WO2020106881A1 - Modified ulinastatin polypeptides - Google Patents

Abstract

Provided are modified ulinastatin polypeptides and related compositions and methods of use, including methods of treating diseases and methods of recombinantly producing the modified ulinastatin polypeptides.

Description

MODIFIED ULINASTATIN POLYPEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Application No. 62/770,099, filed November 20, 2018, which is incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is DIAM_038_01WO_ST25.txt. The text file is about 51 KB, was created on November 20, 2019, and is being submitted electronically via EFS-Web.

BACKGROUND

Technical Field

Embodiments of the present disclosure include modified ulinastatin polypeptides and related compositions and methods of use, including methods of treating diseases and methods of

recombinantly producing the modified ulinastatin polypeptides.

Description of the Related Art

Ulinastatin (also urinary -trypsin inhibitor) is a glycoprotein proteinase inhibitor derived from human urine which inhibits the activity of trypsin, chymotrypsin, lactate, lipase, hyaluronidase, and various pancreatic enzymes. Highly-purified ulinastatin has been used clinically for the treatment of acute pancreatitis, chronic pancreatitis, Stevens-Johnson syndrome, bums, septic shock, toxic epidermal necrolysis (TEN), and other diseases.

Ulinastatin is approved for human use for a variety of conditions, including pancreatitis. However, large quantities of ulinastatin are required because it is a serpin, a potent protease inhibitor that reacts irreversibly with the active site of the protease and is thus typically consumed in a 1 : 1 stoichiometry with its target. This property, coupled with the relatively low in vivo exposures achieved after systemic dosing, creates challenges in generating therapeutics from the native ulinastatin protein.

Therefore, there is a need in the art for modified ulinastatin polypeptides having improved characteristics related to their therapeutic utility and/or recombinant production.

BRIEF SUMMARY

Embodiments of the present disclosure include modified ulinastatin polypeptides, comprising, consisting, or consisting essentially of: (a) a variant of SEQ ID NO: 1 that has at least one at least one modification to an O- linked glycosylation and at least one ulinastatin activity;

(b) a fragment of SEQ ID NO: 1 or (a) that has at least one ulinastatin activity; or

(c) a variant of (a) or (b) that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to (a) or (b) and has at least one ulinastatin activity, excluding SEQ ID NO: 1 (wild-type ulinastatin).

In some embodiments, (a) comprises a substitution or deletion at the O-linked glycosylation site of residues 8-11 of SEQ ID NO: 1, optionally a substitution or deletion at residue S 10 of SEQ ID NO: 1, optionally an S 10A substitution, or wherein (b) comprises, consists, or consists essentially of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or

130 contiguous amino acids of SEQ ID NO: 1.

In some embodiments, (b) comprises, consists, or consists essentially of about 20-130, 20- 120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-130, 30-120, 30-110, 30- 100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-130, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-130, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90-110, 90-100, 100-130, 100-120, or 100-110 contiguous amino acids of SEQ ID NO: 1.

In some embodiments, (b) comprises, consists, or consists essentially of Domain 1 of SEQ ID 1. In some embodiments, (b) comprises, consists, or consists essentially of Domain 2 of SEQ ID

Certain modified ulinastatin polypeptides comprise, consist, or consist essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 (UTIACS), which retains a substitution at position S 10 of SEQ ID NO: 2, optionally a S 10A substitution.

Also included are modified ulinastatin polypeptides, comprising a ulinastatin polypeptide fused to an Fc region, to form an ulinastatin-Fc fusion polypeptide, wherein the ulinastatin-Fc fusion polypeptide has at least one ulinastatin activity.

In some embodiments, the ulinastatin polypeptide comprises, consists, or consists essentially of:

(a) a variant of SEQ ID NO: 1 that has at least one at least one modification to an O- linked glycosylation and at least one ulinastatin activity;

(b) SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 or (a) that has at least one ulinastatin activity; or

(c) a variant of (a) or (b) that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to (a) or (b) and has at least one ulinastatin activity.

In some embodiments, (a) comprises a substitution or deletion at the O-linked glycosylation site of residues 8-11 of SEQ ID NO: 1, optionally a substitution or deletion at residue S 10 of SEQ ID NO: 1, optionally an S 10A substitution, or wherein (b) comprises, consists, or consists essentially of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or

130 contiguous amino acids of SEQ ID NO: 1.

In some embodiments, (b) comprises, consists, or consists essentially of SEQ ID NO: 1 or about 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-130, SO HO, 30-110, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-130, 40-120, 40-110, 40-100, 40- 90, 40-80, 40-70, 40-60, 40-50, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-130, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90-110, 90-100, 100-130, 100-120, or 100-110 contiguous amino acids of SEQ ID NO: 1.

In some embodiments, (b) comprises, consists, or consists essentially of Domain 1 of SEQ ID NO: 1. In some embodiments, wherein (b) comprises, consists, or consists essentially of Domain 2 of SEQ ID NO: 1.

In some embodiments, the ulinastatin-Fc fusion polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from Table U3, or wherein (a) comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2, which retains a substitution at position S 10 of SEQ ID NO: 2, optionally a S10A substitution.

In some embodiments, the Fc region comprises, consists, or consists essentially of CEE region, CEE region, CEE region, and/or hinge region(s) from a IgA, IgD, IgE, IgG, or IgM

immunoglobulin heavy chain. In some embodiments, the Fc region comprises, consists, or consists essentially of one or more of the human Fc region amino acid sequences of Table FI, including variants, fragments, homologs, orthologs, paralogs, and combinations thereof. In some embodiments, the ulinastatin-Fc fusion polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from Table U3.

In some embodiments, the modified ulinastatin polypeptide has a specific activity of about or at least about 1000-3000 U/mg, or about or at least about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 U/mg, wherein one unit (U) is an amount of the modified ulinastatin polypeptide that inhibits the activity of 2 pg trypsin by 50%. In some embodiments, the at least one ulinastatin activity is selected from one or more of protease inhibitor activities, anti-inflammatory activities, and anti-metastatic activities.

In some embodiments, the modified ulinastatin polypeptide has one or more improved biological, physical, and/or pharmacokinetic properties, relative to wild-type ulinastatin (SEQ ID NO: l). In some embodiments, the modified ulinastatin polypeptide has at least one increased ulinastatin activity relative to wild-type ulinastatin (SEQ ID NO: 1), optionally at least one increased protease inhibitor activity, anti-inflammatory activity, and/or anti-metastatic activity, optionally wherein the at least one activity is increased by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more relative to wild-type ulinastatin (SEQ ID NO: l).

In some embodiments, the modified ulinastatin polypeptide has equivalent or increased

optionally wherein the modified ulinastatin polypeptide has equivalent or increased therapeutic efficacy at a dosage that is about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,

50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000,

7000, 8000, 9000, 10,000-fold lower than that of wild-type ulinastatin (SEQ ID NO: 1).

Also included are therapeutic compositions, comprising a pharmaceutically-acceptable carrier and a modified ulinastatin polypeptide described herein. In some embodiments, the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis and is substantially aggregate-free, optionally less than about 10, 9, 8, 7, 6, or 5% aggregated, and wherein the composition is substantially endotoxin-free. In some embodiments, the composition has less than about 1 EU endotoxin/mg protein, less that about 100 ng host cell protein/mg protein, less than about 10 pg host cell DNA/mg protein, and/or greater than about 95% single peak purity by SEC-HPLC.

Also included are methods of treating an inflammatory disease or condition in a subject in need thereof, comprising administering to the subject a modified ulinastatin polypeptide and/or a therapeutic composition described herein, thereby treating the inflammatory disease or condition in the subject.

In some embodiments, the inflammatory disease or condition is selected from one or more of pancreatitis (e.g., acute pancreatitis, chronic pancreatitis, endoscopic retrograde

cholangiopancreatography (ERCP) -induced pancreatitis), systemic inflammation, colitis, autoimmune encephalomyelitis, Stevens-Johnson syndrome, arthritis, renal failure, bums, sepsis/septic shock including severe sepsis and related pro-inflammatory/secondary conditions (e.g., organ failure), systemic inflammatory response syndrome (SIRS), toxic epidermal necrolysis (TEN), Kawasaki disease, kidney disease (e.g., acute kidney failure, chronic kidney disease), ischemic conditions (e.g., ischemia-reperfusion injury in the liver, kidney, heart, lungs, brain), lung inflammation and inflammatory lung conditions (e.g., pulmonary infection, pneumonia, including infectious interstitial pneumonia associated with mixed connective tissue disease, pulmonary fibrosis, acute respiratory distress syndrome), liver inflammation including hepatitis, anaphylaxis, post-operative or post- surgical complications (e.g., renal function, cardiac surgery, lung surgery, cognitive dysfunction, liver transplantation), lipopolysaccharide (LPS)-induced inflammation or tissue injury (e.g., lungs, liver, brain), inflammation or dysfunction secondary to diabetes (e.g., diabetes-induced cardiac dysfunction), bum injury, heat stroke, inflammatory or neuropathic pain, acute poisoning, hyperlipidemia-associated inflammation, autoimmunity-associated inflammation, allogeneic transplant or blood transfusion-associated inflammation, neuroinflammation, and cancer-associated inflammation.

In some embodiments, administering the modified ulinastatin polypeptide reduces one or more of protease activity, endothelial activation/damage, proinflammatory cytokine and chemokine production/release (optionally, IL-Ib, MIP-la, MCP-1, and/or CXCL1), fibrinogen synthesis, neutrophil recruitment into organs, and/or organ injury in the subject.

Also included are methods of treating, ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof, comprising administering to the subject a modified ulinastatin polypeptide and/or therapeutic composition described herein, thereby treating, ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof.

In some embodiments, the cancer is selected from one or more of melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

In some embodiments, the cancer is a metastatic cancer, optionally wherein administering the modified ulinastatin polypeptide reduces cancer cell invasion and/or angiogenesis. In some embodiments, the metastatic cancer is selected from one or more of:

(a) a bladder cancer which has metastasized to the bone, liver, and/or lungs;

(b) a breast cancer which has metastasized to the bone, brain, liver, and/or lungs;

(c) a colorectal cancer which has metastasized to the liver, lungs, and/or peritoneum;

(d) a kidney cancer which has metastasized to the adrenal glands, bone, brain, liver, and/or lungs;

(e) a lung cancer which has metastasized to the adrenal glands, bone, brain, liver, and/or other lung sites;

(f) a melanoma which has metastasized to the bone, brain, liver, lung, and/or skin/muscle;

(g) a ovarian cancer which has metastasized to the liver, lung, and/or peritoneum;

(h) a pancreatic cancer which has metastasized to the liver, lung, and/or peritoneum; (i) a prostate cancer which has metastasized to the adrenal glands, bone, liver, and/or lungs;

(j) a stomach cancer which has metastasized to the liver, lung, and/or peritoneum;

(l) a thyroid cancer which has metastasized to the bone, liver, and/or lungs; and

(m) a uterine cancer which has metastasized to the bone, liver, lung, vagina, and/or peritoneum.

Some embodiments include polynucleotide (s) encoding a modified ulinastatin polypeptide described herein, or a vector comprising the polynucleotide. Certain embodiments include recombinant host cell(s), comprising a polynucleotide or vector described herein.

Also included are methods for recombinantly-producing a modified ulinastatin polypeptide, comprising

(a) expressing the modified ulinastatin polypeptide in a recombinant host cell described herein; and

(b) isolating the modified ulinastatin polypeptide from the host cell,

thereby recombinantly-producing the modified ulinastatin polypeptide.

Certain embodiments further comprise measuring at least one ulinastatin activity of the modified ulinastatin polypeptide under physiological conditions, optionally of temperature, salinity, and/or pH.

In certain embodiments, the modified ulinastatin polypeptide has at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more of the ulinastatin activity of wild- type ulinastatin (SEQ ID NO: 1) under comparable physiological conditions.

Certain embodiments further comprise preparing a therapeutic composition that comprises the modified ulinastatin polypeptide, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis, and wherein the composition is substantially aggregate-free and substantially endotoxin-free.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the molecular structure of ulinastatin (urinary trypsin inhibitor; SEQ ID

NO: l)

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods, materials, compositions, reagents, cells, similar or equivalent similar or equivalent to those described herein can be used in the practice or testing of the subject matter of the present disclosure, preferred methods and materials are described. All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

The practice of the present disclosure will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g. , Current Protocols in Protein Science, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al, Short Protocols in Molecular Biology, 3^rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984): Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

For the purposes of the present disclosure, the following terms are defined below.

The articles“a” and“an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

By“about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. As used herein, the term“amino acid” is intended to mean both naturally occurring and non- naturally-occurring amino acids as well as amino acid analogs and mimetics. Naturally-occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxy lysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

“Biocompatible” refers to materials or compounds which are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic and disease states.

By“coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does not directly contribute to the code for the polypeptide product of a gene.

Throughout this disclosure, unless the context requires otherwise, the words“comprise,” “comprises,” and“comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

By“consisting of’ is meant including, and limited to, whatever follows the phrase“consisting of.” Thus, the phrase“consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By“consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase“consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

The term“endotoxin free” or“substantially endotoxin free” relates generally to compositions, solvents, and/or vessels that contain at most trace amounts (e.g., amounts having no clinically adverse physiological effects to a subject) of endotoxin, and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain micro-organisms, such as bacteria, typically gram negative bacteria, although endotoxins may be found in gram-positive bacteria, such as Listeria monocytogenes . The most prevalent endotoxins are lipopolysaccharides (LPS) or lipo-oligo- saccharides (LOS) found in the outer membrane of various Gram-negative bacteria, and which represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans may produce fever, a lowering of the blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from drug products and/or drug containers, because even small amounts may cause adverse effects in humans. A depyrogenation oven may be used for this purpose, as temperatures in excess of 300°C are typically required to break down most endotoxins. For instance, based on primary packaging material such as syringes or vials, the combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxins are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art.

Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction.

Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin free, endotoxin levels may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mg of active compound. Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

The“half-life” of a polypeptide can refer to the time it takes for the polypeptide to lose half of its pharmacologic, physiologic, or other activity, relative to such activity at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point.“Half-life” can also refer to the time it takes for the amount or concentration of a polypeptide to be reduced by half of a starting amount administered into the serum or tissue of an organism, relative to such amount or concentration at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. The half-life can be measured in serum and/or any one or more selected tissues.

The terms“modulating” and“altering” include“increasing,”“enhancing” or“stimulating,” as well as“decreasing” or“reducing,” typically in a statistically significant or a physiologically significant amount or degree relative to a control. An“increased,”“stimulated” or“enhanced” amount is typically a“statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and ranges in between e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no composition (e.g., the absence of agent) or a control composition. A“decreased” or“reduced” amount is typically a“statistically significant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease (including all integers and ranges in between) in the amount produced by no composition (e.g., the absence of an agent) or a control composition. Examples of comparisons and“statistically significant” amounts are described herein.

The terms“polypeptide,”“protein” and“peptide” are used interchangeably and mean a polymer of amino acids not limited to any particular length. The term“enzyme” includes polypeptide or protein catalysts, and with respect to ulinastatin is used interchangeably with protein, polypeptide, or peptide. The terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms“polypeptide” or“protein” means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms“polypeptide” and“protein” specifically encompass the ulinastatin proteins described herein, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of the ulinastatin proteins. In certain embodiments, the polypeptide is a“recombinant” polypeptide, which is produced by recombinant cell that comprises one or more recombinant DNA molecules, which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the cell.

The term“isolated” polypeptide or protein referred to herein means that a subject protein (1) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or non-covalent interaction) with portions of a protein with which the “isolated protein” is associated in nature, (6) is operably associated (by covalent or non-covalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise). In certain embodiments, the“purity” of any given agent (e.g., modified ulinastatin polypeptide) in a composition may be specifically defined. For instance, certain compositions may comprise an agent that is at least 70, 75 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure (for example, on a protein basis), including all decimals and ranges in between, as measured, for example, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

The term“reference sequence” refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing.

The terms“sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by -amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al, Nucl. Acids Res. 25:3389, 1997.

The term“solubility” refers to the property of an agent (e.g., modified ulinastatin

polypeptide) provided herein to dissolve in a liquid solvent and form a homogeneous solution.

Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration. The maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaP). In specific embodiments, solubility is measured at relatively lower pH (e.g., pH 6.0) and relatively higher salt (e.g., 500mM NaCl and lOmM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain embodiments, an agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/ml at room temperature or at 37°C.

A“subject” or a“subject in need thereof’ or a“patient” or a“patient in need thereof’ includes a mammalian subject such as a human subject.

“Substantially” or“essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

By“statistically significant,” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.

“Therapeutic response” refers to improvement of symptoms (whether or not sustained) based on administration of one or more therapeutic agents.

As used herein,“treatment” of a subject (e.g. a mammal, such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent. Also included are“prophylactic” treatments, which can be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset.“Treatment” or“prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.

The term“wild-type” refers to a gene or gene product (e.g., a polypeptide) that is most frequently observed in a population and is thus arbitrarily designed the“normal” or“wild-type” form of the gene.

Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

Modified Ulinastatin Polypeptides

Certain embodiments relate to“modified ulinastatin polypeptides”, including variants and/or fragments of wild-type human ulinastatin, and ulinastatin-Fc fusion polypeptides.“Ulinastatin” (also referred to as urinary trypsin inhibitor (UTI), HI-30, ASPI, or bikunin) is an acidic glycoprotein with a molecular weight of about 30 kDa by SDS-polyacrylamide gel electrophoresis. Wild-type human ulinastatin is a multivalent Kunitz-type serine protease inhibitor found in human urine and blood that is composed of 143 amino acid residues includes two Kunitz-type domains (see Figure 1 and Table Ul). It is produced by hepatocytes as a precursor in which ulinastatin is linked to al-microgloblin. In hepatocytes, different types of ulinastatin-containing proteins are formed by the assembly of ulinastatin, with one or two of the three evolutionarily related heavy chains (HC) 1, HC 2, and HC 3, through a chondroitin sulfate chain; these proteins comprise inter-a-inhibitor (Ial) family members, including Ial, pre-a-inhibitor (Pal), inter- a-like inhibitor (IaLI), and free ulinastatin. Ial, pal, and IaLI are composed of HC1 + HC2 + UTI, HC3 + UTI, and HC2 + UTI, respectively.

During inflammation, ulinastatin is cleaved from Ial family proteins through proteolytic cleavage by neutrophil elastase in the peripheral circulation or at the inflammatory site, and plasma ulinastatin levels and gene expression are altered in severe inflammatory conditions. Thus, plasma ulinastatin is considered to be one of the acute phase reactions. Further, ulinastatin is rapidly released into urine when infection occurs and is an excellent inflammatory marker, constituting most of the urinary anti-trypsin activity. Various serine proteases such as trypsin, chymotrypsin, kallikrein, plasmin, granulocyte elastase, cathepsin, thrombin, and Factors IXa, Xa, XIa, and Xlla are inhibited by ulinastatin. Furthermore, ulinastatin can suppress urokinase-type plasminogen activator (uPA) expression through the inhibition of protein kinase C (PKC). Ulinastatin appears to prevent organ injury by inhibiting the activity of these proteases.

Beyond its inhibition of inflammatory proteases mentioned above, ulinastatin exhibits anti inflammatory activity and suppresses the infdtration of neutrophils and release of elastase and chemical mediators from them. Likewise, ulinastatin inhibits the production of tumor necrosis factor (TNF)-a and interleukin (IL)-l in LPS-stimulated human monocytes and LPS- or neutrophil elastase- stimulated IL-8 gene expression in HL60 cells or bronchial epithelial cells in vitro. It has also been shown to inhibit LPS-induced TNF-a and subsequent IL-Ib and IL-6 induction by macrophages, at least partly, through the suppression of mitogen-activated protein kinase (MAPK) signaling pathways such as ERK1/2, JNK, and p38 in vitro. Ulinastatin also inhibits neutrophil-mediated endothelial cell injury in vitro, suggesting that it can act directly/indirectly on neutrophils and suppress their production and secretion of activated elastase. Furthermore, ulinastatin down-regulates stimulated arachidonic acid metabolism such as thromboxane B2 production in vitro, which plays a role in the pathogenesis of sepsis.

Thus, in certain instances, a modified ulinastatin polypeptide has at least one“ulinastatin activity”. The term“ulinastatin activity” includes (a) protease inhibitor activities, which include reducing the protease activity of one or more of trypsin, chymotrypsin, kallikrein, plasmin, granulocyte elastase, cathepsin, thrombin, and/or factors IXa, Xa, XIa, and Xlla; (b) anti inflammatory activities, which include reducing inflammation and/or cytokine -depending signaling pathways, for instance, to reduce organ injury during severe inflammation; and (c) anti-metastatic activities, which include reducing tumor invasion and metastasis, for example, by reducing cathepsin B activity and/or reducing CD44 dimerization, at least the latter of which suppresses the MAP kinase signaling cascade and reduces extracellular matrix (ECM) degradation, tumor cell invasion, and/or angiogenesis.

In certain embodiments, a modified ulinastatin polypeptide has a“specific activity” of about or at least about 500-5000 U/mg, or about or at least about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000 U/mg polypeptide, wherein one unit (U) is an amount of the modified ulinastatin polypeptide that inhibits the activity of 2 pg trypsin by 50%.

In certain instances, a modified ulinastatin polypeptide has one or more improved characteristics relative to wild-type ulinastatin (SEQ ID NO: 1), which are related to its therapeutic use and/or recombinant production. Examples of such characteristics include improved biological, physical, and/or pharmacokinetic properties, such as increased absolute biological activity (e.g., ulinastatin activity, specific activity), increased stability in solution and/or solid form (e.g., half-life, kinetic or thermal stability, functional stability, susceptibility to oxidation), increased clarity in solution (e.g., reduced turbidity, opalescence), reduced aggregate formation in solution, increased homogeneity or monodispersion in solution or solid form (e.g., altered ratio of monomeric/dimeric or monomeric/oligomeric forms, altered levels of interchain disulfide bond formation), reduced immunogenicity in vivo, reduced cross reactivity, improved recombinant expression in host cells, for example, in yeast, mammalian cells, or bacteria such as E. coli, (e.g. reduced endotoxin

contamination, improved homogeneity, improved charge homogeneity), improved yield of soluble protein, reduced endotoxin binding, reduced degree of degradation in solution and/or solid form, increased bioavailability (the fraction of a drug that is absorbed), increased half-life in vivo, improved tissue distribution, volume of distribution (apparent volume in which a drug is distributed

immediately after it has been injected intravenously and equilibrated between plasma and the surrounding tissues), concentration (initial or steady-state concentration of drug in plasma), elimination rate constant (rate at which drugs are removed from the body), elimination rate (rate of infusion required to balance elimination), area under the curve (AUC; integral of the concentration time curve, after a single dose or in steady state), clearance (volume of plasma cleared of the drug per unit time), C_max (peak plasma concentration of a drug after oral administration), t_max (time to reach C_max), C_min (lowest concentration that a drug reaches before the next dose is administered), and fluctuation (peak trough fluctuation within one dosing interval at steady state). In certain

embodiments, any one or more of the foregoing characteristics are improved (i.e., increased or decreased, as appropriate) by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more relative to wild-type ulinastatin (SEQ ID NO: 1). In particular embodiments, a modified ulinastatin polypeptide has at least one increased ulinastatin activity relative to wild-type ulinastatin (SEQ ID NO: 1), including increased protease inhibitor activities, anti-inflammatory activities, and anti-metastatic activities, also including increased“specific activity”, as described herein. In some embodiments, the activity is increased by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more relative to wild-type ulinastatin (SEQ ID NO: 1).

In specific embodiments, a modified ulinastatin polypeptide has equivalent or increased therapeutic efficacy at a lower dosage relative to that of wild-type ulinastatin (SEQ ID NO: 1). For example, in some embodiments, a modified ulinastatin polypeptide has equivalent or increased therapeutic efficacy at a dosage that is about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,

35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,

5000, 6000, 7000, 8000, 9000, 10,000-fold lower than that of wild-type ulinastatin (SEQ ID NO: 1).

In some instances, as noted above, a modified ulinastatin polypeptide consists or consists essentially of a variant and/or fragment of wild-type human ulinastatin. The amino acid sequence of wild-type human ulinastatin is provided in Table U1 below.

Thus, in certain embodiments, a modified ulinastatin polypeptide comprises, consists, or consists essentially of (a) a variant of SEQ ID NO: 1 that has at least one at least one modification to an O-linked glycosylation and at least one ulinastatin activity; (b) a fragment of SEQ ID NO: 1 or (a) that has at least one ulinastatin activity; or (c) a variant of (a) or (b) that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to (a) or (b) and has at least one ulinastatin activity, excluding SEQ ID NO: 1 (wild-type ulinastatin).

Certain modified ulinastatin polypeptides have a modified O-linked glycosylation. Here, serine 10 has a chondroitin sulfate (CS) chain attached at a well-conserved Glu-Gly-Ser-Gly (SEQ ID NO:8) O-linked glycosylation site. The CS chain is relatively short (Mwt ~ 8000) with 12-18 disaccharide repeats (GlcUA l,3-GalNacl,4-) and a conventional linkage region (GlcUA l-3Gal 1- 3Gal 1- 4Xyl l)-0-Ser. About 30% of the GalNAc, usually those near the linkage region, are sulfated at C-4 hydroxyl groups. CS chains synthesized during inflammations are shorter with decreased sulfation. Thus, in some instances, a modified ulinastatin polypeptide comprises at least one substitution and/or deletion at one or more of the Glu-Gly-Ser-Gly (SEQ ID NO: 8) residues of SEQ ID NO: 1, which reduces glycosylation at the O-linked glycosylation site. In specific embodiments, a modified ulinastatin polypeptide comprises a substitution or deletion at position S10 of SEQ ID NO:

1, for example, an S10A substitution or other conservative substitution. In some instances, a modified ulinastatin polypeptide comprises, consists, or consists essentially of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 contiguous amino acids of SEQ ID NO: 1. In some instances, a modified ulinastatin polypeptide comprises, consists, or consists essentially of about 20-130, 20-120, 20-110, 20-100, 20- 90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-130, 30-120, 30-110, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-130, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-130, SO HO, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-130, 60-120, 60-110, 60-100, 60-90, 60-80, 60- 70, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90-110, 90-100, 100-130, 100-120, or 100-110 contiguous amino acids of SEQ ID NO: 1.

In some embodiments, a modified ulinastatin polypeptide comprises, consists, or consists essentially of Domain 1 of SEQ ID NO: 1 or an active variant thereof. In certain embodiments, a modified ulinastatin polypeptide comprises, consists, or consists essentially of Domain 2 of SEQ ID NO: 1 or an active variant thereof.

The amino acid sequences of exemplary modified ulinastatin polypeptides are provided in

Table U2 below.

Thus, in some embodiments, a modified ulinastatin polypeptide comprises, consists, or consists essentially of an amino acid sequence selected from Table U2 that has at least one ulinastatin activity, including active variants and/or fragments thereof. For example, in certain embodiments, a modified ulinastatin polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a reference amino sequence selected from Table U2 and has at least one ulinastatin activity, excluding SEQ ID NO: 1. Specific embodiments include a modified ulinastatin polypeptide that comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 (UTIACS), and which retains a substitution at position S 10 of SEQ ID NO: 2, for example, a S10A substitution or other conservative substitution.

A“variant” sequence refers to a polypeptide or polynucleotide sequence that differs from a reference sequence by one or more substitutions, deletions (e.g., truncations), additions, and/or insertions. Certain variants thus include fragments of a reference sequence described herein. Variant polypeptides are biologically active, that is, they continue to possess the enzymatic or binding activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism and/or from human manipulation.

In some instances, a variant comprises one or more“conservative” changes or substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present disclosure and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide described herein, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their utility.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference).

It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (- 4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, . e.. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U. S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine;

glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

A variant may also, or alternatively, contain non-conservative changes. In some

embodiments, variant polypeptides differ from a native or reference sequence by substitution, deletion or addition of about or fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure, enzymatic activity, and/or hydropathic nature of the polypeptide. In certain embodiments, a polypeptide sequence is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, or 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,

900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids in length, including all integers in between, and which may comprise all or a portion of a reference sequence (see, e.g., Table Ul, Table U2, Sequence Listing).

In some embodiments, a polypeptide sequence consists of about or no more than about 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,

320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,

520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,

720, 730, 740, 750, 760, 770, 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,

900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids, including all integers in between, and which may comprise all or a portion of a reference sequence (see, e.g., Table Ul, Table U2, Sequence Listing).

In certain embodiments, a polypeptide sequence is about 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 20-1000, 20-900, 20- 800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 100-900, 100-800, 100- 700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600,

200-500, 200-400, or 200-300 contiguous amino acids, including all ranges in between, and comprises all or a portion of a reference sequence (see, e.g., Table Ul, Table U2 Sequence Listing). In certain embodiments, the C-terminal or N-terminal region of any reference polypeptide may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more amino acids, or by about 10-50, 20-50, 50-100 or more amino acids, including all integers and ranges in between (e.g., 101, 102, 103, 104, 105), so long as the truncated polypeptide retains the binding properties and/or activity of the reference polypeptide (see, e.g., Table Ul, Table U2, Sequence Listing). Typically, the biologically-active fragment has no less than about 1%, about 5%, about 10%, about 25%, or about 50% of an activity of the biologically-active reference polypeptide from which it is derived.

In general, variants will display at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology to a reference polypeptide sequence (see, e.g., Table Ul, Table U2, Sequence Listing). Moreover, sequences differing from the native or parent sequences by the addition (e.g., C- terminal addition, N-terminal addition, both), deletion, truncation, insertion, or substitution (e.g., conservative substitution) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,

49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids (including all integers and ranges in between) but which retain the properties or activities of a parent or reference polypeptide sequence are contemplated (see, e.g., Table Ul, Table U2, Sequence Listing).

In some embodiments, variant polypeptides differ from reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In certain embodiments, variant polypeptides differ from a reference sequence by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity.“Looped” out sequences from deletions or insertions, or mismatches, are considered differences.)

Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (./ Mol. Biol. 48: 444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ( Cabios . 4: 11-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The sequences described herein can be used as a“query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, ./ Mol. Biol, 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules described herein. BLAST protein searches can be performed with the

XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

In some embodiments, as noted above, polynucleotides and/or polypeptides can be evaluated using a BLAST alignment tool. A local alignment consists simply of a pair of sequence segments, one from each of the sequences being compared. A modification of Smith-Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high- scoring segment pairs (HSPs). The results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone.

The raw score, S, is calculated from the number of gaps and substitutions associated with each aligned sequence wherein higher similarity scores indicate a more significant alignment.

Substitution scores are given by a look-up table (see PAM, BLOSUM).

Gap scores are typically calculated as the sum of G, the gap opening penalty and L, the gap extension penalty. Lor a gap of length n, the gap cost would be G+Ln. The choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g., 11, and a low value for L (1-2 ) e.g„ 1.

The bit score, S’, is derived from the raw alignment score S in which the statistical properties of the scoring system used have been taken into account. Bit scores are normalized with respect to the scoring system, therefore they can be used to compare alignment scores from different searches. The terms“bit score” and“similarity score” are used interchangeably. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment. The E-Value, or expected value, describes the likelihood that a sequence with a similar score will occur in the database by chance. It is a prediction of the number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance. The smaller the E-Value, the more significant the alignment. For example, an alignment having an E value of e ¹¹⁷ means that a sequence with a similar score is very unlikely to occur simply by chance.

Additionally, the expected score for aligning a random pair of amino acids is required to be negative, otherwise long alignments would tend to have high score independently of whether the segments aligned were related. Additionally, the BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid and for gapped alignments uses gap creation and extension penalties. For example, BLAST alignment and comparison of polypeptide sequences are typically done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.

In some embodiments, sequence similarity scores are reported from BLAST analyses done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.

In a particular embodiment, sequence identity/similarity scores provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89: 10915-10919, 1992). GAP uses the algorithm of Needleman and Wunsch (JMol Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

In particular embodiments, the variant polypeptide comprises an amino acid sequence that can be optimally aligned with a reference polypeptide sequence (see, e.g.. Table Ul, Table U2, Sequence Listing) to generate a BLAST bit scores or sequence similarity scores of at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,

280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,

480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,

680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,

880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, or more, including all integers and ranges in between, wherein the BLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

As noted above, a reference polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492, 1985); Kunkel el al.,

( Methods in Enzymol. 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D. et al., (“Molecular Biology of the Gene,” Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

Methods for screening gene products of combinatorial libraries made by such modifications, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of reference polypeptides. As one example, recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify polypeptide variants (Arkin and Y ourvan, PNAS USA 89: 7811-7815, 1992; Delgrave et al., Protein Engineering. 6: 327-331, 1993).

Certain“modified ulinastatin polypeptides” include“ulinastatin-Fc fusion polypeptides”, which comprise at least one ulinastatin polypeptide that is fused to at least one Fc region. In some embodiments, the ulinastatin polypeptide portion of the ulinastatin-Fc fusion polypeptide comprises, consists, or consists essentially of (a) a variant of SEQ ID NO: 1 that has at least one at least one modification to an O-linked glycosylation and at least one ulinastatin activity; (b) SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 or (a) that has at least one ulinastatin activity; or (c) a variant of (a) or (b) that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to (a) or (b) and has at least one ulinastatin activity.

In some embodiments, the ulinastatin polypeptide portion of the“ulinastatin-Fc fusion polypeptides” comprises, consists, or consists essentially of SEQ ID NO: 1 or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 contiguous amino acids of SEQ ID NO: 1. In some embodiments, the ulinastatin polypeptide portion of the“ulinastatin-Fc fusion polypeptides” comprises, consists, or consists essentially of SEQ ID NO: 1 or about 20-130, 20- 120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-130, 30-120, 30-110, 30- 100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-130, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-130, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90-110, 90-100, 100-130, 100-120, or 100-110 contiguous amino acids of SEQ ID NO: 1. In some instances, the ulinastatin polypeptide portion of the“ulinastatin-Fc fusion polypeptides” comprises, consists, or consists essentially of Domain 1 of SEQ ID NO: 1 or an active variant thereof. In some instances, the ulinastatin polypeptide portion of the“ulinastatin-Fc fusion polypeptides” comprises, consists, or consists essentially of Domain 2 of SEQ ID NO: 1 or an active variant thereof.

Exemplary ulinastatin-Fc fusion polypeptides are provided in Table U3 below.

Table U3. Exemplary Ulinastatin-Fc fusion polypeptides

Thus, in some embodiments, the ulinastatin-Fc fusion polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from Table U3 and has at least one ulinastatin activity.

A wide variety of Fc region sequences may be employed in the ulinastatin-Fc fusion polypeptides, including wild-type Fc sequences, as well as variants, fragments, hybrids, and chemically modified forms thereof. The ulinastatin-Fc polypeptides may also (optionally) comprise one or more linkers, which typically separate the Fc region(s) from the ulinastatin polypeptide(s), including peptide linkers, as described herein and known in the art. It will be appreciated that in any of these ulinastatin-Fc fusion polypeptides the native N or C terminal amino acid of the ulinastatin polypeptides, or native N or C- amino acid in the Fc domain, may be deleted and/or replaced with non-native amino acid(s), for example, to facilitate expression and or cloning or to serve as a linker sequence between the two proteins.

“Fusion proteins” or“fusion polypeptides” are well known in the art, as are methods of making the same (see, e.g., U.S. Patent Nos. 5,116,964; 5,428,130; 5,455,165; 5,514,582; 6,406,697; 6,291,212; and 6,300,099 for general disclosure and methods related to Fc fusion polypeptides). In a ulinastatin-Fc fusion polypeptide, the Fc region can be fused to the N-terminus of the ulinastatin polypeptide, the C-terminus, or both. In some embodiments, one or more Fc regions can be fused internally relative to ulinastatin sequences, for instance, by placing an Fc region between a first ulinastatin sequence (e.g., domain) and a second ulinastatin sequence (e.g., domain), where the first ulinastatin sequence is fused to the N-terminus of the Fc region and the second ulinastatin sequence is fused to the C-terminus of the Fc region. In specific embodiments, the first and second ulinastatin sequences are identical. In some embodiments, the first and second ulinastatin sequences are different (e.g., they include different functional domains of the ulinastatin polypeptide). Certain ulinastatin-Fc fusion polypeptides can also include additional heterologous protein sequences, that is, non-Fc region and non-ulinastatin polypeptide sequences.

The term“ulinastatin-Fc” can indicate, but does not necessarily indicate, the N-terminal or C- terminal attachment of the Fc region to the ulinastatin polypeptide. For instance, in certain instances the term“Fc-ulinastatin” indicates fusion of the Fc region to the N-terminus of the ulinastatin polypeptide, and the term“ulinastatin-Fc” indicates fusion of the Fc region to the C-terminus of the ulinastatin polypeptide. However, either term can be used more generally to refer to any fusion polypeptide of an Fc region and a ulinastatin polypeptide.

The“Fc region” of a ulinastatin-Fc fusion polypeptide provided herein is usually derived from the heavy chain of an immunoglobulin (Ig) molecule. A typical Ig molecule is composed of two heavy chains and two light chains. The heavy chains can be divided into at least three functional regions: the Fd region, the Fc region (fragment crystallizable region), and the hinge region, the latter being found only in IgG, IgA, and IgD immunoglobulins. The Fd region comprises the variable (VH) and constant (CHi) domains of the heavy chains, and together with the variable (VL) and constant (CL) domains of the light chains forms the antigen-binding fragment or Fab region.

The Fc region of IgG, IgA, and IgD immunoglobulins comprises the heavy chain constant domains 2 and 3, designated respectively as CTT and C¾ regions; and the Fc region of IgE and IgM immunoglobulins comprises the heavy chain constant domains 2, 3, and 4, designated respectively as C¾, C¾, and CH4 regions. The Fc region is mainly responsible for the immunoglobulin effector functions, which include, for example, complement fixation and binding to cognate Fc receptors of effector cells.

The hinge region (found in IgG, IgA, and IgD) acts as a flexible spacer that allows the Fab portion to move freely in space relative to the Fc region. In contrast to the constant regions, the hinge regions are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. The hinge region may also contain one or more glycosylation site(s), which include a number of structurally distinct types of sites for carbohydrate attachment. For example, IgAl contains five glycosylation sites within a 17 amino acid segment of the hinge region, conferring significant resistance of the hinge region polypeptide to intestinal proteases. Residues in the hinge proximal region of the C¾ domain can also influence the specificity of the interaction between an

immunoglobulin and its respective Fc receptor(s) (see, e.g., Shin et al., Intern. Rev. Immunol. 10: ni ne, 1993).

The term“Fc region” or“Fc fragment” or“Fc” as used herein, thus refers to a protein that contains one or more of a C¾ region, a C¾ region, and/or a CH* region from one or more selected immunoglobulin(s), including fragments and variants and combinations thereof. An“Fc region” may also include one or more hinge region(s) of the heavy chain constant region of an immunoglobulin. In certain embodiments, the Fc region does not contain one or more of the CHi, CL, VL, and/or VH regions of an immunoglobulin. The Fc region can be derived from the CEE region, CEE region, CEE region, and/or hinge region(s) of any one or more immunoglobulin classes, including but not limited to IgA, IgD, IgE, IgG, IgM, including subclasses and combinations thereof. In some embodiments, the Fc region is derived from an IgA immunoglobulin, including subclasses IgAl and/or IgA2. In certain embodiments, the Fc region is derived from an IgD immunoglobulin. In particular embodiments, the Fc region is derived from an IgE immunoglobulin. In some embodiments, the Fc region is derived from an IgG

immunoglobulin, including subclasses IgGl, IgG2, IgG2, IgG3, and/or IgG4. In certain embodiments, the Fc region is derived from an IgM immunoglobulin.

Certain Fc regions demonstrate specific binding for one or more Fc-receptors (FcRs).

Examples of classes of Fc receptors include Fey receptors (FcyR), Fca receptors (FcaR), Fee receptors (FceR), and the neonatal Fc receptor (FcRn). For instance, certain Fc regions have increased binding to (or affinity for) one or more FcyRs, relative to FcaRs, FceRs, and/or FcRn. In some embodiments, Fc regions have increased binding to FcaRs, relative to one or more FcyRs, FceRs, and/or FcRn. In some embodiments, Fc regions have increased binding to FceRs (e.g., FcaRI), relative to one or more FcyRs, FcaRs, and/or FcRn. In particular embodiments, Fc regions have increased binding to FcRn, relative to one or more FcyRs, FcaRs, and/or FceRs. In certain embodiments, the binding (or affinity) of an Fc region to one or more selected FcR(s) is increased relative to its binding to (or affinity for) one or more different FcR(s), typically by about 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, lOx, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, lOOOx or more (including all integers in between).

Examples of FcyRs include FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb. FcyRI (CD64) is expressed on macrophages and dendritic cells and plays a role in phagocytosis, respiratory burst, cytokine stimulation, and dendritic cell endocytic transport. Expression of FcyRI is upregulated by both GM-CSF and g-interferon (g-IFN) and downregulated by interleukin-4 (IL-4). FcyRIIa is expressed on polymorphonuclear leukocytes (PMN), macrophages, dendritic cells, and mast cells. FcyRIIa plays a role in phagocytosis, respiratory burst, and cytokine stimulation. Expression of FcyRIIa is upregulated by GM-CSF and g-IFN, and decreased by IL-4. Fcyllb is expressed on B cells, PMN, macrophages, and mast cells. Fcyllb inhibits immunoreceptor tyrosine-based activation motif (ITAM) mediated responses, and is thus an inhibitory receptor. Expression of FcyRIIc is upregulated by intravenous immunoglobulin (IVIG) and IL-4 and decreased by g-IFN. FcyRIIc is expressed on NK cells. FcyRIIIa is expressed on natural killer (NK) cells, macrophages, mast cells, and platelets. This receptor participates in phagocytosis, respiratory burst, cytokine stimulation, platelet aggregation and degranulation, and NK-mediated ADCC. Expression of FcyRIII is upregulated by C5a, TGF-b, and g-IFN and downregulated by IL-4. Fc g RHIb is a GPI-linked receptor expressed on PMN.

Certain Fc regions have increased binding to FcyRI, relative to FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and/or FcyRIIIb. Some embodiments have increased binding to FcyRIIa, relative to FcyRI, FcyRIIb, FcyRIIc, FcyRIIIa, and/or FcyRIIIb. Particular Fc regions have increased binding to FcyRIIb. relative to FcyRI. FcyRIIa. FcyRIIc, FcyRIIIa. and/or FcyRIIIb. Certain Fc regions have increased binding to FcyRIIc, relative to FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, and/or FcyRIIIb. Some Fc regions have increased binding to FcyRIIIa, relative to FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, and/or FcyRIIIb. Specific Fc regions have increased binding to FcyRIIIb, relative to FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, and/or FcyRIIIa.

FcaRs include FcaRI (CD89). FcaRI is found on the surface of neutrophils, eosinophils, monocytes, certain macrophages (e.g., Kupffer cells), and certain dendritic cells. FcaRI is composed of two extracellular Ig-like domains, is a member of both the immunoglobulin superfamily and the multi-chain immune recognition receptor (MIRR) family, and signals by associating with two FcRy signaling chains.

FceRs include FceRI and FceRII. The high-affinity receptor FceRI is a member of the immunoglobulin superfamily, is expressed on epidermal Langerhans cells, eosinophils, mast cells and basophils, and plays a major role in controlling allergic responses. FceRI is also expressed on antigen- presenting cells, and regulates the production pro-inflammatory cytokines. The low-affinity receptor FceRII (CD23) is a C-type lectin that can function as a membrane -bound or soluble receptor. FceRII regulates B cell growth and differentiation, and blocks IgE -binding of eosinophils, monocytes, and basophils. Certain Fc regions have increased binding to FceRI, relative to FceRII. Other Fc regions have increased binding to FceRII, relative to FceRI.

The amino acid sequences of CEE, CEE, CEE, and hinge regions from exemplary, wild-type human IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, and IgM immunoglobulins are shown in

Table FI below.

An Fc region of a ulinastatin-Fc fusion polypeptide can thus comprise, consist of, or consist essentially of one or more of the human Fc region amino acid sequences of Table FI, including variants, fragments, homologs, orthologs, paralogs, and combinations thereof. Certain illustrative embodiments comprise an Fc region that ranges in size from about 20-50, 20-100, 20-150, 20-200, 20-250, 20-300, 20-400, 50-100, 50-150, 50-200, 50-250, 50-300, 50-400, 100-150, 100-200, 100- 250, 100-300, 100-350, 100-400, 200-250, 200-300, 200-350, or 200-400 amino acids in length, and optionally comprises, consists of, or consists essentially of any one or more of the sequences in Table FI. Certain embodiments comprise an Fc region of up to about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400 or more amino acids, which optionally comprises, consists of, or consists essentially of any one or more of the amino acid sequences of Table FI.

In some instances, a ulinastatin-Fc fusion polypeptide provides a variety of advantages relative to its“corresponding ulinastatin polypeptide”, i.e., a ulinastatin polypeptide of the same sequence having no Fc region(s) attached thereto. Merely by way of illustration, in some instances the covalent attachment of one or more Fc regions alters (e.g., increases, decreases) the ulinastatin polypeptide’s solubility, half-life (e.g., in serum, in a selected tissue, in a test tube under storage conditions, for example, at room temperature or under refrigeration), dimerization or multimerization properties, biological activity or activities, for instance, by providing Fc-region-associated effector functions (e.g., activation of the classical complement cascade, interaction with immune effector cells via the Fc receptor (FcR), compartmentalization of immunoglobulins), cellular uptake, intracellular transport, tissue distribution, and/or bioavailability, relative to an unmodified ulinastatin polypeptide having the same or similar sequence. In certain aspects, Fc regions can confer effector functions relating to complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and/or antibody-dependent cell-mediated phagocytocis (ADCP), which are believed to play a role in clearing specific target cells such as tumor cells and infected cells.

Thus, in some instances, a ulinastatin-Fc fusion polypeptide has altered (e.g., improved, increased, decreased) pharmacokinetic properties and/or ulinastatin activities relative to its corresponding ulinastatin polypeptide. Examples of pharmacokinetic properties include stability or half-life, bioavailability (the fraction of a drug that is absorbed), tissue distribution, volume of distribution (apparent volume in which a drug is distributed immediately after it has been injected intravenously and equilibrated between plasma and the surrounding tissues), concentration (initial or steady-state concentration of drug in plasma), elimination rate constant (rate at which drugs are removed from the body), elimination rate (rate of infusion required to balance elimination), area under the curve (AUC or exposure; integral of the concentration-time curve, after a single dose or in steady state), clearance (volume of plasma cleared of the drug per unit time), C_max (peak plasma

concentration of a drug after oral administration), t_max (time to reach C_max), C_min (lowest concentration that a drug reaches before the next dose is administered), and fluctuation (peak trough fluctuation within one dosing interval at steady state). In some aspects, these improved properties are achieved without significantly altering the secondary structure and/or reducing the ulinastatin activity of the ulinastatin polypeptide. In some instances, a ulinastatin-Fc fusion polypeptide has increased ulinastatin activity, as described herein, relative to its corresponding ulinastatin polypeptide.

In some embodiments, a ulinastatin-Fc fusion polypeptide has a plasma or sera

pharmacokinetic AUC profde at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, or 500-fold greater than its corresponding ulinastatin polypeptide when administered to a mammal under the same or comparable conditions. In certain embodiments, a ulinastatin-Fc fusion polypeptide has a stability (e.g., as measured by half-life) which is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% greater than its corresponding ulinastatin polypeptide when compared under similar conditions at room temperature, for example, in PBS at pH 7.4 for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or 1, 2, 3, 4 weeks or so.

In particular embodiments, a ulinastatin-Fc fusion polypeptide has a biological half life at pH 7.4, 25°C, e.g., a physiological pH, human body temperature (e.g., in vivo, in serum, in a given tissue, in a given species such as rat, mouse, monkey, or human), of about or at least about 30 minutes, about 1 hour, about 2 hour, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours, or about 144 hours or more or any intervening half-life.

In certain embodiments, a ulinastatin-Fc fusion polypeptide has greater bioavailability after subcutaneous (SC) administration relative to its corresponding ulinastatin polypeptide. In certain embodiments, a ulinastatin-Fc fusion polypeptide has at least about 20%, at least about 30%, at least about 40%„ at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100 %, or more bioavailability relative to its corresponding ulinastatin polypeptide.

In certain embodiments, variant or otherwise modified Fc regions can be employed, including those having altered properties or biological activities relative to wild-type Fc region(s). Examples of modified Fc regions include those having mutated sequences, for instance, by substitution, insertion, deletion, or truncation of one or more amino acids relative to a wild-type sequence, hybrid Fc polypeptides composed of domains from different immunoglobulin classes/subclasses, Fc

polypeptides having altered glycosylation/sialylation patterns, and Fc polypeptides that are modified or derivatized, for example, by biotinylation (see. e.g., US Application No. 2010/0209424), phosphorylation, sulfation, etc., or any combination of the foregoing. Such modifications can be employed to alter (e.g., increase, decrease) the binding properties of the Fc region to one or more particular FcRs (e.g., FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, FcyRIIIb, FcRn), its

pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, volume of distribution, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, Cma_X, tma_X, C_min, fluctuation), its immunogenicity, its complement fixation or activation, and/or the CDC/ADCC/ADCP-related activities of the Fc region, among other properties described herein, relative to a corresponding wild-type Fc sequence.

In certain embodiments, a peptide linker sequence may be employed to separate the ulinastatin polypeptide(s) and the Fc region(s) by a distance sufficient to ensure that each polypeptide folds into its desired secondary and tertiary structures. Such a peptide linker sequence can be incorporated into a fusion polypeptide using standard techniques well known in the art.

Certain peptide linker sequences may be chosen based on the following exemplary factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; (3) their physiological stability; and (4) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes, or other features. See, e.g., George and Heringa, J Protein Eng.

15:871-879, 2002.

The linker sequence may generally be from 1 to about 200 amino acids in length. Particular linkers can have an overall amino acid length of about 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17, 18, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 60, 70, 80, 90, 100 or more amino acids.

A peptide linker may employ any one or more naturally-occurring amino acids, non-naturally occurring amino acid(s), amino acid analogs, and/or amino acid mimetics as described elsewhere herein and known in the art. Certain amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et ah, Gene 40:39-46, 1985; Murphy et ah, PNAS USA. 83:8258- 8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. Particular peptide linker sequences contain Gly, Ser, and/or Asn residues. Other near neutral amino acids, such as Thr and Ala may also be employed in the peptide linker sequence, if desired.

Certain exemplary linkers include Gly, Ser and/or Asn-containing linkers, as follows: [G]_x, [S]_x, [N]_x, [GS]_X, [GGS]_X, [GSS]_X, [GSGS]_X (SEQ ID NO:38), [GGSG]_X (SEQ ID NO: 39), [GGGS]_X (SEQ ID NO: 40), [GGGGS]_X (SEQ ID NO: 41), [GN]_X, [GGN]_X, [GNN]_X, [GNGN]_X (SEQ ID NO: 42), [GGNG]_X (SEQ ID NO: 43), [GGGN]_X (SEQ ID NO: 44), [GGGGN]_X (SEQ ID NO: 45) linkers, where _x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids will be apparent to persons skilled in the art.

Additional examples of linker peptides include, but are not limited to the following amino acid sequences: Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 46); Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser- (SEQ ID NO: 47); Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly- Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 48); Asp-Ala-Ala-Ala-Lys-Glu- Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-Ala- Ala-Ala- Arg-Asp-Ala-Ala-Ala-Lys-(SEQ ID NO: 49); and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg-(SEQ ID NO: 50).

Further non-limiting examples of linker peptides include DGGGS (SEQ ID NO: 51); TGEKP (SEQ ID NO: 52) (see, e.g., Liu et al., PNAS. 94:5525-5530, 1997); GGRR (SEQ ID NO: 53) (Pomerantz et al. 1995); (GGGGS)_n (SEQ ID NO: 41) (Kim et al., PNAS. 93: 1156-1160, 1996); EGKSSGSGSESKVD (SEQ ID NO: 54) (Chaudhary et al., PNAS. 87: 1066-1070, 1990);

KESGSVSSEQLAQFRSLD (SEQ ID NO: 55) (Bird et al, Science. 242:423-426, 1988),

GGRRGGGS (SEQ ID NO: 56); LRQRDGERP (SEQ ID NO: 57); LRQKDGGGSERP (SEQ ID NO: 58); LRQKd(GGGS)2 ERP (SEQ ID NO: 59). In specific embodiments, the linker sequence comprises a Gly3 linker sequence, which includes three glycine residues. In particular embodiments, flexible linkers can be rationally designed using a computer program capable of modeling both DNA- binding sites and the peptides themselves (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91: 11099-11103, 1994) or by phage display methods.

The peptide linkers may be physiologically stable or may include a releasable linker such as a physiologically degradable or enzymatically cleavable linker (e.g., proteolytically cleavable linker). In certain embodiments, one or more releasable linkers can result in a shorter half-life and more rapid clearance of the conjugate. These and related embodiments can be used, for example, to enhance the solubility and blood circulation lifetime of ulinastatin polypeptides in the bloodstream, while also delivering a ulinastatin polypeptide into the bloodstream that, subsequent to linker degradation, is substantially free of the Fc region(s). These aspects are especially useful in those cases where ulinastatin polypeptides, when permanently conjugated to an Fc region, demonstrate reduced activity. By using the linkers as provided herein, such ulinastatin polypeptides can maintain their therapeutic activity when in conjugated form. As another example, a large and relatively inert ulinastatin-Fc fusion polypeptide may be administered, which is then degraded in vivo (via the degradable linker) to generate a bioactive ulinastatin polypeptide possessing a portion of the Fc region or lacking the Fc region entirely. In these and other ways, the properties of the ulinastatin-Fc fusion polypeptide can be more effectively tailored to balance the bioactivity and circulating half-life of the ulinastatin polypeptide over time.

In particular embodiments, the linker peptide comprises an autocatalytic or self-cleaving peptide cleavage site. In a particular embodiment, self-cleaving peptides include those polypeptide sequences obtained from potyvirus and cardiovirus 2A peptides, FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thosea asigna virus and porcine teschovirus. In certain embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence or domain (Donnelly et al., J. Gen. Virol. 82: 1027-1041, 2001). Exemplary 2A sites include the following sequences:

LLNFDLLKLAGDVESNPGP (SEQ ID NO: 60); TLNFDLLKLAGDVESNPGP (SEQ ID NO: 61); LLKLAGDVESNPGP (SEQ ID NO: 62); NFDLLKLAGDVESNPGP (SEQ ID NO: 63); QLLNFDLLKLAGDVESNPGP (SEQ ID NO: 64); APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 65); VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT (SEQ ID NO: 66);

LNFDLLKLAGDVESNPGP (SEQ ID NO: 67);

LLAIHPTEARHKQKIV APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 68); and

EARHKQKIV APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 69). In some embodiments, the autocatalytic peptide cleavage site comprises a translational 2A signal sequence, such as, e.g., the 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein, which is an 18 amino acid sequence. Additional examples of 2A-like sequences that may be used include insect virus polyproteins, the NS34 protein of type C rotaviruses, and repeated sequences in Trypanosoma spp., as described, for example, in Donnelly et al, Journal of General Virology. 82: 1027-1041, 2001.

Suitable protease cleavages sites and self-cleaving peptides are known to the skilled person (see, e.g., Ryan et al, J. Gener. Virol. 78:699-722, 1997; and Scymczak et al., Nature Biotech. 5:589- 594, 2004). Exemplary protease cleavage sites include, but are not limited to the cleavage sites of potyvirus NIa proteases (e.g., tobacco etch virus protease), potyvirus HC proteases, potyvirus PI (P35) proteases, byovirus NIa proteases, byovirus RNA-2 -encoded proteases, aphthovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picoma 3C proteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase. Due to its high cleavage stringency, TEV (tobacco etch virus) protease cleavage sites are included in some embodiments, e.g., EXXYXQ(G/S) (SEQ ID NO: 70), for example, ENLYFQG (SEQ ID NO: 71) and ENLYFQS (SEQ ID NO: 72), wherein X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).

Further examples of enzymatically degradable linkers suitable for use in particular embodiments include, but are not limited to: an amino acid sequence cleaved by a serine protease such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or substilisin. Illustrative examples of thrombin-cleavable amino acid sequences include, but are not limited to: -Gly-Arg-Gly-Asp-(SEQ ID NO: 73), -Gly-Gly-Arg-, -Gly- Arg-Gly-Asp-Asn-Pro-(SEQ ID NO: 74), -Gly-Arg-Gly-Asp-Ser- (SEQ ID NO: 75), -Gly-Arg-Gly-Asp-Ser-Pro-Lys-(SEQ ID NO: 76), -Gly-Pro- Arg-, -Val-Pro-Arg-, and -Phe- Val -Arg-. Illustrative examples of elastase-cleavable amino acid sequences include, but are not limited to: -Ala-Ala-Ala-, -Ala-Ala-Pro-Val-(SEQ ID NO: 77), -Ala-Ala-Pro-Leu-(SEQ ID NO: 78), -Ala-Ala-Pro-Phe-(SEQ ID NO:79), -Ala-Ala-Pro-Ala-(SEQ ID NO:80), and -Ala-Tyr-Leu-Val- (SEQ ID NO: 81).

Enzymatically degradable linkers also include amino acid sequences that can be cleaved by a matrix metalloproteinase such as collagenase, stromelysin, and gelatinase. Illustrative examples of matrix metalloproteinase-cleavable amino acid sequences include, but are not limited to: -Gly-Pro-Y- Gly-Pro-Z-(SEQ ID NO: 82), -Gly-Pro-, Leu-Gly-Pro-Z-(SEQ ID NO: 83), -Gly-Pro-Ile-Gly-Pro-Z- (SEQ ID NO: 84), and -Ala-Pro-Gly-Leu-Z-(SEQ ID NO: 85), where Y and Z are amino acids. Illustrative examples of collagenase-cleavable amino acid sequences include, but are not limited to: - Pro-Leu-Gly-Pro-D-Arg-Z-(SEQ ID NO: 86), -Pro- Leu-Gly-Leu-Leu-Gly-Z-(SEQ ID NO: 87), -Pro- Gln-Gly-Ile-Ala-Gly-Trp-(SEQ ID NO: 88), -Pro-Leu-Gly-Cys(Me)-His-(SEQ ID NO: 89), -Pro-Leu- Gly-Leu-Tyr-Ala-(SEQ ID NO: 90), -Pro-Leu-Ala-Leu-Trp-Ala-Arg-(SEQ ID NO: 91), and -Pro- Leu-Ala-Tyr-Trp-Ala-Arg-(SEQ ID NO: 92), where Z is an amino acid. An illustrative example of a stromelysin-cleavable amino acid sequence is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg-(SEQ ID NO: 93); and an example of a gelatinase-cleavable amino acid sequence is -Pro-Leu-Gly-Met-Tyr- Ser-Arg-(SEQ ID NO: 94).

Enzymatically degradable linkers suitable for use in particular embodiments include amino acid sequences that can be cleaved by an angiotensin converting enzyme, such as, for example, -Asp- Lys-Pro-, -Gly-Asp-Lys-Pro-(SEQ ID NO: 95), and -Gly-Ser-Asp-Lys-Pro-(SEQ ID NO: 96).

Enzymatically degradable linkers suitable for use in particular embodiments include amino acid sequences that can be degraded by cathepsin B, such as, for example, Val-Cit, Ala-Leu-Ala-Leu- (SEQ ID NO: 97), Gly-Phe-Leu-Gly-(SEQ ID NO: 98) and Phe-Lys.

In particular embodiments, a releasable linker has a half life at pH 7.4, 25°C, e.g., a physiological pH, human body temperature (e.g., in vivo, in serum, in a given tissue), of about 30 minutes, about 1 hour, about 2 hour, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours or more or any intervening half-life. One having skill in the art would appreciate that the half life of a ulinastatin-Fc fusion polypeptide can be finely tailored by using a particular releasable linker.

In certain embodiments, however, any one or more of the peptide linkers are optional. For instance, linker sequences may not required when the first and second polypeptides have non-essential N-terminal and/or C-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

The modified ulinastatin polypeptides can be used in any of the compositions, methods, and/or kits described herein.

Compositions and Methods of Use

Certain embodiments include therapeutic compositions comprising a modified ulinastatin polypeptide described herein, and methods of using the same for the treatment of various diseases.

For example, certain embodiments include compositions, for example, therapeutic or pharmaceutical compositions, comprising a modified ulinastatin polypeptide described herein, and a pharmaceutically-acceptable carrier. Certain compositions are substantially pure on a protein basis or weight-basis. For instance, certain compositions have a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis and are substantially aggregate-free, for example, less than about 10, 9, 8, 7, 6, or 5% aggregated. Certain compositions are substantially endotoxin-free, as described herein. The compositions may be prepared by methodology well known in the pharmaceutical art.

For example, a composition intended to be administered by injection can be prepared by combining a composition that comprises a modified ulinastatin polypeptide, as described herein, and optionally one or more of buffers or excipients, optionally with sterile, distilled water so as to form a solution. Certain compositions comprise a physiological saline solution (e.g., 0.9% normal saline) or dextrose (e.g., about 1-10% dextrose, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% dextrose). A surfactant can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the the modified ulinastatin polypeptide in the composition so as to facilitate dissolution or homogeneous suspension of the polypeptide in the aqueous delivery system.

Certain compositions are at a pharmaceutically-acceptable pH. For instance, in certain embodiments, the pharmaceutically-acceptable pH is about 5.0 to about 8.0 (±0.01 to ±0.1), or about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1. 6.2. 6.3. 6.4. 6.5. 6.6. 6.7. 6.8. 6.9. 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 (±0.01 to ±0.1), including all integers and ranges in between.

In certain embodiments, a modified ulinastatin polypeptide has at least one ulinastatin activity at a pH close to the physiological pH of human blood. Thus, in some embodiments, a modified ulinastatin polypeptide has at least one ulinastatin activity at a pH of about 4 to about 10.8, or about 6 to about 8, or about 6.5 to about 7.5. In certain embodiments, a modified ulinastatin polypeptide has effective ulinastatin activity at about pH 7.4.

In specific embodiments, the composition has one or more of the following determinations of purity: less than about 1 EU endotoxin/mg protein, less that about 100 ng host cell protein/mg protein, less than about 10 pg host cell DNA/mg protein, and/or greater than about 95% single peak purity by SEC HPLC.

Also included are methods of treating, ameliorating the symptoms of, or inhibiting the progression of, a disease in a subject in need thereof, comprising administering to the subject a composition comprising at least one modified ulinastatin polypeptide, as described herein.

The methods and compositions described herein can be used in the treatment of any variety of diseases or conditions. For instance, certain embodiments include methods of treating an

inflammatory disease or condition in a subject in need thereof, comprising administering to the subject a therapeutic composition comprising a modified ulinastatin polypeptide, as described herein.

Exemplary inflammatory diseases or conditions include pancreatitis (e.g., acute pancreatitis, chronic pancreatitis, endoscopic retrograde cholangiopancreatography (ERCP)-induced pancreatitis), systemic inflammation, colitis, autoimmune encephalomyelitis, Stevens-Johnson syndrome, arthritis, renal failure, bums, sepsis/septic shock including severe sepsis and related pro- inflammatory/secondary conditions (e.g., organ failure), systemic inflammatory response syndrome (SIRS), toxic epidermal necrolysis (TEN), Kawasaki disease, kidney disease (e.g., acute kidney failure, chronic kidney disease), ischemic conditions (e.g., ischemia-reperfusion injury in the liver, kidney, heart, lungs, brain), lung inflammation and inflammatory lung conditions (e.g., pulmonary infection, pneumonia, including infectious interstitial pneumonia associated with mixed connective tissue disease, pulmonary fibrosis, acute respiratory distress syndrome), liver inflammation including hepatitis, anaphylaxis, post-operative or post-surgical complications (e.g., renal function, cardiac surgery, lung surgery, cognitive dysfunction, liver transplantation), lipopolysaccharide (LPS)-induced inflammation or tissue injury (e.g., lungs, liver, brain), inflammation or dysfunction secondary to diabetes (e.g., diabetes-induced cardiac dysfunction), bum injury, heat stroke, inflammatory or neuropathic pain, acute poisoning, hyperlipidemia-associated inflammation, autoimmunity-associated inflammation, allogeneic transplant or blood transfusion-associated inflammation, and

neuroinflammation .

Also included are methods of treating, ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein. The methods and therapeutic compositions described herein can be used in the treatment of any variety of cancers or tumors. In some embodiments, the cancer is a primary cancer, i.e., a cancer growing at the anatomical site where tumor progression began and yielded a cancerous mass. In some embodiments, the cancer is a secondary or metastatic cancer, i.e., a cancer which has spread from the primary site or tissue of origin into one or more different sites or tissues. In some embodiments, the subject has a cancer selected from one or more of melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

In some embodiments, as noted above, the cancer or tumor is a metastatic cancer. Further to the above cancers, exemplary metastatic cancers include, without limitation, bladder cancers which have metastasized to the bone, liver, and/or lungs; breast cancers which have metastasized to the bone, brain, liver, and/or lungs; colorectal cancers which have metastasized to the liver, lungs, and/or peritoneum; kidney cancers which have metastasized to the adrenal glands, bone, brain, liver, and/or lungs; lung cancers which have metastasized to the adrenal glands, bone, brain, liver, and/or other lung sites; melanomas which have metastasized to the bone, brain, liver, lung, and/or skin/muscle; ovarian cancers which have metastasized to the liver, lung, and/or peritoneum; pancreatic cancers which have metastasized to the liver, lung, and/or peritoneum; prostate cancers which have metastasized to the adrenal glands, bone, liver, and/or lungs; stomach cancers which have metastasized to the liver, lung, and/or peritoneum; thyroid cancers which have metastasized to the bone, liver, and/or lungs; and uterine cancers which have metastasized to the bone, liver, lung, peritoneum, and/or vagina; among others.

In some instances, administration of a modified ulinastatin polypeptide reduces inflammation or one or more inflammatory responses in the subject. For example, in some instances the administration a modified ulinastatin polypeptide reduces one or more of endothelial

activation/damage, proinflammatory cytokine and chemokine production/release (for example, IL-Ib, MIP-la, MCP-1, and CXCL1), fibrinogen synthesis, neutrophil recruitment into organs, and/or organ injury in the subject.

In some embodiments, the methods or compositions described herein increase median survival time of a patient by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certain embodiments, the methods or compositions described herein increase median survival time of a patient by 1 year, 2 years, 3 years, or longer.

In certain embodiments, for example, in the treatment of cancer, the composition

administered is sufficient to result in tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least a 10%, 20%, 30%, 40%, 50% or greater decrease in tumor mass, or by altered (e.g., decreased with statistical significance) scan dimensions. In certain embodiments, the composition administered is sufficient to result in stable disease. In certain embodiments, the composition administered is sufficient to result in stabilization or clinically relevant reduction in symptoms of a particular disease indication known to the skilled clinician.

Methods for identifying subjects with one or more of the diseases or conditions described herein are known in the art.

Administration may be achieved by a variety of different routes. Modes of administration depend upon the nature of the condition to be treated or prevented. For example, a composition can be administered orally, intranasally, intraperitoneally, parenterally, intravenously, intralymphatically, intratumorly, intramuscularly, interstitially, intraintestinally, intra-arterially, subcutaneously, intraocularly, intrasynovial, transepithelial, and/or transdermally. Particular embodiments include administration by IV infusion.

The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated. A

pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need. In some embodiments, a therapeutically effective amount or therapeutic dosage of a composition described herein is an amount that is effective to reduce inflammation or an

inflammatory response in a subject. In certain instances, treatment is initiated with small dosages which can be increased by small increments until the optimum effect under the circumstances is achieved.

In some embodiments, a dosage is administered from about once a day to about once every two or three weeks. For example, in certain embodiments, a dosage is administered about once every 1, 2, 3, 4, 5, 6, or 7 days, or about once a week, or about twice a week, or about three times a week, or about once every two or three weeks.

Certain embodiments comprise administering a modified ulinastatin polypeptide at a dosage (e.g., a daily dosage) of about lxlO⁴ U to about 1 ^c 10⁵ U to about lOOxlO⁵ U, or about lxlO⁴ U, 2xl0⁴ U, 3xl0⁴ U, 4xl0⁴ U, 5xl0⁴ U, 6xl0⁴ U, 7xl0⁴ U, 8xl0⁴ U, 9xl0⁴ U, 1 ^c 10⁵ U, 2x 10⁵ U, 3x 10⁵ U, 4x l0⁵ U, 5 x l0⁵ U, 6xl0⁵ U, 7xl0⁵ U, 8xl0⁵ U, 9xl0⁵ U, lOxlO⁵ U, l lxl0⁵ U, 12xl0⁵ U, 15xl0⁵ U, 20x10⁵ U, 30X10⁵U, 40X10⁵ U, 50xl0⁵ U, 60xl0⁵ U, 70xl0⁵ U, 80xl0⁵ U, or lOOxlO⁵ U, including all ranges and integers in between. Some embodiments comprise infusing the daily dosage intravenously over about a 1, 2, or 3 hour period. Particular embodiments comprise infusing the daily dosage over about a 1, 2, or 3 hour period, optionally about 1, 2, or 3 times per day, and optionally for about 2, 3,

4, 5, 6, or 7 or more days in a row.

Also included are patient care kits, comprising one or more compositions or modified ulinastatin polypeptides described herein. Certain kits also comprise one or more pharmaceutically- acceptable diluents or solvents, such as water (e.g., sterile water) or saline. In some embodiments, the compositions or modified ulinastatin polypeptides are stored in vials, cartridges, dual chamber syringes, and/or pre-filled mixing systems.

The kits herein may also include a one or more additional therapeutic agents or other components suitable or desired for the indication being treated, or for the desired diagnostic application. The kits herein can also include one or more syringes or other components necessary or desired to facilitate an intended mode of delivery (e.g., stents, implantable depots, etc.).

Polynucleotides, Expression Vectors, and Host Cells

Certain embodiments relate to polynucleotides that encode a modified ulinastatin polypeptide, as described herein. Thus, certain embodiments include a polynucleotide that encodes any one or more of the individual ulinastatin polypeptides in Table U2 or Table U3, including variants and/or fragments thereof. For instance, certain polynucleotides encode a modified ulinastatin polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a reference amino sequence selected from Table U2 or Table U3.

Among other uses, these and related embodiments may be utilized to recombinantly produce modified ulinastatin polypeptide in a host cell. It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide described herein. Some of these polynucleotides may bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated, for example, polynucleotides that are optimized for human, yeast, or bacterial codon selection.

As will be recognized by the skilled artisan, polynucleotides may be single-stranded (coding or antisense) or double -stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a modified ulinastatin polypeptide) or may comprise a variant, or a biological functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as described herein, preferably such that the activity of the variant polypeptide is not substantially diminished relative to the unmodified polypeptide.

Additional coding or non-coding sequences may, but need not, be present within a polynucleotide, and a polynucleotide may, but need not, be linked to other molecules and/or support materials. Hence, the polynucleotides, regardless of the length of the coding sequence itself, may be combined with other DNA or RNA sequences, such as promoters, enhances, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.

The polynucleotide sequences may also be of mixed genomic, cDNA, RNA, and that of synthetic origin. For example, a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the polypeptide, after which the DNA or RNA sequence may be modified at a site by inserting synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures or preferably generating the desired sequence by PCR using suitable oligonucleotides. In some embodiments a signal sequence can be included before the coding sequence. This sequence encodes a signal peptide N-terminal to the coding sequence which communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media. Typically the signal peptide is clipped off by the host cell before the protein leaves the cell. Signal peptides can be found in variety of proteins in prokaryotes and eukaryotes.

One or multiple polynucleotides can encode a modified ulinastatin polypeptide described herein. Moreover, the polynucleotide sequence can be manipulated for various reasons. Examples include but are not limited to the incorporation of preferred codons to enhance the expression of the polynucleotide in various organisms (see generally Nakamura et ah, Nuc. Acid. Res. 28:292, 2000).

Also included are expression vectors that comprise the polynucleotides, and host cells that comprise the polynucleotides and/or expression vectors. Modified ulinastatin polypeptides can be produced by expressing a DNA or RNA sequence encoding the polypeptide in a suitable host cell by well-known techniques. The term“host cell” is used to refer to a cell into which has been introduced, or which is capable of having introduced into it, a nucleic acid sequence encoding one or more of the polypeptides described herein, and which further expresses or is capable of expressing a polypeptide of interest, such as a polynucleotide encoding any herein described polypeptide. The term includes the progeny of the parent cell, whether or not the progeny are identical in morphology or in genetic make up to the original parent, so long as the selected gene is present. Host cells may be chosen for certain characteristics, for instance, the expression of a formylglycine generating enzyme (FGE) to convert a cysteine or serine residue within a sulfatase motif into a formylglycine (FGly) residue, or the expression of aminoacyl tRNA synthetase(s) that can incorporate unnatural amino acids into the polypeptide, including unnatural amino acids with an azide side-chain, alkyne side-chain, or other desired side-chain, to facilitate chemical conjugation or modification.

In some instances, a polynucleotide or expression vector comprises additional non-coding sequences. For example, the“control elements” or“regulatory sequences” present in an expression vector are non-translated regions of the vector, including enhancers, promoters, 5' and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used.

A variety of expression vector/host systems are known and may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with an expression vector, for example, a recombinant bacteriophage, plasmid, or cosmid DNA expression vector; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cell and more specifically human cell systems transformed with viral, plasmid, episomal or integrating expression vectors. Certain embodiments therefore include an expression vector, comprising a polynucleotide sequence that encodes a polypeptide described herein, for example, a modified ulinastatin protein. Also included are host cells that comprise the polynucleotides and/or expression vectors.

Certain embodiments employ E. coli-based expression systems (see, e.g., Structural

Genomics Consortium et al., Nature Methods. 5: 135-146, 2008). These and related embodiments may optionally utilize ligation-independent cloning (LIC) to produce a suitable expression vector. In specific embodiments, protein expression may be controlled by a T7 RNA polymerase (e.g., pET vector series), or modified pET vectors with alternate promoters, including for example the TAC promoter. These and related embodiments may utilize the expression host strain BL21(DE3), a ZDE3 lysogen of BL21 that supports T7 -mediated expression and is deficient in Ion and ompT proteases for improved target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coli, such as ROSETTA^™ (DE3) and Rosetta 2 (DE3) strains. In some embodiments, other E. coli strains may be utilized, including other E. coli K-12 strains such as W3110 (F lambda IN(rmD-rmE)l rph-1), and UT5600 (F, araC14, leuB6(Am), secA206(aziR), lacYl, proC14, tsx67, A(ompTfepC)266, entA403, glnX44(AS), l , trpE38, rfbCl, rpsL109(strR), xylA5, mtl-1, thiEl), which can result in reduced levels of post-translational modifications during fermentation. Cell lysis and sample handling may also be improved using reagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER® Protein Extraction Reagent. For cell culture, auto-inducing media can improve the efficiency of many expression systems, including high- throughput expression systems. Media of this type (e.g., OVERNIGHT EXPRESS™ Autoinduction System) gradually elicit protein expression through metabolic shift without the addition of artificial inducing agents such as IPTG.

Particular embodiments employ hexahistidine tags (such as those sold under the trademark HIS_*TAG® fusions), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques. In certain aspects, however, clinical grade proteins can be isolated from E. coli inclusion bodies, without or without the use of affinity tags (see, e.g., Shimp et ak, Protein Expr Purif. 50:58-67, 2006).

Also included are high-density bacterial fermentation systems. For example, high cell density cultivation of Ralstonia eutropha allows protein production at cell densities of over 150 g/L, and the expression of recombinant proteins at titers exceeding 10 g/L. In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al, Methods Enzymol. 753:516-544 (1987). Also included are Pichia pandoris expression systems (see, e.g., Li et al, Nature Biotechnology. 24, 210 - 215, 2006; and Hamilton et al, Science, 301: 1244, 2003).

Certain embodiments include yeast systems, for example, which are engineered to selectively glycosylate proteins, including yeast that have humanized N-glycosylation pathways, among others (see, e.g., Hamilton et al, Science. 313: 1441-1443, 2006; Wildt et al, Nature Reviews Microbiol. 3: 119-28, 2005; and Gemgross et al, Nature-Biotechnology. 22: 1409 -1414, 2004; U.S. Patent Nos. 7,629,163; 7,326,681; and 7,029,872). Merely by way of example, recombinant yeast cultures can be grown in Fembach Flasks or 15L, 50L, 100L, and 200L fermentors, among others.

In mammalian host cells, a number of expression systems are well known in the art and commercially available. Exemplary mammalian vector systems include for example, pCEP4, pREP4, and pREP7 from Invitrogen, the PerC6 system from Crucell, and Lentiviral based systems such as pLPl from Invitrogen, and others. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus

transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659, 1984). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells sub-cloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));

monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al. , Annals N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; LS4 cells; and a human hepatoma line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHLR-CHO cells (Urlaub et al, PNAS USA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0. Lor a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu. Methods in Molecular Biology, Vol. 248 (B. K.C Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268. Certain preferred mammalian cell expression systems include CHO and HEK293-cell based expression systems. Mammalian expression systems can utilize attached cell lines, for example, in T-flasks, roller bottles, or cell factories, or suspension cultures, for example, in 1L and 5L spinners, 5L, 14L, 40L, 100L and 200L stir tank bioreactors, or 20/50L and 100/200L WAVE bioreactors, among others known in the art.

Also included are methods of cell-free protein expression. These and related embodiments typically utilize purified RNA polymerase, ribosomes, tRNA, and ribonucleotides. Such reagents can be produced, for example, by extraction from cells or from a cell-based expression system.

In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation, or the insertion of non- naturally-occurring amino acids (see generally US Patent Nos; US 7,939,496; US 7,816,320; US 7,947,473; US 7,883,866; US 7838,265; US 7,829,310; US 7,820,766; US 7,820,766; US7, 7737, 226, US 7,736,872; US 7,638,299; US 7,632,924; and US 7,230,068). Post-translational processing which cleaves a“prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition to bacterial cells, which have or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein. Also included are methods for recombinantly-producing a modified ulinastatin polypeptide, as described herein. In some embodiments, a polynucleotide encoding a modified ulinastatin polypeptide is introduced directly into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded protein(s). The polypeptide sequences of this disclosure may be prepared using standard techniques well known to those of skill in the art in combination with the polypeptide and nucleic acid sequences provided herein.

Therefore, according to certain embodiments, there is provided a recombinant host cell that comprises a polynucleotide or a fusion polynucleotide which encodes a modified ulinastatin polypeptide described herein. Expression of a modified ulinastatin polypeptide in the host cell may be achieved by culturing under appropriate conditions recombinant host cells containing the

polynucleotide. Following production by expression, the modified ulinastatin polypeptide may be isolated and/or purified using any suitable technique, and then used as desired.

The modified ulinastatin polypeptides produced by a recombinant host cell can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid

chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-performance liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration, reverse- phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. See also the Examples.

Also included is assessing or measuring the activity of the modified ulinastatin polypeptide under physiological conditions, optionally of temperature and pH, wherein the modified ulinastatin polypeptide HAS activity under the physiological conditions. In some embodiments, the modified ulinastatin polypeptide has at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more of the activity relative to an ulistatin polypeptide of SEQ ID NO: 1 (FL human ulinastatin) under comparable physiological conditions.

Certain aspects further comprise preparing a composition that comprises the modified ulinastatin polypeptide, for example, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis, and wherein the composition is substantially aggregate-free and substantially endotoxin-free.

All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

EXAMPLES

Example 1

Preparation and Testing of Modified Ulinastatin Polypeptides Certain of the modified ulinasatin polypeptides in Table U2 and Table U3 were evaluated using in silico methodology for manufacturing feasibility and were found to be generally amenable to high level recombinant expression.

Initial biochemical studies are performed to determine the ability of each modified ulinasatin polypeptide to inhibit trypsin. An in vivo screening paradigm is then employed in which the partially purified polypeptides are evaluated for the ability to prevent E. coli lipopolysaccharide (LPS)-induced sepsis in mice. Later stage studies are then performed to determine the activity profile of the modified ulinasatin polypeptides against a variety of target proteases, and also to test the polypeptides in animal models of acute pancreatitis, a disease indication of interest.

Claims

1. A modified ulinastatin polypeptide, comprising, consisting, or consisting essentially of:

(a) a variant of SEQ ID NO: 1 that has at least one at least one modification to an O- linked glycosylation and at least one ulinastatin activity;

(b) a fragment of SEQ ID NO: 1 or (a) that has at least one ulinastatin activity; or

(c) a variant of (a) or (b) that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to (a) or (b) and has at least one ulinastatin activity, excluding SEQ ID NO: 1 (wild-type ulinastatin).

2. The modified ulinastatin polypeptide of claim 1, wherein (a) comprises a substitution or deletion at the O-linked glycosylation site of residues 8-11 of SEQ ID NO: 1, optionally a substitution or deletion at residue S 10 of SEQ ID NO: 1, optionally an S 10A substitution, or wherein (b) comprises, consists, or consists essentially of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 contiguous amino acids of SEQ ID NO: l .

3. The modified ulinastatin polypeptide of claim 1 or 2, wherein (b) comprises, consists, or consists essentially of about 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-130, 30-120, 30-110, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-130, 40- 120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-130, 50-120, 50-110, 50-100, 50-90, SO SO, 50-70, 50-60, 60-130, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-130, 70-120, 70-110, 70- 100, 70-90, 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90-110, 90-100, 100-130, 100-120, or 100-110 contiguous amino acids of SEQ ID NO: 1.

4. The modified ulinastatin polypeptide of any one of claims 1-3, wherein (b) comprises, consists, or consists essentially of Domain 1 of SEQ ID NO: 1.

5. The modified ulinastatin polypeptide of any one of claims 1-3, wherein (b) comprises, consists, or consists essentially of Domain 2 of SEQ ID NO: 1.

6. The modified ulinastatin polypeptide of any one of claims 1-5, comprising, consisting, or consisting essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2 (UTIACS), which retains a substitution at position S 10 of SEQ ID NO: 2, optionally a S 10A substitution.

7. A modified ulinastatin polypeptide, comprising a ulinastatin polypeptide fused to an Fc region, to form an ulinastatin-Fc fusion polypeptide, wherein the ulinastatin-Fc fusion polypeptide has at least one ulinastatin activity.

8. The modified ulinastatin polypeptide of claim 7, wherein the ulinastatin polypeptide comprises, consists, or consists essentially of:

(a) a variant of SEQ ID NO: 1 that has at least one at least one modification to an O- linked glycosylation and at least one ulinastatin activity;

(b) SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 or (a) that has at least one ulinastatin activity; or

(c) a variant of (a) or (b) that is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to (a) or (b) and has at least one ulinastatin activity.

9. The modified ulinastatin polypeptide of claim 8, wherein (a) comprises a substitution or deletion at the O-linked glycosylation site of residues 8-11 of SEQ ID NO: 1, optionally a substitution or deletion at residue S 10 of SEQ ID NO: 1, optionally an S 10A substitution, or wherein (b) comprises, consists, or consists essentially of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 contiguous amino acids of SEQ ID NO: l .

10. The modified ulinastatin polypeptide of claim 8 or 9, wherein (b) comprises, consists, or consists essentially of SEQ ID NO: 1 or about 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20- 70, 20-60, 20-50, 20-40, 20-30, 30-130, 30-120, 30-110, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-130, 40-120, 40-110, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-130, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90- 110, 90-100, 100-130, 100-120, or 100-110 contiguous amino acids of SEQ ID NO: 1.

11. The modified ulinastatin polypeptide of any one of claims 8-10, wherein (b) comprises, consists, or consists essentially of Domain 1 of SEQ ID NO: 1.

12. The modified ulinastatin polypeptide of any one of claims 8-10, wherein (b) comprises, consists, or consists essentially of Domain 2 of SEQ ID NO: 1.

13. The modified ulinastatin polypeptide of any one of claims 8-12, wherein the ulinastatin-Fc fusion polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from Table U3, or wherein (a) comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 2, which retains a substitution at position S10 of SEQ ID NO: 2, optionally a S 10A substitution.

14. The modified ulinastatin polypeptide of any one of claims 8-13, wherein the Fc region comprises, consists, or consists essentially of CEE region, CEE region, CEE region, and/or hinge region(s) from a IgA, IgD, IgE, IgG, or IgM immunoglobulin heavy chain.

15. The modified ulinastatin polypeptide of claim 14, wherein the Fc region comprises, consists, or consists essentially of one or more of the human Fc region amino acid sequences of Table FI, including variants, fragments, homologs, orthologs, paralogs, and combinations thereof.

16. The modified ulinastatin polypeptide of any one of claims 8-15, wherein the ulinastatin-Fc fusion polypeptide comprises, consists, or consists essentially of an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a sequence selected from

Table U3.

17. The modified ulinastatin polypeptide of any one of claims 1-16, which has a specific activity of about or at least about 1000-3000 U/mg, or about or at least about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 U/mg, wherein one unit (U) is an amount of the modified ulinastatin polypeptide that inhibits the activity of 2 pg trypsin by 50%.

18. The modified ulinastatin polypeptide of any one of claims 1-17, wherein the at least one ulinastatin activity is selected from one or more of protease inhibitor activities, anti-inflammatory activities, and anti-metastatic activities.

19. The modified ulinastatin polypeptide of any one of claims 1-18, which has one or more improved biological, physical, and/or pharmacokinetic properties, relative to wild-type ulinastatin (SEQ ID NO: 1).

20. The modified ulinastatin polypeptide of claim 19, which has at least one increased ulinastatin activity relative to wild-type ulinastatin (SEQ ID NO: 1), optionally at least one increased protease inhibitor activity, anti-inflammatory activity, and/or anti-metastatic activity, optionally wherein the at least one activity is increased by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more relative to wild-type ulinastatin (SEQ ID NO: l).

21. The modified ulinastatin polypeptide of any one of claims 1-20, which has equivalent or increased therapeutic efficacy at a lower dosage relative to that of wild-type ulinastatin (SEQ ID NO: 1), optionally wherein the modified ulinastatin polypeptide has equivalent or increased therapeutic efficacy at a dosage that is about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,

35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,

5000, 6000, 7000, 8000, 9000, 10,000-fold lower than that of wild-type ulinastatin (SEQ ID NO: 1).

22. A therapeutic composition, comprising a pharmaceutically -acceptable carrier and a modified ulinastatin polypeptide of any one of claims 1-21.

23. The therapeutic composition of claim 22, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis and is substantially aggregate-free, optionally less than about 10, 9, 8, 7, 6, or 5% aggregated, and wherein the composition is substantially endotoxin-free.

24. The therapeutic composition of claim 22 or 23, wherein the composition has less than about 1 EU endotoxin/mg protein, less that about 100 ng host cell protein/mg protein, less than about 10 pg host cell DNA/mg protein, and/or greater than about 95% single peak purity by SEC-HPLC.

25. A method of treating an inflammatory disease or condition in a subject in need thereof, comprising administering to the subject a therapeutic composition of any one of claims 22-24, thereby treating the inflammatory disease or condition in the subject.

26. The method of claim 25, wherein the inflammatory disease or condition is selected from one or more of pancreatitis (e.g., acute pancreatitis, chronic pancreatitis, endoscopic retrograde cholangiopancreatography (ERCP) -induced pancreatitis), systemic inflammation, colitis, autoimmune encephalomyelitis, Stevens-Johnson syndrome, arthritis, renal failure, bums, sepsis/septic shock including severe sepsis and related pro-inflammatory/secondary conditions (e.g., organ failure), systemic inflammatory response syndrome (SIRS), toxic epidermal necrolysis (TEN), Kawasaki disease, kidney disease (e.g., acute kidney failure, chronic kidney disease), ischemic conditions (e.g., ischemia-reperfusion injury in the liver, kidney, heart, lungs, brain), lung inflammation and inflammatory lung conditions (e.g., pulmonary infection, pneumonia, including infectious interstitial pneumonia associated with mixed connective tissue disease, pulmonary fibrosis, acute respiratory distress syndrome), liver inflammation including hepatitis, anaphylaxis, post-operative or post- surgical complications (e.g., renal function, cardiac surgery, lung surgery, cognitive dysfunction, liver transplantation), lipopolysaccharide (LPS)-induced inflammation or tissue injury (e.g., lungs, liver, brain), inflammation or dysfunction secondary to diabetes (e.g., diabetes-induced cardiac

dysfunction), bum injury, heat stroke, inflammatory or neuropathic pain, acute poisoning, hyperlipidemia-associated inflammation, autoimmunity-associated inflammation, allogeneic transplant or blood transfusion-associated inflammation, neuroinflammation, and cancer-associated inflammation.

27. The method of claim 25 or 26, wherein administering the modified ulinastatin polypeptide reduces one or more of protease activity, endothelial activation/damage, proinflammatory cytokine and chemokine production/release (optionally, IL-Ib, MIP-la, MCP-1, and/or CXCL1), fibrinogen synthesis, neutrophil recruitment into organs, and/or organ injury in the subject.

28. A method of treating, ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof, comprising administering to the subject a therapeutic composition of any one of claims 22-24, thereby treating, ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof.

29. The method of claim 28, wherein the cancer is selected from one or more of melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor

(medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, thyroid cancer, and stomach cancer.

30. The method of claim 28 or 29, wherein the cancer is a metastatic cancer, optionally wherein administering the modified ulinastatin polypeptide reduces cancer cell invasion and/or angiogenesis.

31. The method of claim 30, wherein the metastatic cancer is selected from one or more of:

(a) a bladder cancer which has metastasized to the bone, liver, and/or lungs;

(b) a breast cancer which has metastasized to the bone, brain, liver, and/or lungs;

(c) a colorectal cancer which has metastasized to the liver, lungs, and/or peritoneum;

(d) a kidney cancer which has metastasized to the adrenal glands, bone, brain, liver, and/or lungs;

(e) a lung cancer which has metastasized to the adrenal glands, bone, brain, liver, and/or other lung sites; (f) a melanoma which has metastasized to the bone, brain, liver, lung, and/or skin/muscle;

(g) a ovarian cancer which has metastasized to the liver, lung, and/or peritoneum;

(h) a pancreatic cancer which has metastasized to the liver, lung, and/or peritoneum;

(ⁱ) a prostate cancer which has metastasized to the adrenal glands, bone, liver, and/or lungs;

(j) a stomach cancer which has metastasized to the liver, lung, and/or peritoneum;

(1) a thyroid cancer which has metastasized to the bone, liver, and/or lungs; and (m) a uterine cancer which has metastasized to the bone, liver, lung, vagina, and/or peritoneum.

32. A polynucleotide encoding a modified ulinastatin polypeptide of any one of claims 1- 21, or a vector comprising the polynucleotide.

33. A recombinant host cell, comprising a polynucleotide or vector of claim 32.

34. A method for recombinantly-producing a modified ulinastatin polypeptide, comprising

(a) expressing the modified ulinastatin polypeptide in a recombinant host cell of claim

34; and

(b) isolating the modified ulinastatin polypeptide from the host cell,

thereby recombinantly-producing the modified ulinastatin polypeptide.

35. The method of claim 34, further comprising measuring at least one ulinastatin activity of the modified ulinastatin polypeptide under physiological conditions, optionally of temperature, salinity, and/or pH.

36. The method of claim 36, wherein the modified ulinastatin polypeptide has at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more of the ulinastatin activity of wild-type ulinastatin (SEQ ID NO: 1) under comparable physiological conditions.

37. The method of any one of claims 34-36, further comprising preparing a therapeutic composition that comprises the modified ulinastatin polypeptide, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis or a weight-weight basis, and wherein the composition is substantially aggregate-free and substantially endotoxin-free.