kit (Molecular Devices, Sunnyvale, CA, USA). Briefly, 50,000 cells / well of 293F / NPR-A cultured in 96-well plates were mixed with 80 μl pre-stimulation buffer (10 mM glucose, pH 7.4, 15 nM sodium bicarbonate, and 0 Washed in Krebs-Ringer Bicarbonate Buffer) containing .75 mM 3-isobutyl-1-methylxatin. BNP-HSA or recombinant BNP in 40 μl pre-stimulation buffer was added to the cells for 10 minutes at 37 ° C. Cells were lysed with 40 μl Lysis Buffer for 10 minutes with shaking. The amount of cGMP in the lysate was quantified according to the instruction manual and the EC50 value was determined. In this assay, higher cGMP levels result in lower signals (relative fluorescence units or RFUs).
Production of construct ID # 3796

Construct ID # 3796, pSAC35: HSA. BNP (1-32) is the HSA-treated, matured, fused to HSAsp / KEX2 leader sequence followed by the N-terminus (amino acids 1-32) of the pretreated mature form of the BNP peptide in the yeast expression vector pSAC35. It contains a DNA encoding a BNP albumin fusion protein (see SEQ ID NO: 214 for construct 3796 in Table 2).
Cloning of BNP cDNA for structure 3796

  The polypeptide encoding BNP was PCR amplified using primers BNP-102689 and BNP-102692, described below, cut with Bsu36I / AscI, ligated into Bsu36I / AscI cut pSAC-NEC and the result As the structure ID # 3796. The template for PCR amplification was a polynucleotide encoding all mature BNP (1-32 sequences).

Two oligonucleotides, BNP-102689 and BNP-102692, were synthesized suitable for PCR amplification of polynucleotides encoding BNP activity, processed form.
BNP-102689: 5′-AAGCTG CCTTAGG CTTAAGCCCCAAGATGGTGCAAGGGTC-3 ′ (SEQ ID NO: 378)
BNP-102692: 5′-GCACC GGGCGCGCC TTAATGCCCCCTCAGCACTTTGCAGC-3 ′ (SEQ ID NO: 379)

  BNP-102689 contains a Bsu36I cloning site (underlined), a polynucleotide encoding the last 5 amino acids of HSA (italicized), and a polynucleotide encoding the first 8 amino acid sequence of BNP. In BNP-102692, the underlined sequence is an AscI site, and the subsequent polynucleotide contains the reverse complement of DNA (bold) and the termination codon (italicized) encoding the last 8 amino acids of BNP. Using these two primers, the HSA / BNP fusion region was PCR amplified. Annealing and extension temperatures and times must be determined empirically for each particular primer pair and template.

PCR products were purified (eg, using the Wizard PCR Preps DNA Purification System (Promega Corp) and then digested with Bsu36I and Asc I. After further purification of the Bsu36I / Asc I fragment by gel electrophoresis, The product was cloned into Bsu36I / AscI digested pSAC35-NEC to produce construct ID # 3796. The expression structure was sequence verified.
Expression and purification of Construct ID 3796 Expression in cerevisiae

Construct ID # 3796, pSAC35: HSA. BNP (1-32) was transfected into BXP10 by methods known in the art (see Example 4).
Purification from BXP10 cell supernatant

After approximately 84 hours, the culture medium was collected and the cells were clarified via centrifugation and 0.2 μm filtration. Recombinant protein was captured by a 5 ml Blue Sepharose fast flow column (GE Healthcare) and eluted with a mixture of sodium chloride and sodium octanoate. The preparation was completed by binding the protein to a ceramic hydroxyapatite column (BioRad) and eluting with increasing concentrations of phosphoric acid. Another preparation was bound to a DEAE Sepharose fast flow column (GE Healthcare) and eluted with a linear sodium chloride gradient. The elution pool was then bound to a Q Sepharose high performance column (GE Healthcare) and eluted with a sodium chloride gradient. The final product was concentrated and exchanged in formulation buffer by final flow filtration.
Production and cloning of construct ID # 3959

  Construct ID # 3959, pSAC35: HSA. BNP (1-29) was fused to the N-terminus of the pretreated mature form of the BNP peptide lacking the HSAsp / KEX2 leader sequence followed by the last 3 amino acids (S1-L29) in the yeast expression vector pSAC35. DNA encoding the BNP albumin fusion protein with the processed, mature form of (see SEQ ID NO: 501 for structure 3959 in Table 2).

  A polynucleotide encoding BNP is PCR amplified using the primers BNP-103801 and BNP-104315 described below, cleaves Bsu36I / AscI, ligates into Bsu36I / AscI, and cleaves pSAC-NEC. The result construct ID # 3959 is obtained. The template for PCR amplification was primer construct ID3796 (see below).

Two oligonucleotides, BNP-103801 and BNP-104315, were synthesized suitable for PCR amplification of polynucleotides encoding BNP activity, processed form.
BNP-103801: 5'-CAGGAGCC CCTTAGG CTTAAGCCCCAAGATGGTGCAAGGGTCT-3 '(SEQ ID NO: 578)
BNP-104315: 5′-CCTCACTC GGCGCGCC TTACAGCACTTTGCAGCCCAGCCCACTGGA-3 ′ (SEQ ID NO: 579)

  BNP-103801 contains a Bsu36I cloning site (underlined), a polynucleotide encoding the last 5 amino acids of HSA (italicized), and a polynucleotide encoding the first 8 amino acid sequence of BNP (bold). Including. In BNP-104315, the underlined sequence is the Asc I site, and the subsequent polynucleotide contains the reverse complement of DNA (bold) and the stop codon (italics) encoding the S21-L29 amino acids of BNP. Using these two primers, the HSA / BNP fusion region was PCR amplified. Annealing and extension temperatures and times must be determined empirically for each particular primer pair and template.

The PCR product was purified (eg, using the Wizard PCR Preps DNA Purification System (Promega Corp) and then digested with Bsu36I and Asc I. After further purification of the Bsu36I / Asc I fragment by gel electrophoresis, The product was cloned into Bsu36I / AscI digested pSAC35-NEC to produce construct ID # 3959. The expression structure was sequence verified.
Expression and purification of construct ID 3959 Expression in cerevisiae

Construct ID # 3959, pSAC35: HSA. BNP (S1-L29) was transfected into BXP10 by methods known in the art (see Example 4).
Purification from BXP10 cell supernatant

After approximately 84 hours, the culture medium was collected and the cells were clarified via centrifugation and 0.2 μm filtration. Recombinant protein was captured by a 5 ml Blue Sepharose fast flow column (GE Healthcare) and eluted with a mixture of sodium chloride and sodium octanoate. The preparation was completed by binding the protein to a ceramic hydroxyapatite column (BioRad) and eluting with increasing concentrations of phosphoric acid. Another preparation was bound to a DEAE Sepharose fast flow column (GE Healthcare) and eluted with a linear sodium chloride gradient. The elution pool was then bound to a Q Sepharose high performance column (GE Healthcare) and eluted with a sodium chloride gradient. The final product was concentrated and exchanged in formulation buffer by final flow filtration.
result

Dose-response relationships of HSA-BNP (1-29) and HSA-BNP (1-32) and recombinant BNP were measured (see FIG. 10). The EC50 of HSA-BNP (1-29) and HSA-BNP (1-32) was several times greater than the EC50 value of recombinant BNP (467.9, 45.06, and 0.2227, respectively). Furthermore, the HSA-BNP (1-29) fusion protein had an EC50 10 times greater than the HSA-BNP (1-32) fusion protein.

Example 81: In vitro assay for degradation of ANP and BNP
Background and method

  Neprilysin, also known as MME, CALLA, CD10, common acute lymphocytic leukemia antigen, enkephalinase, EPN, NEP, neutral endopeptidase, or neutral endopeptidase 24.11. It is a 743 amino acid (MW 90,000-110,000 kD) cell surface metallopeptidase expressed by numerous tissues including liver, intestine, uterus, endometrium, adrenal gland, and lung. Neprilysin includes, but is not limited to, cleaving the amino side of hydrophobic residues, including but not limited to ANP, BNP, CNP, substance P, bradykinin, oxytocin, Leu- and Met-enkephalin, neurotensin, bombesin, endothelin-1 and Inactivates various physiologically active peptides, including bombesin-like peptides.

The relative sensitivity to neprilysin hydrolysis was determined to be approximately 4-5 minutes for CNP, 8 minutes for ANP, and 2 hours for BNP (Kenny, AJ et al.,). Biochem J. 291 (1): 83-8 (1993)).
Effects of neprilysin on ANP and BNP peptides

ANP and BNP peptides were assayed for activity in the CatPoint cGMP assay (Molecular Devices) with or without exposure to the protease neprilysin. In particular, 5 μM ANP or BNP was incubated for 24 hours at 37 ° C. in 10 nM neprilysin (R & D Systems MES buffer (0.1 M 2- (N-morpholino) ethanesulfonic acid (Sigma)). 293F cells stably transfected with NPR-A were then worked with various concentrations of protease-treated ANP or BNP, as was well-known in Example 80. Cell lysates were then Catch point cGMP assay (Molecular Devices) was used for analysis of cGMP activation as described at 80.
result

Dose-response curves were calculated for BNP and ANP with or without incubation with neprilysin (see FIG. 11A). BNP with or without incubation with neprilysin showed similar BNP activity with EC50 values of 0.2966 and 0.2702, respectively. However, ANP neprilysin incubation resulted in a significant decrease in ANP activity compared to untreated ANP, with EC50 values of 0.2965 and 60.47, respectively. Selected samples were further analyzed by reverse phase HPLC using techniques known in the art. The percent comparison is for samples incubated in the absence of neprilysin for a similar period of time (see FIG. 10). Although ANP proteolysis occurs within 20 minutes of treatment with neprilysin, no significant BNP proteolysis is observed even after 24 hours of incubation with neprilysin.
Effect of neprilysin on ANP-HSA fusion protein

ANP and ANP-HSA (CID 3484) were added with or without 10 nM neprilysin (R & D Systems) for 20 minutes, 1 hour or 24 hours at 37 ° C. (0.1M 2- ( N-morpholino) ethanesulfonic acid (Sigma)). 293F cells stably transfected with NPR-A as described in Example 80 were stimulated with various concentrations of protease-treated ANP or ANP-HSA. Cell lysates were then analyzed for cGMP activation using the CatchPoint cGMP assay (Molecular Devices) as described in Example 80.
result

/ Time-response curves were calculated for ANP and ANP-HSA with or without incubation with neprilysin (see FIGS. 11B and 11C, respectively). The ANP peptide showed a significant decrease in activity within 1 hour of treatment with neprilysin. However, ANP-HSA (CID 3484) did not show a significant decrease in activity even after 24 hours of treatment with neprilysin. Selected samples were further analyzed by reverse phase HPLC using techniques known in the art. The percent comparison is for samples incubated in the absence of neprilysin for a similar period of time (see FIG. 11D). Although ANP proteolysis occurs within 20 minutes of treatment with neprilysin, proteolysis of ANP-HSA (CID 3484) is not observed even after 24 hours of incubation with neprilysin.

Example 82: Antiviral activity of HSA-IFNα2b in combination with ribavirin in genotype 1, interferon-naive hepatitis C (HCV) patients
background

Conventional treatments for genotype 1, interferon-naive (IFN-naive) HCV patients utilize interferon alpha in combination with ribavirin (RBV) for 48 weeks. However, this treatment has significant implementation limitations. Because of the well-known side effects of current interferon therapy, the patient's quality of life is essentially reduced after each dose of interferon. A large number of patients resulted in discontinuation of treatment, with some studies reporting discontinuation rates greater than 50%. Furthermore, current interferon therapy also has a significant rate of significant hematologic reduction, requires a reduction in RBV / times, or more clearly, until the hematological value is normalized, A temporary stop may be necessary. Thus, there is a clear need for improved therapeutic protocols for the treatment of genotype 1 HCV in INF-naïve patients.
principle

HSA-IFNa2b was produced at its C-terminus by genetically fusing mature albumin to the N-terminus of mature interferon α-2b. The safety and efficacy of treatment with HSA-IFNa2b in combination with RBV was evaluated in an aggressive controlled clinical study in genotype 1, IFN-naïve HCV human patients, and in combination with RBV as an active control , Compared with conventional treatment with PEG-IFNa-2a (PEG-IFN).
Method

Human HCV patients who were genotype 1, IFN-naive were treated with either HSA-IFNa2b or an active control PEG-IFN in combination with ribavirin (RBV). More specifically, 458 human subjects were randomized into 4 subcutaneous (sc) treatment groups. (A) PEG-IFN administered at 180 μg once a week, (b) HSA-IFNa2B administered at 900 μg once every two weeks (Q2w), (c) Q2w, HSA-IFNa2b administered at 1200 μg, Or (d) HSA-IFNa2b administered at 1200 μg once every four weeks (Q4w).

Each subject in each treatment group also received 1000-1200 mg / day RBV based on body weight. The basics for stratification are body weight index (BMI) (<25 kg / m ² or = 25 kg / m ² ) and HCV RNA titer (<800,000 IU / ml or = 800,000 IU / ml). Included.

  The treatment period for the study is 48 weeks and 24 weeks follow-up. The primary efficacy endpoint for this study is sustained virological response (SVR).

  HCV RNA titers were analyzed in real-time PCR assay, Quantasure ™ (Labcorp, at a sensitivity range of 43 IU to 69 million IU / mL (quantification level (LOQ)) and a detection level (LOD) of 10 IU / mL. )). Hematological effects including alanine transferase (ALT) and absolute neutrophil count (ANC), hemoglobin and platelet count were measured using standard techniques known in the art.

Inclusive analysis (ITT) patients were defined as all randomized and treated subjects in each treatment group regardless of whether the patient missed a data point or dropped out of the study. Modified global analysis (MITT) patients are defined as patients who have been visited for 24 weeks whenever possible, based on those days of study enrollment.
Results and discussion

  The target layer, antiviral response and hematologic reduction were summarized in Table 10 (preliminary interim analysis) and Table 11 (final interim analysis). Overall, all four treatment protocols were well tolerated and there were no significant differences between treatment groups with regard to grade 3-4 lab values or withdrawal due to side effects.

  Antiviral response prediction of SVR was defined as negative HCV RNA titer (ie with HCV RNA titer <LOQ) at 12 weeks of treatment with a second phase slope> 0.6 log / wk (2nd slope). The phase of the antiviral response curve slope is an indicator of two activities. The first phase shows a direct antiviral activity of the response. The second phase predicts destruction of HCV infected cells by the treated compound. Therefore, the value of the second phase slope at> 0.6 log / wk is a good predictor of SVR (positive predictive value (PPV)> 90%).

  Antiviral response prediction of SVR at 12-week ITT is 75/114 subjects or 65.8% ((final interim analysis) (70/112 or 62.5% (preliminary interim analysis)) in PEG-IFN control treatment )) And 49%, 82/110 subjects or 74.5% ((final interim analysis) (77/104 or 74.0% (preliminary analysis))) had HCV RNA negative levels ( That is, it was highest in the HSA-IFNa2b 1200 μg Q2w treatment group, where LOQ (levels below <43 IU / mL)) and 58% showed a second phase slope of> 0.6 log / wk. These data suggest that the HSA-IFNa2b 1200 μg Q2w treatment protocol has antiviral activity that is at least comparable to conventional treatment with PEG-IFN at 12 weeks. In the HSA-IFNa2b 1200 μg Q2w treatment protocol, RNA negative (ie, the number of patients with HCV RNA titers is at or below LOQ level) at 12 weeks is approximately 9% (final interim analysis) and 12% or more (Preliminary interim analysis), phase II slope is approximately 9% or higher compared to PEG-IFN control treatment (both final and preliminary interim analysis), HSA-IFNa2b 1200 μg Q2w treatment protocol results As a result, it can be an effect superior to the conventional PEG-IFN treatment. The number of patients with HCV RNA negative in HSA-IFNa2b 900 μg Q2w and HSA-IFNa2b 1200 μg Q4w treatment groups was similar to conventional treatment with PEG-IFN.

Antiviral response predictions for SVR at 20 and 24 weeks are shown as subjects with HCV RNA titers below the detection level (LOD) (ie, <10 IU / mL). The antiviral response prediction of SVR at 20 weeks is highest in the HSA-IFNa2b 1200 μg Q2w treatment group, compared to 77/114 or 67.5% (final interim analysis) in the PEG-IFN control therapy, 82 / 110 subjects or 75% (final interim analysis) had undetectable HCV RNA levels (ie <10 IU / mL). Similarly, the expected antiviral response of SVR at 24 weeks is highest in the HSA-IFNa2b 1200 μg Q2w treatment group, with 64/91 or 70.3% (final interim analysis) having undetectable HCV RNA levels, On the other hand, the PEG-IFN control treatment had undetectable HCV RNA levels of 57/90 or 63.3%. Both weekly 20 and 24 data suggest that the HSA-IFNa2b 1200 μg Q2w treatment protocol has antiviral activity and is safe, at least comparable to conventional treatment with an improved dosing schedule of PEG-IFN. Yes.
Normalization of ALT level

A common measure of liver function in patients is the level of alanine transferase (ALT). One of the hallmarks of HCV infected patients is antiserum ALT levels, an indicator of liver damage. Thus, normalization of ALT levels correlates with improved liver function and has a favorable prognosis for responding to treatment. All treatment protocols showed the ability to normalize ALT levels, but the most dramatic effect was seen with the HSA-IFNa2b 1200 μg Q4w treatment protocol compared to conventional treatment with PEG-IFN. More than twice as many patients (final and preliminary interim analysis) achieved normalization of ALT levels. Thus, in normalizing liver function in genotype 1, IFN-naive HCV patients, HSA-IFNa2b administered at 1200 μg is surprisingly more effective than conventional PEG-IFN treatment.
Hematological effect

  Ascertaining the compliance and exposure to full doses of IFN and RBV in combination treatment protocols is critical to maximize SVR rates.

  Hematologic decline is common during combination treatment with IFN and RBV. Reduction of hemoglobin (Hb) due to RBV-induced hemolysis requires a decrease in RBV. In particular, Hb <12 g / dL requires a reduction in RBV of 1000-1200 mg / day to 800 mg / day. RBV / time is extremely important to prevent HCV recurrence. The HSA-IFNa2b 1200 μg Q4W treatment protocol surprisingly had a significantly less reduction of Hb <12 g / dL (52% vs 65% for PEG-IFN (final interim analysis), 49.1 vs PEG-IFN) 64% (preliminary interim analysis)). This is considered to be a lower recurrence rate with the HSA-IFNa2b 1200 μg Q4W treatment protocol, allowing for improved SVR.

A reduction of ANC <750 / mm ³ requires a dose reduction of the IFN component of the combination therapy. Surprisingly, the HSA-IFNa2b 1200 μg Q4W treatment protocol had significantly lower ANC <750 / mm ³ compared to PEG-IFN (6% vs 20.2% (final interim analysis), respectively), 4 .3% vs. 17.5% (preliminary interim analysis)). This again results in a higher SVE rate that gives less dose reduction as required by the HSA-IFNa2b 1200 μg Q4W treatment protocol.

Similarly, hematologic reduction occurred across the HSA-IFNa2b Q2w and PEG-IFN treatment groups at 12 weeks. Surprisingly, however, at 12 weeks, the hematologic reduction observed in the HSA-IFNa2b 1200 μg Q4w treatment group is approximately 75% lower than that observed in the PEG-IFN treatment group. These results suggest that HSA-IFNa2b Q4w may provide a better safety profile and improved relapse rate compared to PEG-IFN treatment.
Conclusion

  At 12 weeks, maximal antiviral activity in genotype 1, IFN-naive HCV was observed in the HSA-IFNa2b 1200 μg Q2w treatment group. Similar effects on hematologic reduction were also observed in the HSA-IFNa2b 1200 μg Q2w and PEG-IFN treated groups. In addition, at 20 and 24 weeks, maximal antiviral activity was also observed in the HSA-IFNa2b 1200 μg Q2w treatment group. A comparable antiviral activity continued to be observed in the HSA-IFNa2b 900 μg Q2w treatment group at the 20 and 24 week time points. Thus, HSA-IFNa2b 900 μg Q2w can provide at least comparable efficacy and safety profiles with PEG-IFN with an improved dosing schedule, which is the current treatment standard, and greater convenience for patients It becomes. Furthermore, 1200 μg Q2w may provide at least a comparable safety profile with PEG-IFN at the current treatment standard, potentially superior efficacy and improved dosing schedule, Greater convenience.

  Treatment with the HSA-IFNa2b 1200 μg Q4w protocol surprisingly, although subjects receiving conventional treatment with PEG-IFN are receiving three additional doses per schedule, the conventional PEG -Compared to IFN treatment, showed a comparable effect at 12 weeks. Compared to conventional PEG-IFN treatment, comparable efficacy of HSA-IFNa2b 1200 μg Q4w continued throughout 20 and 24 weeks. Improved ability to stabilize liver function and reduce hematological factors dramatically was also observed in the HSA-IFNa2b 1200 μg Q4w treatment group at 12 weeks, indicating that these patients Suggests improved liver damage and / or reduced or temporary termination, and possibly reduced incidence of recurrence after treatment. Thus, these results indicate that treatment with HSA-IFNa2b 1200 μg Q4w has improved dosing schedule, greater ability to normalize liver function, and reduced hematologic decline, resulting in greater compliance for patients It suggests that it may provide a comparable effect to the combination treatment with PEG-IFN, with the advantage of being convenient and more favorable post-treatment results. In summary, given recent advances in the area of HCV therapy, the HSA-IFNa2b Q4W treatment protocol is ideally suited to become an interferon-selective skeleton for interferon-antiviral combination therapy (eg, comparable). (Effectiveness, better tolerability, better convenience resulting in better compliance).

Taken together, these results show that the combined treatment of genotype 1 INF-naive HCV patients with HSA-IFNa2b and RBV is at least as effective as the combined treatment of PEG-IFN and RBV with the advantage of an improved dosing schedule. It suggests that there is. Notably, these results show that HSA-IFNa2b in combination with RBV is similar in safety compared to an improved or very favorable dosing schedule with conventional HSA-IFNa2b PEG-IFN combination therapy with RBV. It suggests that it can have excellent efficacy with a profile, similar effectiveness with a good safety profile, or both good efficacy and safety profiles.

Example 83: Response of chronic hepatitis C (HCV) non-responder patients to HSA-IFNα2b in combination with ribavirin
background

  Over 4 million people are infected with hepatitis C virus (HCV) in the United States, and the virus is the most common cause of liver disease in the United States. Interferon alpha (INFα) with or without concurrent treatment with an antiviral molecule, ribavirin (RBV), has historically been recognized as the most effective treatment for patients. More recently, a pegylated form of interferon alpha has been approved for the treatment of HCV in combination with RBV. These pegylated interferons have been shown to be more effective in treating HCV than standard interferon or interferon in combination with ribavirin therapy, and has become the standard treatment for HCV .

  However, treatment has obvious implementation limitations. In addition to the well-known side effects that essentially reduce the patient's quality of life during treatment, and a significant rate of significant hematologic reduction associated with standard treatment, standard treatment Ineffective for a large proportion of patients receiving treatment. A clinical study of the treatment of HCV patients who have not previously received IFNa-RBV has shown that approximately 45% of patients who have begun standard therapy fail to remove HCV and remain chronically infected (eg non-responders) . The proportion of patients responding to treatment is clearly small in the population.

The clinical investigator responds to the request of a non-responder population of HCV patients who have failed to respond to previous treatments in combination with IFNα or RBV, treating them with pegylated IFNα and RBV standard treatment Have responded by re-treating with. Shiffman et al. , “Peginterferon alfa-2a and ribavirin in patients with chronic hepatitis C hor fre ate in the patients with chronic hepatitis C who have failed prior treatment.” -23 (2004). Many of these patients who relapsed after treatment were discontinued, although 35% enrolled in this study had no evidence of HCV RNA after 20 weeks of standard treatment retreatment. Thus, only 18% of patients actually achieved sustained virological response (SVR) and HCV was cured. Similarly, when non-responder patients who failed prior treatment with standard pegylated IFNα therapy were retreated with other pegylated IFNα, only ˜5-10% of these patients were based on case evidence SVR could be achieved. Thus, the population of patients not only failing one interferon treatment but also failing all current interferon treatments continues to increase significantly. Thus, there is a clear need not only for general HCV treatment but also for patients previously treated with interferon therapy (eg, IFNα treatment-experience) and other therapies for treatment of non-responders Sex is present, and in particular non-responder patients (eg, patients who have failed previous treatments or have failed re-treatment with current standard treatments) are the most difficult to treat, and other treatments are desired.
principle

HSA-IFNa2b was produced by genetically fusing mature albumin at the C-terminus to the N-terminus of mature interferon α-2b. The safety, tolerability and efficacy of treatment with HSA-IFNa2b in combination with RBV was evaluated in a randomized, open-label clinical study in IFNα treated experience, non-responder HCV human patients. For the purposes of this study, patients who stopped prior treatment at 12 weeks due to non-responders failing to achieve a 2-log reduction in HCV RNA levels (eg, early virological work) Efficacy, 12 weeks, or EVR12), or patients who failed to achieve SVR after completion of the treatment protocol. Patients who relapsed after discontinuation of treatment were excluded from the study. Furthermore, at least 50% of patients in this study previously failed the pegylated IFNα treatment protocol.
Method

  Human IFNα-treated, non-responder HCV patients who previously failed at least one IFNα treatment protocol were randomized into 3 subcutaneous (sc) treatment groups. (A) 900 μg once every two weeks (Q2w), (b) 1200 μg Q2w, and (c) 1200 μg once every four weeks (Q4w). Each subject in each treatment group will also receive 1000-1200 mg / day RBV. After evaluation of safety data from these initial 3 cohorts, HSA-IFNa2b was added in succession by adding two additional cohorts administered either at 1500 μg Q2w or 1800 μg Q2w in combination with 1000-1200 mg / day RBV. -The dose of IFNa2b was increased.

  The treatment period for the study is 48 and 24 weeks follow-up. The primary efficacy endpoint of this study is persistent virological response (SVR).

HCV RNA titers were measured using a real-time PCR assay, Quanture ™ (Labcorp) with a sensitivity range (quantification level (LOQ)) of 10 IU to 100 million IU / mL. Hematological effects including alanine transferase (ALT) and absolute neutrophil count (ANC), hemoglobin and platelet count were measured using standard techniques known in the art.
Results and discussion composition

  A number of constitutive features were identified for use as independent indicators of patients with a prevalence that was unresponsive to treatment. These key non-responsive pretreatment predictions included (1) genotype 1, (2) high baseline median HCV RNA levels, (3) previous non-response to PEG + RBV therapy, (4) Africa American Americans, (5) F3-F4 progressive fibrosis levels (using the METAVIR® classification), and (6) high BMI (eg, = 25 mg / kg)). Perhaps the best overall measure for non-responsiveness is with respect to the previous failure of PEG-RBV treatment and the number of regimens based on the previous IFN that failed.

  The target composition is summarized in Table 12. Overall, all target configurations were similar in all treatment groups. The main subject was exposed to one or more IFNα-containing regimens and failed prior treatment with PEG-RBV. Furthermore, the baseline disease characteristics were comparable across the 5 treatment groups, while the 1800 μg Q2w treatment group had significantly higher pretreatment HCV RNA and the highest proportion of prePEG-RBV failure. Thus, subjects in the 1800 μg Q2w treatment group represent a supplementally intractable patient population.

有効性と生物学的活性

Efficacy and biological activity

  The decrease in HCV RNA from pretreatment levels over the treatment period is shown in Table 13 for genotype 1, PEG-RBV non-responders, most refractory HCV patient populations. At weeks 2-12, the degree of HCV RNA reduction was similar across the 900-1500 μg treatment group. However, maximum viral load reduction was observed in the 1800 μg treatment group. This is surprising given the higher levels of pretreatment HCV and the highest percentage of PEG + RBV failures in this treatment group. The degree of antiviral response over the first 12 weeks of treatment reflects the second phase slope of virus kinetics and is a positive predictor of SVR.

  As shown in Table 12, the slope of HCV RNA reduction is similar for genotype 1, PEG + RBV non-responders at 900 weeks versus 900-1500 μg treatment group. Surprisingly, the extent of HCV RNA reduction is greatest in the 1800 μg treatment group. The HVC RNA reduction for the 1500 and 1800 μg treatment groups is comparable at 24 weeks in this subgroup of patients.

  At 24 weeks, the proportion of HCV RNA negative subjects was similar across the 900-1500 μg treatment group. Subjects can be discontinued at 24 weeks due to lack of efficacy in the judgment of the attending physician, and the cumulative data from the interferon-based regimen shows a high negative predictive value for lack of EVR12 and 24 weeks for SVR. RNA negative. The total treatment termination response (ETR, HCV negative at 48 weeks) was 30% (22/73) for the 900-1200 μg treatment group. Thus, a high proportion of subjects became HCV RNA negative at 12 weeks (eg, EVR12) or achieved ETR at 24 weeks. In addition, the main subject (13/22) continued to be negative for HCV RNA at 12 weeks follow-up after 48 weeks of treatment. This suggests that the potential SVR following treatment with HSA-IFNα2b / RBV is 18%.

  In summary, these data are significant and comparable as a result of a high percentage of PEG + RBV failure with 900-1200 μg HSA-IFNa2b in combination with RBV, resulting in IFNα treatment-experienced, treatment of non-responder HCV patients Antiviral activity. Low virus breakthrough (eg, HCV RNA undetected but essentially positive at 2 or more time points) and recurrence rates were also observed in this treated refractory non-responder population. In addition, a significantly greater decrease was also observed in the 1800 μg treatment group over the first 12 weeks of treatment, indicating that patients treated with this dose of HSA-IFNa2b in combination with ribavirin were significant in SVR rate This suggests that it can have a significant increase.

血液学的効果

Hematological effect

  Ascertaining the compliance and exposure to full doses of IFN and RBV in combination treatment protocols is critical to maximize SVR rates.

Hematologic decline is common during combination treatment with IFN and RBV. However, a reduction in hemoglobin (Hb) and platelet (PLT) numbers due to RBV-induced hemolysis requires a dose reduction of RBV. Absolute neutrophil count (ANC) <750 / mm ³ requires a dose reduction of the IFN component of the combination therapy.

  Although ANC and PLT reductions were observed, these reductions were comparable across all Q2w and reached a plateau around weeks 4-8. Similarly, Hb reduction from baseline was comparable across all Q2w treatment groups (including the 1800 μg treatment group) up to 12 weeks and beyond. The decrease in hematology was less in the Q4w treated group. Overall, 12/115 subjects were reduced / times due to side effect management. Most / times reduction in HSA-IFNa2b is the result of a reduction in ANC as outlined in the treatment protocol. No responsiveness was observed between Q2w treatment groups. Therefore, no further need for reduction / times in the higher dose treatment group was observed.

In summary, although some reductions in hematologic values were observed with HSA-IFNa2b treatment in combination with RBV, these reductions were comparable across treatment groups and in combination with RBV. There was no significant difference in safety between treating IFNα treated-experienced unresponsive HCV patients with 900-1800 μg HSA-IFNa2b.
Conclusion

Taken together, these results show that treatment with HSA-IFNa2b and RBV achieves SVR and therefore a significant proportion of IFNα treatment-experienced, non-responder HCV, including patients who previously failed the PEG + RBV treatment protocol. It can be effective in extinguishing HCV in patients. Notably, these results can result in treatment with HSA-IFNa2b 900-1200 μg in combination with RBV, even after a previously failed PEG + RBV, 18% of patients achieve SVR. It suggests. In addition, the 1800 μg treatment group showed the highest 24-week HCV RNA negative in most recurrent patient populations, suggesting that this value may result in a greater SVR rate for these patients. Furthermore, the safety profile was similar across all treatment groups of HSA-IFNa2b. Furthermore, these results suggest that HSA-IFNa2b is effectively administered every 2-4 weeks, providing an improved dosing schedule. Thus, these results indicate that patients treated with HSA-IFNa2b in combination with RBV have failed interferon-based therapy, particularly standard pegylated interferon-RBV therapy currently lacking in the art. It suggests providing an effective and safe treatment alternative for patients who have failed, which is highly advantageous and has an improved dosing schedule.

Example 84: Antiviral activity of HSA-IFNα2b in combination with ribavirin in genotype 2 or 3, interferon-naive hepatitis C (HCV) patients
background

  More than 170 hundred patients are infected with hepatitis C virus (HCV) worldwide, and this virus has emerged as an obvious public health problem and has rapidly become the most common liver disease worldwide. It became a cause. Acute HCV infection is usually asymptomatic and the problem is to make an early diagnosis. In fact, HCV infection tends to be a chronic condition, with approximately 70% of acute infections persisting. Thus, although the incidence of new infections is decreasing, the prevalence of HCV infection is expected to remain constant in the near future.

  Interferon alpha (IFNα) with or without concurrent treatment with the antiviral molecule, ribavirin (RBV) has historically been recognized as the most effective treatment for patients with chronic hepatitis C (CHC) . More recently, a pegylated form of interferon alpha has been approved for treatment of HCV in combination with RBV. These pegylated interferons have been shown to be more effective in treating HCV than interferons in combination with standard interferon or ribavirin therapy.

  Total sustained virological response (SVR) to currently recommended treatments varies greatly in CHC patients, depending on the characteristics of the virus and the host, especially the viral genotype. For example, SVR rates range from approximately 42-46% in more common genotype 1 patients. On the other hand, patients with favorite gastric genotype 2 or 3 experience SVR rates at 76-80%. Furthermore, genotype 2 or 3 patients, which are much more difficult to treat than genotype 1 patients, can be treated with lower doses of ribavirin in a shorter treatment period.

Currently recommended treatment for pegylated interferon in combination with RBV for genotype 2 or 3 in 24 weeks and the following 24-hour follow-up period results in a clear proportion of patients with SVR As will be achieved, the treatment protocol still leaves clear and obvious implementation limitations, which are common for IFN-extensive treatment. In particular, currently recommended treatments remain fragile after each administration due to side effects that substantially reduce the patient's quality of life. Thus, there is a continuing need for new treatment regimens that are effective and more tolerated by patients infected with genotype 2 or 3 HCV.
principle

HSA-IFNa2b was produced by genetically fusing mature albumin at the C-terminus to the N-terminus of mature interferon α-2b. The safety, tolerability and efficacy of treatment with HSA-IFNa2b in combination with RBV was evaluated in a randomized, open-label clinical study in genotype 2 or 3, IFN-naive CHC human patients.
Method

  43 human HCV patients with either genotype 2 or 3 IFN-naive were randomized into two subcutaneous (sc) HSA-IFNa2b treatment groups. (A) 1500 μg (Q2w) administered every 2 weeks (Q2w) or (2) 1500 μg administered every 4 weeks (Q4w). Each subject in each treatment group also received 800 mg / day of RBV. The primary basis for stratification included genotype (2 or 3) and HCV RNA (<800,000 IU / mL or ≧ 800,000 IU / mL).

  The treatment period for this study is 24 weeks and 24 weeks follow-up. The primary efficacy measure is persistent virological response (SVR).

  HCV RNA titers were measured using a real-time PCR assay, Quantasure ™ (Labcorp) with a sensitivity range of 43 IU to 69 million IU / mL (quantification level (LOQ)). Insulin resistance was assessed using the Homeostasis Assessment Model (HOMA).

Inclusive analysis (ITT) patients were defined as all randomized and treated subjects in each treatment group regardless of whether the patient missed a data point or dropped out of the study. Modified global analysis (MITT) patients are defined as patients who have been visited for 12 weeks, if possible, based on those days of study enrollment.
Results and discussion

  Subject composition and antiviral responses at 4 and 12 weeks are summarized in Table 14. Overall, HSA-IFNa2b was well tolerated in both treatment groups.

  The degree of HCV RNA reduction and the proportion of genotype 2 or 3 patients with HCV RNA <LOQ were comparable for both HSA-IFNa2b Q2w and HSA-IFNa2b Q4w treatment groups. At 4 weeks, the proportion of genotype 2 or 3 patients with HCV RNA <LOQ was 76.2% at 1500 μg Q2w and 68.2% at 1500 μg Q4w. At 12 weeks, a high proportion of genotype 2 or 3 patients in both treatment groups had HCV RNA <LOQ (82.4% at 1500Q2w, 88.9% at 1500Q4w).

Therefore, these results suggest that treatment of genotype 2 or 3 CHC patients with 1500 mg HSA-IFNa2b in either Q2w or Q4w results in a strong antiviral response rate. Furthermore, treatment with the HSA-IFNa2b 1500 μg Q4w protocol showed efficacy similar to treatment with the HSA-IFNa2b 1500 μg Q2w protocol in genotype 2 or 3 patients. Thus, these results indicate that treatment with 1500 μg HSA-IFNa2b with either Q2w or Q4w is currently present for patients infected with HCV genotype 2 or 3, with the benefit of a much improved dosing schedule. It is at least as effective as the recommended treatment, suggesting that it is inherently well tolerated and convenient for the patient.

Example 85: Quality of Life (QOL) of Genotype 1, Interferon-Naive Chronic Hepatitis C (HCV) Treated with HSA-IFNα2b in Combination with Ribavirin Patients
background

As mentioned earlier, treatment of conventional genotype 1, interferon-naive (IFN-naive) HCV patients with pegylated interferon in combination with 48 hours ribavirin (RBV) has obvious implementation limitations. Because of the well-known side effects of the currently recommended interferon treatment, the patient's quality of life is essentially reduced after each interferon administration. Current protocols require at least weekly administration, resulting in increased periods of reduced quality of life and increased days of disability due to treatment. A large number of patients resulted in discontinuation of treatment, with some studies reporting discontinuation rates greater than 50%. Thus, there is a clear need for an improved therapeutic protocol for the treatment of genotype 1 HCV in IFN-naïve patients with an improved impact on quality of life compared to current standard therapies.
principle

The safety and efficacy of treatment with HSA-IFNa2b in combination with RBV was evaluated in an aggressive controlled clinical trial in genotype 1, IFN-naive HCV human patients and as described in Example 82 As an activity control, compared to conventional treatment with PEG-IFNa-2a (PEG-IFN) in combination with RBV. During the first 12 weeks of treatment, the effect of treatment with HSA-IFNa2b in combination with RBV on the QOL and inability days (for example, days when work failed) was compared with PEG-IFN in combination with RBV Compared with.
Method

  458 human, genotype 1 HCV patients were randomized and treated as described in Example 82. QOL and impossibility dates as determined by the SF-36v2® measurement model (QualityMetric, Lincoln, RI) were assessed before treatment, at 4 and 12 weeks of treatment. In particular, the 8SF-36v2 domain was assessed. Physical Function (PF), Physical Role (RP), Body Pain (BP), General Health (GH), Vitality (VT), Social Function (SF), Mental Role (RE), and Mental Health (MH). The first four domains (PF, RP, BP and GH) are the total investment in Physical Health Component, and the remaining four domains (VT, SF, RE and MH) correspond to the Mental Health Component of the QOL model.

The 8SF-36v2 domain conversion (raw) score, as well as the standard based physical component summary (PCS) score, and neural component summary (MCS) score were evaluated over 12 weeks of treatment.
Results and discussion

  At 12 weeks, the QOL of patients receiving 900 μg HSA-IFNa2b Q2w improved compared to PEG-IFN for each measurement, with statistics on MCS and PCS, and 5 out of 8 individual domains. Achieved significant significance. In the 1200 μg Q2w and 1200 μg Q4w HSA-IFNa2b treatment groups, QOL worsening was effectively reduced in each measurement compared to PEG-IFN, and clinically significant differences were observed in both body pain and mental health It was done.

  Overall, (HDW) days when genotype 1 HCV patients in any HSA-IFNa2b treatment group were unable to work due to their HCV infection and subsequent therapy compared to genotype HCV patients in the PEG-IFN treatment group Less (MDW). In particular, 75% less MDW in patients receiving 900 μg HSA-IFNa2b Q2w compared to patients receiving PEG-IFN, 25% less in patients receiving 1200 μg HSA-IFNa2b Q2w or 1200 μg HSA-IFNa2b Q4w MDW.

Taken together with the antiviral activity of HSA-IFNa2b shown in Example 82, these results indicate that combined treatment of genotype 1, IFN-naive HCV patients with HSA-IFNa2b and RBV has improved dosing schedule Suggests that it has at least the same efficacy as treatment with conventional PEG-IFN and RBV combination treatments, with the benefits of improved QOL. In particular, these results indicate that HSA-IFNa2b in combination with RBV is less likely to be obtained with PEG-IFN combination therapy with RBV and the patient has reduced quality of life and reduced days of inactivity Suggesting that it can provide an improved and very convenient treatment protocol over conventional PEG-IFN combination treatment with RBV.

Example 86: In vivo induction of cGMP by HSA-BNP (construct ID # 3959) in a normal pig model
principle

The ability of brain (type B) natriuretic peptide (BNP) to mediate cellular effects such as altered renal function and vascular tone is well known in the art. The activity of BNP depends on its binding to natriuretic receptor A (NPR-A), a guanylyl cyclase, and subsequent activation. Activation of NPR-A by BNP leads to an increase in intracellular cGMP levels that can be measured by assays known in the art, such as ELISA. The ability of HSA-BNP (CID 3959) to induce cGMP production in vivo was tested in a normal pig model.
Method

  HSA-BNP (CID 3959) was produced by genetically fusing mature albumin at its C-terminus to the N-terminus of the C-terminal shortened form of BNP (amino acids 1-29).

Normal blood pressure, healthy pigs (n = 4-6 / group) were dosed at 0 time with a single bolus of 5 mg / kg HSA-BNP (CID 3959) or formulation vehicle only. Plasma and urine were collected at 1, 8, 16, 24, 48 and 72 hours after administration. The recovered plasma and urine cGMP levels were measured by a commercially available ELISA (Molecular Dynamics).
result

A single IV bolus of 5 mg / kg HSA-BNP (CID3959) resulted in a significant increase in cGMP levels in both plasma (FIG. 13A) and urine (FIG. 13B) at 1 hour after IV administration. . cGMP levels gradually decreased to baseline levels at 24 hours in plasma and 48-72 hours in urine.

Example 87: Natriuretic activity of a BNP-HSA fusion construct in an in vivo pacing model of heart failure
background

Exogenous BNP administration has been used to promote natriuresis in congestive heart failure (CHF). However, recent research mimics suggest that BNP administration may adversely affect kidney function. The effect of HSA-BNP (CID 3959) in the kidney and left ventricle (LV) was assessed in an in vivo severe CHF porcine model.
Method

  HSA-BNP (CID 3959) was produced by genetically fusing mature albumin at its C-terminus to the N-terminus of the C-terminal shortened form of BNP (amino acids 1-29).

  CHF was induced in 18 pigs by chronic spacing at 240 bpm for 3 weeks. Eight pigs were used as reference controls. The following baseline characteristics were reduced with CHF compared to the reference control. Baseline measurements were performed using techniques known in the art.

Following baseline measurements, animals with signs of heart failure (eg, LV expansion occurred with a subsequent decline in shortening rate) were randomized for this study. Animals were anesthetized (n = 10 / group), administered either vehicle, 2 mg / kg HSA-BNP (CID3959) or 6 mg / kg HSA-BNP (CID3959) IV and monitored for 4 hours. Left ventricular end-diastolic diameter was measured by electrocardiogram.
Results and conclusions

  HSA-BNP (CID 3959) had a significant effect on heart rate, mean arterial blood pressure, left ventricular end-diastolic pressure, mean pulmonary artery pressure, left ventricular peak pressure, peak positive dp / dt or cardiac output (data is Not shown). No change in coronary blood flow was seen in animals infused with any dose of HSA-BNP (CID 3959) (data not shown). In addition, animals injected with 6 mg / kg HSA-BNP (CID 3959) resulted in increased creatinine clearance and a slight sodium excretion 30 minutes after injection, which was statistically significant on baseline. Not reached (data not shown). Similarly, infusion of 6 mg / kg HSA-BNP (CID 3959) resulted in a non-significant decrease in plasma renin activity and endothelin plasma levels compared to vehicle (data not shown).

  Significant increase in sodium clearance relative to vehicle (492 ± 281% at 30 minutes post-injection, 950 ± 483% at 60 minutes post-injection), 6 mg / kg HSA-BNP (CID 3959) Seen in animals injected with Furthermore, changes in left ventricular end-diastolic diameter caused by pacing were significantly reduced after administration of either HSA-BNP (CID3959) compared to vehicle (FIG. 14A). Furthermore, the change in left ventricular shortening rate caused by pacing was reduced after administration of either HSA-BNP (CID3959) compared to vehicle, and 2 mg / kg / dose HSA-BNP (CID3959) The phenomenon caused by is significant for excipients (FIG. 14B).

Taken together, these results demonstrate that acute infusion of HSA-BNP (CID3959) can induce natriuresis in the in vivo CHF model without adversely affecting left ventricular or renal function. is doing.

Example 88: Effect of HSA-BNP (construct ID 3959) on cardiorenal function in anesthetized dogs
principle

The ability of brain (type B) natriuretic peptide (BNP) to mediate cellular effects such as altered renal function and vascular tone is well known in the art. In particular, a useful model for studying the effects of BNP on cardiorenal function is the dog. Therefore, extensive assessment of cardiorenal effects of HSA-BNP (CID 3959), including hemodynamics, kidney and hormonal effects, was performed in an anesthetized normal dog model.
Method

  HSA-BNP (CID 3959) was produced by genetically fusing mature albumin at its C-terminus to the N-terminus of the C-terminal shortened form of BNP (amino acids 1-29).

  Hemodynamics of cardiorenal function, kidney and hormone parameters were evaluated in this study. In particular, hemodynamic parameters included measurements of cardiac output, mean arterial pressure, pulmonary capillary wedge pressure and pulmonary artery pressure. Kidney parameters included urine flow, sodium excretion, glomerular filtration rate (GFR) and renal blood flow. Hormonal parameters included measurements of plasma cGMP, renin, angiotensin II, aldosterone and urine cGMP.

  Normal healthy mongrel dogs (n = 8 / group) were fed an immobilized sodium diet for 5 days prior to the start of the study. The night before the acute experiment, animals were fasted and given 300 mg lithium carbonate for assessment of renal tubular function. On the day of the acute experiment, dogs were anesthetized with sodium pentobarbital (15 mg / kg) via IV, intubated, and mechanically oxygenated.

  A thermodilution catheter with a flow directed balloon tip was advanced through the external jugular vein into the pulmonary artery for cardiac hemodynamic measurements. The femoral artery was cannulated for blood pressure monitoring, blood sampling, and insulin and normal saline infusion. The left kidney ureter was cannulated for urine collection. A calibrated electromagnetic airflow probe was placed around the renal artery to measure renal blood flow (RBF).

  On the day of the acute experiment, dogs were administered a single IV bolus of HSA-BNP (CID 3959) at either 0.5 mg / kg or 5 mg / kg (n = 8 / group). The effect on cardiorenal function was monitored for 4.5 hours.

  Measured cardiovascular parameters included mean arterial pressure (MAP), renal arterial pressure (RAP), pulmonary artery pressure (PAP), cardiac output (CO) and pulmonary capillary wedge pressure (PCWP). CO was measured by a thermodilution method. MAP was assessed via direct measurement from the femoral artery catheter. GFR was measured by insulin clearance.

Cardiovascular hemodynamics were measured at the beginning of each clearance. Arterial blood was collected in heparin and EDTA tubes and immediately placed on ice halfway through each clearance. After centrifugation at 2,5000 rpm and 4 ° C., the plasma was decanted and stored at −20 ° C. until analysis. Urine was collected on ice for the entire duration of each clearance for assessment of urine dose, electrolytes and insulin. Urine collected for cGMP analysis was heated above 90 ° C. prior to storage.
Results and discussion

Effects on hemodynamics

  FIGS. 15A-H show 0.5 mg / kg (FIGS. 15A, C, E and G) or 5 mg / kg (FIGS. 15B, D, in hemodynamic performance over 4.5 hours post-infusion compared to baseline readings. Figure 8 shows the effect of HSA-BNP (CID 3959) administered in F and H). Hemodynamic parameters were measured at baseline before IV bolus of HSA-BNP (CID 3959) and at 30, 60, 90, 150, 210 and 270 time points after injection.

  The haemodynamic effect of HSA-BNP (CID 3959) was maintained over a 4.5 hour observation period. Both 0.5 and 5 mg / kg IV bolus of HSA-BNP (CID 3959) resulted in a statistically significant and sustained decrease in lung capillary wedge pressure (PCWP) (FIG. 15E). And F). The extent of PCWP reduction at these two doses was not significantly different.

  The effect of HSA-BNP (CID 3959) was dose related in terms of pulmonary artery pressure (PAP) (Figures 15G and H), mean arterial pressure (MAP (Figures 15C and D). Similarly, a significant effect on MAP was observed at 270 minutes post-injection in the 5 mg / kg treatment group (FIG. 15D) when 5 mg / kg was administered (FIG. 15H).

Effects on the kidney

  FIGS. 16A-H show 0.5 mg / kg (FIGS. 16A, C, E and G) or 5 mg / kg (FIG. 16B) in renal output and blood pressure over 4.5 hours after infusion compared to baseline readings. , D, F and H) shows the effect of HSA-BNP (CID 3959) administered. Renal performance parameters were measured at the pre-baseline IV bolus of HSA-BNP (CID 3959) and at time points 30, 60, 90, 150, 210 and 270 after infusion.

  Administration of HSA-BNP (CID 3959) resulted in a significant effect on kidney function. Significant increases in renal blood flow (Figures 16E and F), diuresis (Figures 16A and B), natriuresis (Figures C and d) were observed at both 0.5 and 5 mg / kg HSA-BNP (CID 3959). It was. A dose-related trend in increasing GFR was also evident (FIGS. 16G and H).

  The time to maximum effect of HSA-BNP (CID 3959) on renal parameters tended to be slightly slower compared to the hemodynamic effect. Furthermore, the degree of increase in natriuresis and diuresis was slightly higher in the 5 mg / kg treatment group than in the 0.5 mg / kg treatment group.

Effects on hormones

  FIGS. 17A-F show 0.5 mg / kg (FIGS. 17A, C and E) or 5 mg / kg (FIGS. 17B, D, and 4.5) in RAAS hormone over 4.5 hours after infusion compared to baseline readings. (B) shows the effect of HSA-BNP (CID 3959) administered in F). Plasma aldosterone, renin and angiotensin II levels were measured at the baseline before the IV bolus of HSA-BNP (CID 3959) and at 30, 60, 90, 150, 210 and 270 time points after infusion.

Administration of both 0.5 and 5 mg / kg IV bolus of HSA-BNP (CID 3959) resulted in a decrease in renin, angiotensin and aldosterone levels during the 4.5 hour post-infusion observation period. The effect on aldosterone levels was significant and was maintained throughout the study for 270 minutes. The effects on renin and angiotensin II were significant between 30 and 90 minutes after administration of HSA-BNP (CID 3959) in both / times, but rebounded at the end of the observation period.
Conclusion

  This study showed that HSA-BNP (CID 3959) behaved in a pharmacological manner similar to unfused BNP. Administration of HSA-BNP (CID 3959) in a single IV bolus at 0.5 and 5 mg / kg results in a number of cardiorenal parameters consistent with its activity as long-acting BNP / Time-dependent, significant and sustained changes. In particular, HSA-BNP (CID 3959) results in increased plasma and urine cGNP levels, decreased PCWP and PAP, natriuresis, diuresis, renal blood flow and glomerular filtration rate at 5 mg / kg / dose, There was a decrease in plasma aldosterone, renin and angiotensin II, and a slight decrease in MAP. The different effects on the elements of the renin-angiotensin-aldosterone system are somewhat unexpected results and may be a favorable property of HSA-BNP (CID 3959).

Taken together, these results suggest that HSA-BNP (CID 3959) can be administered at doses that improve cardiorenal function without an essentially undesired decrease in systemic blood pressure.

Example 89: Effect of HSA-BNP (CID 3959) on blood pressure in telemetry beagle
principle

The ability of brain (type B) natriuretic peptide (BNP) to mediate vascular tone is well known in the art. As mentioned earlier, a particularly useful model for studying the effects of BNP on cardiovascular function, including blood pressure, is a dog. Therefore, the effect of HSA-BNP (CID 3959) intravenous (IV) administration on systolic and mean arterial blood pressure and cardiovascular function including heart rate was evaluated in conscious normal beagle dogs. In addition, the effect and duration of the subcutaneous (SC) response of HSA-BNP (CID 3959) was also evaluated.
Method

  Healthy beagles (n = 4 / group) were surgically implanted with a Data Sciences International radio telemetry transmitter with systemic arterial blood pressure, heart rate and ECG data collection capability. After transplantation, dogs were given either 0.1, 0.5, or 5 mg / kg HSA-BNP (CID 3959) or a single IV bolus of vehicle.

  Continuous recordings of ECG parameters and systemic blood pressure were monitored for 48 hours after infusion. The dog was followed for an additional 9 days (total time = 11 days) with intermittent monitoring.

The effect and duration of subcutaneous administration of HSA-BNP (CID 3959) on systemic blood pressure, the effect of IV bolus administration of unfused BNP (0.02 mg / kg), HSA-BNP (CID 3959) (10 mg / kg) was evaluated by comparing to the administration of SC administration.
Results and discussion

Effect of HSA-BNP (CID 3959) on systolic and mean arterial blood pressure

  18A-C show the effect of a single IV bolus of HSA-BNP (CID 3959) on systolic and mean arterial blood pressure in conscious dogs. Administration of 5 mg / kg HSA-BNP (CID 3959) resulted in a gradual decrease in systolic blood pressure that had a peak effect at approximately 16 hours and returned to baseline by 48 hours. The maintenance of a decrease in systolic blood pressure of approximately 15 mm Hg was initiated 8 hours after drug administration and continued up to 20 hours after administration.

  Administration of HSA-BNP (CID 3959) had no apparent effect on diastolic blood pressure or heart rate over the same observation period (data not shown).

  Lower doses (eg 0.5 and 0.1 mg / kg) of HSA-BNP (CID 3959) showed no apparent effect on either blood pressure or heart rate. Furthermore, no treatment-related changes in EOG parameters were observed with any / time HSA-BNP (CID 3959).

Comparison of IV-administered unfused BNP peptide with SC-administered HSA-BNP (CID 3959)

  FIGS. 19A and B show the effect of an IV bolus of unfused BNP on systemic blood pressure in normal healthy beagle as compared to SC injection of HSA-BNP (CID 3959). Both HSA-BNP (CID 3959) and unfused BNP reduced systolic blood pressure in healthy beagle dogs. The effect of unfused BNP was maximal at approximately 30 minutes and returned to baseline within hours. On the other hand, the effect of HSA-BNP (CID 3959) appeared approximately 10 hours after SC administration, reached a maximum at approximately 40 hours and returned to baseline between 48 and 72 hours after injection. The slow onset effect of HSA-BNP (CID 3959) in blood pressure is consistent with its slow absorption (Tmax-36 hours in dogs). The long-term effect of HSA-BNP (CID 3959) is consistent with its long half-life (72 hours in dogs).

  Taken together, these results suggest that HSA-BNP (CID 3959) can be administered to heart failure patients at low enough / times to improve cardiorenal function without affecting systemic blood pressure. ing.

  The full disclosure of each cited document (including patents, patent specifications, patent specifications, academic papers, abstracts, laboratory manuals, books or other disclosures) cited herein, as well as GenBank, Information available through identifiers specific to databases such as GeneSeq or CAS Registry is incorporated herein by reference in its entirety.

In addition, the specifications and sequence listings of each of the following international and US patent specifications are all hereby incorporated by reference: International Patent Specification No. PCT / US02 / 40891 filed on December 23, 2002, International Patent Specification No. 2004/001369 filed on January 20, 2004, February 9, 2005 International Patent Specification No. PCT / US2005 / 004041, filed on February 11, 2004, US Patent Specification No. 10 / 775,204 filed on February 11, 2004, filed July 7, 2005 US Patent Specification No. 11 / 175,690, US Patent Specification No. 11 / 429,373, filed May 8, 2006, US Patent Specification, filed May 8, 2006 No. 11 / 429,276, US Patent Specification No. 11 / 429,374 filed on May 8, 2006, and US filed on August 12, 2005 Provisional application specification No. 60 / 707,521, 60 / 712,386 filed on August 31, 2005, 60 / 732,724 filed on November 3, 2005, 2006 No. 60 / 776,914 filed February 28, 2006, No. 60 / 781,361 filed March 13, 2006, and No. 60/810 filed June 2, 2006 No. 182 and No. 60 / 813,682, filed on June 6, 2006.

Labeling on contracted biological substances (PCT rule 13bis)
A. The label produced below relates to the contracted biological material referenced in the description table 1A.
B. Contract display

Further contracts are identified on additional sheets

Contractor Name: American Type Culture Collection
Trustee Address: 10801 University Boulevard Manassas, Virginia 20110-2209 United States of America

Accession number Accession date 1 PTA-3663 October 4, 2001 2 PTA-3940 December 19, 2001 3 PTA-3942 December 19, 2001 4 PTA-3939 December 19, 2001 5 PTA-4670 2002 September 16

Canada The Applicant is the Director General of the Patent Office until a Canadian patent is granted based on the specification or until the specification is rejected or abandoned and not subject to further reinstatement or destruction. Only require that the samples of deposited biological material cited herein be allowed to be supplied to independent experts designated by the Commissioner, Report to the International Bureau from time to time before the technical preparation for publication is complete.

The Norwegian Applicant is here that the specification has been finalized by the Norwegian Patent Office (by the Norwegian Patent Office), either publicly available or without public access, and the sample supply is subject to expert knowledge in the art. Require that only be done. A request for this entry must be filed by the applicant with the Norwegian Patent Office before the specification becomes generally available under Articles 22 and 33 (3) of the Norwegian Patent Law. If such a request is filed by the applicant, the request for sample supply from a third party should be directed by an expert in use. The expert is a person listed on the list of recognized experts listed by the Norwegian Patent Office or, in each case, a person authorized by the applicant.

Australia Applicant declares that the supply of microbial samples to recipients who are not interested in the present invention will only take place before the grant of the patent or prior to the expiry, rejection or destruction of the specification (Australia) Patent Law Rule 3.25 (3))

Finnish applicants will not supply samples until the specification is publicly available (by the Patent and Regulatory Committee) or until it is finalized by the Patent and Regulatory Committee without being publicly available. Require only technical experts to do it.

The UK applicant here requires that the supply of a sample of microorganisms is made only by specialists. A request for this entry shall be made to the International Bureau before the technical preparation for the issuance of the international specification is completed.

The Danish Applicant is here that the supply of the sample will remain in the technical field until the specification is publicly available (by the Danish Patent Office) or until it is finalized by the Danish Patent Office without public access. Require only professionals to do it. A request for this effect must be filed by the applicant with the Danish Patent Office before the specification becomes generally available under Articles 22 and 33 (3) of the Danish Patent Act. If such a request is filed by the applicant, the request for sample supply from a third party should be directed by an expert in use. The expert is a person listed on the list of recognized experts listed by the Norwegian Patent Office or, in each case, a person authorized by the applicant.

The Swedish applicant here will supply the sample until the specification is publicly available (by the Swedish Patent Office) or until the Swedish Patent Office has finalized the specification without being publicly available. Require only professionals to do it. A request for this effect will be filed with the International Bureau before the expiry date of 16 months from the priority date (preferably on Form PCT / RO / 134 copied in Volume I, Annex Z of PCT Applicants' Guide). It must be applied by a person. If such a request is filed by the applicant, the request for sample supply from a third party should be directed by an expert in use. The expert is a person listed on the list of recognized experts listed by the Swedish Patent Office or, in each case, a person authorized by the applicant.

The Netherlands Applicant shall only serve as an example, as a sample, until the date of grant of the Dutch patent and until the date on which the specification is rejected or abandoned or expires, as microorganisms are provided in Patent Act 31F (1). Requires that it should only be available. Requests for this effect must be made by the applicant to the Dutch Industrial Property Office before the date on which the specification under Article 22C or Article 25 of the Dutch Patent Act is published or earlier.

Claims (30)

An albumin fusion protein comprising a member selected from the group consisting of:
(A) Therapeutic protein X or a fragment or variant of therapeutic X and albumin or an albumin fragment or variant thereof;
(B) Therapeutic protein X or therapeutic X fragment or variant and albumin or albumin fragment or variant thereof, wherein the albumin or albumin fragment or variant thereof has the amino acid sequence of SEQ ID NO: 1. Including;
(C) The therapeutic protein X or therapeutic X fragment or variant of (a) or (b) and the albumin or albumin fragment or variant thereof, wherein the therapeutic X fragment or variant is The biological activity of therapeutic protein X, wherein the albumin fragment or variant thereof has albumin activity;
(D) The therapeutic protein X of (c) or a fragment or variant of therapeutic X and albumin or an albumin fragment or variant thereof, wherein said albumin activity is the lifetime of therapeutic protein X in an unfused state The ability to extend the life span of therapeutic protein X compared to
(E) The therapeutic protein X of (c) or a fragment or variant of therapeutic X and albumin or a fragment or variant thereof, wherein the albumin activity is the serum lifetime of therapeutic protein X in an unfused state The ability to prolong the serum life of therapeutic protein X compared to;
(F) The therapeutic protein X or therapeutic X fragment or variant of (a)-(e) and albumin or albumin fragment or variant thereof, wherein said albumin fragment or variant is SEQ ID NO: 1 The amino acid sequence of amino acids 1-387 of
(G) The therapeutic protein X or therapeutic X fragment or variant of (a) to (f) and albumin or albumin fragment or variant thereof, wherein said therapeutic protein X or therapeutic X variant A fragment of is fused to the N-terminus of albumin or the N-terminus of an albumin fragment or variant thereof;
(H) of therapeutic protein X or therapeutic X fragment or variant of (a)-(f) and albumin or albumin fragment or variant thereof, wherein therapeutic protein X or therapeutic X variant The fragment is fused to the C-terminus of albumin, or the C-terminus of an albumin fragment or variant thereof;
(I) of the therapeutic protein X or therapeutic X fragment or variant of (a)-(f) and albumin or albumin fragment or variant thereof, wherein the therapeutic protein X or therapeutic X variant The fragment is fused to the N-terminus and C-terminus of albumin or the N-terminus and C-terminus of the albumin fragment or variant thereof;
(J) The therapeutic protein X or fragment of therapeutic X of (a)-(f) comprising the first therapeutic protein X or fragment or variant thereof and the second therapeutic protein X or fragment or variant thereof or The variant and albumin or albumin fragment or variant thereof, wherein the first therapeutic protein X, or a fragment or variant thereof, is different from the second therapeutic protein X or a fragment or variant thereof;
(K) of therapeutic protein X or therapeutic X fragment or variant of (a)-(j) and albumin or albumin fragment or variant thereof, wherein therapeutic protein X or therapeutic X variant The fragments are separated from albumin or albumin fragments or variants thereof by a linker;
(L) Therapeutic protein X or therapeutic X fragment or variant of (a)-(k) and albumin or albumin fragment or variant thereof, wherein the albumin fusion protein has the following formula:
R1-L-R2; R2-L-R1, or R1-L-R2-L-R1,
Wherein R1 is therapeutic protein X, or a fragment or variant thereof, L is a peptide linker, R2 is albumin comprising the amino acid sequence of SEQ ID NO: 1, or a fragment or variant of albumin is there;
(M) The therapeutic protein X or therapeutic X fragment or variant of (a)-(l) and the albumin or albumin fragment or variant thereof, wherein the lifetime of the albumin fusion protein is Greater than the lifetime of a fragment of therapeutic protein X or a variant of therapeutic X;
(N) The therapeutic protein X of (a)-(l) or a fragment or variant of therapeutic X and albumin or albumin fragment or variant thereof, wherein the serum half-life of the albumin fusion protein is unfused Longer than the serum half-life of a fragment of therapeutic protein X or a variant of therapeutic X at
(O) Therapeutic protein X of (a)-(l) or a fragment or variant of therapeutic X and albumin or albumin fragment or variant thereof, wherein therapeutic protein X or therapeutic treatment of said albumin fusion protein The in vitro biological activity of the fragment of X variant is greater than the in vitro biological activity of the therapeutic protein X or fragment of the therapeutic X variant in the unfused state;
(P) Therapeutic protein X of (a)-(l) or a fragment or variant of therapeutic X and albumin or albumin fragment or variant thereof, where therapeutic protein X or therapeutic of said albumin fusion protein The in vivo biological activity of the fragment of X mutant is greater than the in vivo biological activity of the therapeutic protein X or a fragment of the therapeutic X variant in an unfused state.   2. The albumin fusion protein of claim 1 expressed in a host cell, wherein the host cell is a yeast, mammal or bacterium.   The albumin fusion protein of claim 1, wherein the albumin fusion protein further comprises a secretory leader sequence.   A composition comprising the albumin fusion protein according to claim 1 and a pharmacologically acceptable carrier.   A kit comprising the composition of claim 4.   A method of treating a disease or condition in a patient comprising administering an albumin fusion protein of claim 1.   The method of claim 6, wherein the disease or condition comprises indication Y.   8. The method of claim 7, wherein the disease or condition is hepatitis C infection. The albumin fusion protein is
(A) Construct ID 2249,
(B) construct ID 2343,
(C) Construct ID 2366,
(D) Construct ID 2381,
(E) Construct ID 2382,
(F) Construct ID 2410,
(G) Construct ID 3165,
(H) Construct ID 3422,
(I) Construct ID 3423,
(J) Construct ID 3424,
(K) Construct ID 3476,
(L) Construct ID 3960,
(M) Construct ID 4290,
(N) construct ID 4291,
(O) Construct ID 4292,
(P) Construct ID 4295, and (q) Construct ID 4296
9. The method of claim 8, wherein the method is expressed by a host cell comprising an albumin fusion construct selected from the group consisting of:   10. The method of claim 9, wherein the albumin fusion construct is (d).   10. The method of claim 9, wherein the albumin fusion construct is (g).   The method of claim 9, wherein the albumin fusion construct is (l).   12. The method of claim 11, wherein the patient suffering from hepatitis C infection is a treatment naive or is a treatment experienced person.   The method according to claim 13, wherein the treatment experienced person is a non-responder.   15. The method of claim 14, wherein the non-responder has already failed at least one combination therapy protocol consisting of PEGylated-interferon alpha and ribavirin.   14. The method of claim 13, wherein the hepatitis C infection is genotype 1 or genotype 2/3. The therapeutically effective albumin fusion protein is
(A) about 600 μg / time,
(B) about 900 μg / time,
(C) about 1000 μg / time,
(D) about 1200 μg / time,
17. The method of claim 16, wherein the method is selected from the group consisting of (e) about 1800 μg / dose and (f) about 2000 μg / dose. The albumin fusion protein is
(A) Once a week,
(B) Once every two weeks,
(C) Once every three weeks,
18. The method of claim 17, wherein the method is administered according to a dosing schedule selected from the group consisting of (d) once every four weeks and (e) once every five weeks.   A method of treating a patient suffering from hepatitis C infection with a therapeutically effective amount of an albumin fusion protein comprising mature interferon alpha-2b fused to mature albumin, said mature interferon alpha-2b comprising Fused to the C-terminus of mature albumin; and (1) the patient is treated naive, (2) the hepatitis C infection is genotype 1, and the therapeutically effective amount is about 900 μg / Times to about 1800 μg / dose, wherein the albumin fusion protein is administered once every two weeks. The therapeutically effective amount is
(A) about 900 μg / time,
20. The method of claim 19, wherein the method is selected from the group consisting of (b) about 1200 μg / dose and (c) about 1800 μg / dose.   A method of treating a patient suffering from hepatitis C infection with a therapeutically effective amount of an albumin fusion protein comprising mature interferon alpha-2b fused to mature albumin, said mature interferon alpha-2b comprising Fused to the C-terminus of mature albumin, and (1) the patient is treated naive, (2) the hepatitis C infection is genotype 1, and the therapeutically effective amount is about 900 μg / Times to about 1800 μg / dose, wherein the albumin fusion protein is administered once every four weeks. The therapeutically effective amount is
(A) about 900 μg / time,
24. The method of claim 21, wherein the method is selected from the group consisting of (b) about 1200 μg / dose and (c) about 1800 μg / dose.   A method of treating a patient suffering from hepatitis C infection with a therapeutically effective amount of an albumin fusion protein comprising mature interferon alpha-2b fused to mature albumin, said mature interferon alpha-2b comprising Fused to the C-terminus of mature albumin, and (1) the patient is experienced in treatment, (2) the hepatitis C infection is genotype 1, and the therapeutically effective amount is about 1200 μg / Times to about 1800 μg / dose, wherein the albumin fusion protein is administered once every two weeks. The therapeutically effective amount is
(A) about 1200 μg / time,
24. The method of claim 23, selected from the group consisting of (b) about 1500 μg / dose and (c) about 1800 μg / dose.   A method of treating a patient suffering from hepatitis C infection with a therapeutically effective amount of an albumin fusion protein comprising mature interferon alpha-2b fused to mature albumin, said mature interferon alpha-2b comprising Fused to the C-terminus of mature albumin, and (1) the patient is experienced in treatment, (2) the hepatitis C infection is genotype 1, and the therapeutically effective amount is about 1200 μg / Times to about 1800 μg / dose, wherein the albumin fusion protein is administered once every four weeks. The therapeutically effective amount is
(A) about 1200 μg / time,
26. The method of claim 25, selected from the group consisting of (b) about 1500 μg / dose and (c) about 1800 μg / dose.   The lifetime or serum half-life of a fragment of therapeutic protein X or therapeutic X variant is compared to the lifetime or serum half-life of the fragment of therapeutic protein X or therapeutic X in an unfused state. Of therapeutic protein X or therapeutic X variant comprising fusing a fragment of therapeutic protein X or therapeutic X variant to albumin or albumin fragment or variant thereof, sufficient to extend A method of extending fragment life or serum half-life.   A nucleic acid molecule comprising a polynucleotide sequence encoding the albumin fusion protein of claim 1.   A vector comprising the nucleic acid molecule of claim 28.   A host cell comprising the nucleic acid molecule of claim 28.

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