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WO2015172305A1 - 抑制taci-baff复合物形成的融合蛋白及其制法和用途 - Google Patents

抑制taci-baff复合物形成的融合蛋白及其制法和用途 Download PDF

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WO2015172305A1
WO2015172305A1 PCT/CN2014/077314 CN2014077314W WO2015172305A1 WO 2015172305 A1 WO2015172305 A1 WO 2015172305A1 CN 2014077314 W CN2014077314 W CN 2014077314W WO 2015172305 A1 WO2015172305 A1 WO 2015172305A1
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Prior art keywords
fusion protein
baff
taci
positions
cell
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PCT/CN2014/077314
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English (en)
French (fr)
Inventor
蔡则玲
陈羿
吴珩
方国波
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上海康岱生物医药技术股份有限公司
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Priority to PCT/CN2014/077314 priority Critical patent/WO2015172305A1/zh
Priority to US15/310,703 priority patent/US10562954B2/en
Publication of WO2015172305A1 publication Critical patent/WO2015172305A1/zh

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Definitions

  • Fusion protein for inhibiting formation of TACI-BAFF complex preparation method and use thereof
  • the present invention relates to the field of biology and medicine, and more particularly to a fusion protein (e.g., TACI-Fc fusion protein) which inhibits the formation of a TACI-BAFF complex, and a process and use thereof.
  • a fusion protein e.g., TACI-Fc fusion protein
  • TNF and its receptor superfamily members play an important role in the body's defense, inflammatory response, immune regulation and other processes. They can act in a membrane-bound or soluble form by paracrine, autocrine, and endocrine, and when combined with the corresponding receptor, cause a range of biological effects.
  • the TNF family receptor is usually a type I transmembrane protein consisting of several extracellular regions rich in cysteine domain (CRD) and an intracellular region containing a TRAF protein joining site. In some cases, the intracellular region It also contains a death structure.
  • CCD cysteine domain
  • a TACI transmembrane activator and calcium modulator and cyclophi l in l igand -interactor belongs to the TNFR superfamily and is a type III membrane protein with 293 amino acid residues (166 extracellular functional regions, 10 transmembrane regions, intracellular) 117 functional areas, no signal peptide at the amino terminus. There are two cysteine-rich repeat regions (S33-66 and C70-C104) extracellularly, but lack the "death structure" of the TNF family compared to other TNF receptor superfamily members.
  • TACI is mainly expressed on B cells and activated T cells, and regulates T cell-independent B cell antibody responses, type switching, and B cell homeostasis, which may play a more important role in the transmission of negative signals.
  • BAFF B lymphocyte stimulator factor
  • TNF ligands Two closely related members of the superfamily are directly related to the development of B lymphocytes, T cell activation and humoral immunity, while overexpression of BAFF and APRIL may be involved in the production of autoreactive B cells and autoimmune tolerance. , affecting the immune response, leading to the occurrence of various autoimmune diseases and malignant tumors.
  • BAFF can be combined with receptor BCMA (B cel l maturation antigen) ⁇ TACI (TNFR homology transmernrane activator and calcium modulator and cyclofiolide in temtor) and BAFF-R (BAFF receptor, BR3), all three receptors Type III transmembrane proteins belong to the TNFR superfamily, while BCMA and TACI are also receptors for APRIL.
  • BCMA B cel l maturation antigen
  • TACI TNFR homology transmernrane activator and calcium modulator and cyclofiolide in temtor
  • BAFF-R BAFF receptor, BR3
  • TACI is a receptor for BAFF and APRIL, which recognizes BAFF and acts on APPIL.
  • BAFF/APRRIL heterotrimers appear to be recognized only by TACI.
  • BAFF/APPRIL plays a key role in maintaining B cell homeostasis.
  • BAFF has strong B cell chemotaxis and can induce activated B cells to secrete a large number of immunoglobulins such as IgG, IgA, IgM, etc., enhance humoral immune response, and also act as a stimulator to regulate T cell activation and Answer. In vivo, it promotes the development of T1 ⁇ B cells in the spleen to T2 ⁇ B cells as well as mature B cells. Overexpression of BAFF may involve the production of autoreactive B cells and the destruction of autoimmune tolerance, ie, changes in BAFF signaling may affect humoral immune responses, leading to autoimmune diseases or tumors.
  • the level of BAFF/APPRIL was significantly increased, and the titer of anti-dsDNA antibody was positively correlated. In patients with rheumatoid arthritis, serum BAFF/APPRIL levels are significantly elevated.
  • BAFF/APPRIL Since the overexpression of BAFF/APPRIL is closely related to autoimmune diseases, try to target them. Blocking its activity can reduce the incidence of disease, reduce the symptoms of the disease, and thus achieve the purpose of relieving and treating autoimmune diseases. Antibodies or their related receptors that block the BAFF/APPRIL signal can specifically act on target cells and are expected to become new biological agents for the treatment of autoimmune diseases.
  • a fusion protein comprising the following elements fused together: (a) a TNF receptor or an active fragment thereof; (b) a BAFF receptor or an active fragment thereof, wherein The BAFF receptor b comprises TACI, BCMA, BAFFR or a combination thereof; and (c) an antibody Fc region;
  • the fusion protein has the following functions: inhibition of formation of the TACI-BAFF complex.
  • the fusion protein retains the biological activities of the above elements (a) and (b).
  • the fusion protein further has one or more of the following functions:
  • the fusion protein has the structure represented by the following formula I or I I:
  • X is the first domain of the extracellular domain of the TNF receptor, or the extracellular domain of the TNF receptor containing the first domain (eg, first to second domains, first to third domains, and first to fourth Domain)
  • ⁇ is the full-length TACI, BCMA or BAFFR extracellular domain, or contains at least the 30-119 TACI extracellular domain;
  • Z is the Fc region of no, or optionally human, antibody;
  • any of the X, ⁇ , ⁇ are connected in a head-to-head, head-to-tail, or tail-to-tail manner.
  • the "head” refers to the terminus of a polypeptide or a fragment thereof, particularly the terminal end of a wild type polypeptide or a fragment thereof.
  • the "tail” refers to the C-terminus of a polypeptide or a fragment thereof, particularly the C-terminus of a wild-type polypeptide or a fragment thereof.
  • the peptide linker is a peptide linker of 1-20 lengths.
  • the X contains or has positions 23-76 of the TNF-R2 amino acid sequence; positions 23-118; positions 23-162; positions 23-201; and/or
  • the Y contains or has positions 30-119 of the TACI amino acid sequence; and/or the BAFF receptor BCMA amino acid sequence positions 1-54; and/or the BAFF receptor BAFFR amino acid sequence positions 1-78.
  • said Z contains or has positions 216-447 of the human ⁇ ⁇ amino acid sequence.
  • the fusion protein has the amino acid sequence of positions 1-398, 23-398 or 39-398 of SEQ ID NO.: 5.
  • a nucleic acid molecule encoding the fusion protein of any one of the first aspects of the invention is provided.
  • the nucleic acid molecule has a nucleotide sequence of positions 1-1197, 67-1197 or 115-1197 in the nucleotide sequence shown in SEQ ID NO: 4.
  • a vector comprising the nucleic acid molecule of the second aspect of the invention is provided.
  • a genetically engineered cell comprising the vector of the third aspect of the invention; or the cell genome is integrated with the second aspect of the invention Nucleic acid molecule.
  • a method of producing a fusion protein of the invention comprising the steps of:
  • the host cell of the fourth aspect of the invention is cultured under conditions suitable for expression of the fusion protein, thereby expressing the fusion protein;
  • the fusion protein is isolated or purified.
  • a pharmaceutical composition comprising the fusion protein of the invention and a pharmaceutically acceptable carrier thereof is provided.
  • a fusion protein according to the invention for the preparation of one or more compositions selected from the group consisting of: (a) inhibiting a BAFF/APPRIL signaling pathway composition; a composition for lowering serum or blood IgE, IgM concentration; (c) a composition for treating an immune disease; and (d) a composition for treating a disease associated with an increase in B cells.
  • the composition is a pharmaceutical composition.
  • the immune disease comprises: asthma, systemic lupus erythematosus, rheumatoid arthritis, and the like.
  • the B cell-associated diseases include multiple myeloma, chronic lymphocytic leukemia, macroglobulinemia, and plasma cell leukemia.
  • the composition is also used to reduce the weight of the spleen.
  • a method of (a) lowering serum or blood IgE and/or IgM concentration; (c) a composition for treating an immune disease; or (d) a method for treating a disease associated with B cell growth comprises the steps of: administering to a subject in need thereof the fusion protein of the first aspect of the invention.
  • Figure 1 shows the human TACI amino acid sequence (SEQ ID NO.: 1). Italics are labeled with two cysteine-rich regions (Cysteine-Rich Domain, CRD). Labeled with a frame is a transmembrane sequence. Underlined is the main stem sequence.
  • Figure 2 shows the human TNF-R2 amino acid sequence (SEQ ID NO.: 2).
  • the underlined sequence is the first cysteine-rich region. Labeled with a frame is a transmembrane sequence.
  • Figure 3 shows the wild type Y 1 constant region Fc amino acid sequence of human (SEQ ID NO.: 3). Chain area,
  • the CH2 and CH3 areas are shown in the figure. Since the CH1 area is not part of the Fc, it is not shown in this figure.
  • Figure 4A shows a schematic diagram of the structure of fusion proteins A and B.
  • Figure 4B shows the nucleotide sequence (SEQ ID NO.: 4) encoding the fusion protein A precursor and its encoded amino acid sequence (SEQ ID NO.: 5), wherein the elements are as follows:
  • TNF-R2 element nucleotide sequence: positions 67-228; amino acid sequence: positions 23-76, wherein the cysteine-rich region CRD1 is located at nucleotide positions 115-228; the amino acid sequence is located at 39-76.
  • TACI element nucleotide sequence: positions 229-498; amino acid sequence: positions 77-166
  • Fc region elements nucleotide sequence: positions 499-1194; amino acid sequence: positions 167-398.
  • amino acids 1-22 of the fusion protein A precursor are also derived from amino acids 1-22 of TNF-R2.
  • This amino acid sequence is a transmembrane signal peptide that is cleaved when the protein is secreted outside the cell membrane, so that the mature fusion protein A does not contain this amino acid sequence.
  • Figure 5 shows the transient expression of the fusion protein in transfected cells by Western Blot.
  • the developer is an alkaline phosphatase-labeled anti-human Fc antibody.
  • the lanes are as follows: 1. Untransfected cell culture medium; 2, l u g
  • Figure 6 shows the expression level of the fusion protein by SDS-PAGE. After the cells were grown in a 96-well plate for 13 days, 20 ul of the culture solution was taken out, subjected to SDS-PAGE separation, and protein development was carried out using Coomassie Blue. The expressed fusion proteins are each marked with an arrow.
  • Figure 7 shows a non-reduced electropherogram of fusion proteins A and B after four steps of purification.
  • Figure 8 shows the effect of fusion proteins A and B on the proliferation of RPMI 8226 cells.
  • Figure A shows the comparison of cell viability of fusion protein A/B;
  • Figure B shows the comparison of cell viability between fusion protein A and TACI-Fc (R&D).
  • Figure 9 shows in vitro binding assays of fusion proteins A and B with rhBAFF. The results indicate that the fusion protein retains the activity of binding of TACI to its ligand.
  • Figure 10 shows the plasma concentration versus time curve after a single administration of fusion proteins A and B. The results showed that the half-life of the fusion protein was prolonged and the blood concentration was significantly increased.
  • Figure 11 shows the effect of different doses of fusion proteins A and B on serum IgM concentrations in normal BALB/C mice.
  • Figure 12 shows the effect of different doses of fusion proteins A and B on spleen weight/body weight in normal BALB/C mice.
  • Figure 13 shows the effect of fusion proteins A and B on 0VA-induced serum IgE and IgM concentrations in C57/B6 asthma mice.
  • Figure 14 shows the effect of fusion protein A and B on 0VA-induced spleen weight/body weight in C57/B6 asthmatic mice.
  • the inventors have intensively studied and unexpectedly found that (a) TNF receptor or its active fragment, (b) TACI, The fusion protein of BCMA, BAFFR or its active fragment, and (c) antibody Fc region, the fusion protein obtained has extremely excellent biological activity, and can significantly reduce the serum concentration of IgE. In addition, the fusion protein has good stability and long half-life, thus contributing to the treatment of certain autoimmune diseases.
  • the present invention has been completed on this basis.
  • the inventors prepared an optimized TACI-Fc fusion protein, and the study showed that the fusion protein was biologically active and significantly reduced serum IgM concentration and spleen weight in normal Balb/c mice.
  • the fusion protein of the present invention can significantly reduce serum IgE concentration in C57/B6 asthma mice, which will lay the foundation for the preparation of TACI-Fc fusion protein and its application in disease treatment.
  • TNF-R2, TNFR I I I, hTNFR I I are used interchangeably and refer to human tumor necrosis factor I type I receptors.
  • Fc refers to an Fc fragment of a human immunoglobulin.
  • immunoglobulin Fc region refers to the immunoglobulin chain constant region, particularly the carboxy terminus of the immunoglobulin heavy chain constant region or a portion thereof, for example, the immunoglobulin Fc region may comprise two of the heavy chains CH1, CH2, CH3.
  • the Fc region of the immunoglobulin used comprises at least one immunoglobulin hinge region, one CH2 domain and one CH3 domain, preferably lacking CH1 domain.
  • Human immunoglobulins are known to have various classes, such as IgA, IgD, IgE, IgM, and IgG (including four subclasses of IgG1, IgG2, IgG3, and IgG4), and specific immunity is selected from specific immunoglobulin classes and subclasses.
  • the globin Fc region is within the purview of those skilled in the art.
  • the immunoglobulin Fc region can select a coding sequence comprising an Fc region of a human immunoglobulin IgG4 subclass, wherein one immunodexin is deleted.
  • the globulin heavy chain 1 domain (CH1) but includes the coding region of the hinge region as well as the CH2, CH3, and two domains.
  • containing includes “including”, “consisting essentially of”, “consisting essentially of”, and “Consisting of " ⁇ ”; “mainly composed of ⁇ ”
  • the fusion protein is an isolated protein that is not associated with other proteins, polypeptides or molecules, is a purified product of recombinant host cell culture or is a purified extract.
  • the invention provides a fusion protein comprising the following elements: (a) a TNF receptor or an active fragment thereof, (b)
  • BAFF receptors such as TACI, BCMA and BAFFR
  • active fragments thereof and (c) antibody Fc regions.
  • the elements e.g., between element a and element b, element b or element c
  • the linker sequence is typically a sequence that does not affect two proteins.
  • the fusion protein of the present invention not only has a longer half-life in vivo, but also more effectively inhibits the concentration of antibodies (especially IgE) associated with immune diseases in serum.
  • the fusion protein of the present invention can conveniently prepare by various known methods. These methods are, for example but not limited to, recombinant DNA methods, artificial synthesis, etc. [See Murray KM, Dahl SLAnn; Pharmacother 1997 Nov; 31 (1 1): 1335-8].
  • the gene encoding the fusion protein of the present invention has the SEQ ID NO: 4
  • the sequence shown which is particularly suitable for high expression of the fusion protein of the invention in eukaryotic cells, preferably CH0 cells, including its amino acid sequence as SEQ ID NO.: 5 full length (i.e., positions 1-398) Or an active fragment thereof, such as the polypeptide (fusion protein) shown at positions 23-398 or 39-398.
  • the nucleic acid encoding of the present invention can be conveniently prepared by a variety of known methods based on the nucleotide sequences described herein. These methods are, for example but not limited to: PCR, DNA synthesis, etc. For specific methods, see J. Sambrook, Molecular Cloning Experiment Guide.
  • the nucleic acid sequence of the present invention can be constructed by a method of segmentally synthesizing a nucleotide sequence and performing overlap extension PCR.
  • the invention also provides an expression vector comprising a sequence encoding a fusion protein of the invention and an expression control sequence operably linked thereto.
  • operably linked or “operably linked” is meant a condition in which portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.
  • Expression and cloning vectors may contain one or more screening genes, also referred to as selectable markers.
  • Typical screening gene-encoded proteins can (a) resist antibiotics, etc.; (b) compensate for auxotrophs or (c) provide critical nutrients that are not present in the culture medium.
  • DG44 cells deficient in DHFR dihydrofolate reductase-deficient cells
  • DHFR dihydrofolate reductase-deficient cells
  • Both expression and cloning vectors will typically contain one or more gene transcriptional promoters, either by prokaryotic transcriptional machinery or by eukaryotic transcriptional mechanisms. Promoters for transcription of eukaryotic cells include, but are not limited to, cytomegalovirus (CMV) promoter, retroviral promoter, simian virus 40 (SV40) pre-promoter and the like.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • the expression vector may be a commercially available vector such as, but not limited to, pIRES, pDR, pUC18 or the like which can be used for expression of a eukaryotic cell system.
  • pIRES pIRES
  • pDR pDR
  • pUC18 pUC18
  • One skilled in the art can select a suitable expression vector based on the host cell.
  • the skilled person can insert the coding sequence of the fusion protein of the present invention into a suitable restriction site by restriction enzyme cleavage and splicing according to a conventional method, and obtain the present invention.
  • Recombinant expression vector According to the restriction enzyme map of the known empty-load expression vector, the skilled person can insert the coding sequence of the fusion protein of the present invention into a suitable restriction site by restriction enzyme cleavage and splicing according to a conventional method, and obtain the present invention. Recombinant expression vector.
  • the present invention also provides a host cell expressing a fusion protein of the present invention, which comprises the coding sequence of the fusion protein of the present invention.
  • the host cell is preferably a eukaryotic cell such as, but not limited to, CH0, COS cells, 293 cells, RSF cells and the like.
  • the cell is a CH0 cell which can express the fusion protein of the present invention well, and a fusion protein having good binding activity and good stability can be obtained.
  • the invention also provides a method for producing a fusion protein of the invention using recombinant DNA, the steps of which comprise:
  • Introduction of the coding sequence into a host cell can employ various techniques known in the art such as, but not limited to, calcium phosphate precipitation, protoplast fusion, lipofection, electroporation, microinjection, reverse transcription, phage Transduction method, alkali metal ion method.
  • the cells and debris in the suspension can be removed by centrifugation and the supernatant collected. It can be identified by polyacrylamide gel electrophoresis.
  • the fusion protein obtained as described above can be purified to a substantially uniform nature, such as a single band on SDS-PAGE electrophoresis.
  • a commercially available ultrafiltration membrane can be used to separate the protein, for example, a product of Mi ll ipore, Pell icon, etc., and the expression supernatant is first concentrated.
  • the concentrate may be further purified by gel chromatography or by ion exchange chromatography.
  • anion exchange chromatography (DEAE, etc.) or cation exchange chromatography.
  • the gel matrix may be a matrix commonly used for protein purification such as polyacrylamide, dextran, polyamide, and the like.
  • the Q- or SP- group is a preferred ion exchange group.
  • the purified product may be further purified by hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). All of the above purification steps can utilize different combinations to ultimately achieve a substantially uniform protein purity.
  • the expressed fusion protein can be purified using an affinity chromatography column containing a specific antibody, receptor or ligand of the fusion protein.
  • the fusion polypeptide bound to the affinity column can be eluted by conventional methods such as high salt buffer, pH change, and the like.
  • the amino terminus or carboxy terminus of the fusion protein may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in the present invention.
  • the label may be FLAG, HA, HA1, c-Myc, 6-His or 8-His. These tags can be used to purify the fusion protein.
  • the present invention also provides a composition
  • a composition comprising an effective amount (e.g., 0.0001 to 90% by weight ; preferably 0.1 to 50% by weight ; more preferably 5 to 40% by weight) of the fusion protein of the present invention, And a pharmaceutically acceptable carrier.
  • an effective amount e.g., 0.0001 to 90% by weight ; preferably 0.1 to 50% by weight ; more preferably 5 to 40% by weight
  • a pharmaceutically acceptable carrier e.g., 0.0001 to 90% by weight ; preferably 0.1 to 50% by weight ; more preferably 5 to 40% by weight
  • the fusion proteins of the invention may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8.
  • the term "effective amount” or “effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, a substance having a reasonable benefit/risk ratio .
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise a safe and effective amount of a fusion protein of the invention together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.
  • the effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc.
  • pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.
  • severity of the disease to be treated by the patient body weight of the patient, immune status of the patient, administration Ways, etc.
  • a satisfactory effect can be obtained.
  • several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • the main advantages of the invention are:
  • the fusion protein of the invention has a long half-life; (b) The fusion protein of the invention can significantly reduce the serum concentration of IgE;
  • the fusion protein of the present invention specifically inhibits the formation of the TACI-BAFF complex, thereby terminating the activation of TACI on the membrane, thereby terminating signal transduction.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually produced according to the conditions described in the conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.
  • the fusion protein A precursor (Fig. 4) is composed of the following three partial fragments from the N-terminus to the C-terminus, and the specific amino acid sequence is as follows:
  • the TNF-R2 amino acid sequence is from positions 1 to 76 and contains the first cysteine-rich region ( Figure 2).
  • the TACI amino acid sequence is from position 30 to 119 and contains two cysteine-rich regions and part of the main stem sequence (Fig. 1).
  • the PCR template is a human cDNA prepared by a conventional method. 5' primer
  • AAGCTTGCGGCCGCGAGCTCGGATCCACT (SEQ ID NO.: 6) is the sequence of the plasmid vector. In order to clone into the pT vector, a Not I restriction site was introduced at the 5' end.
  • the primer sequence at the 3' end is GGGGCAGGA TCTCATAGCGTCACACACGGTGTCCGAG (SEQ ID NO.: 7), which contains 19 TACI ribonucleotides for attachment to the TACI cDNA.
  • the purified cDNAs of the two fragments were mixed as a template, and the polymerase was carried out using the primer of the 5' end of the fragment 1 and the primer of the 3' end of the fragment 2/3. Chain reaction.
  • the amplified PCR fragment was purified and cloned into the vector pCR-Blunt II-T0P0 (purchased from Invitrogen).
  • the recombinant plasmid was transfected into competent bacteria XL-1 (purchased from Invitrogen) bacteria, positive colonies were selected, and the recombinant plasmid was purified. The correctness of the recombinant gene was identified by enzyme digestion and sequencing.
  • the recombinant plasmid is cloned into a mammalian cell expression vector.
  • the commercially available expression vector used contains a human CMV promoter and contains a DHFR expression gene, which can be used to screen a stable cell line expressing the fusion protein and amplifying the expression gene copy number.
  • the recombinant plasmid and the expression vector were digested with Notl and Xbal, and the cut recombinant gene and the double-digested vector were purified by gel purification, and then transfected into competent bacteria DH50 (purchased from Invitrogen) in vitro, and positive clones were selected. , Amplification and purification of the TACI-Fc expression plasmid.
  • Example 2 Amplification and purification of the TACI-Fc expression plasmid.
  • the fusion protein B precursor (Fig. 4) is composed of the following three parts from the N-terminus to the C-terminus.
  • the specific amino acid sequence is as follows: Fragment 1. Human CD33 protein signal peptide, containing the first 16 amino acids of the N-terminus.
  • the TACI amino acid sequence from 30 to 119 contains two cysteine-rich regions and part of the main stem sequence (Fig. 1).
  • Fragment 3 The amino acid sequence of human IgG1, from 216 to 447, contains a hinge region and two CH regions (Fig. 3).
  • the recombinant gene encoding fusion protein B was constructed by overlapping polymerase chain reaction (PCR).
  • the template for the PCR reaction is human cDNA prepared by a conventional method.
  • the polymerase chain reaction (PCR) was carried out using Invi trogen's Plantium SuperMix according to the manufacturer's instructions.
  • Each PCR fragment was purified using a kit of Qiagen's gel-purified DNA fragments. Since TACI itself does not have a protein secretion signal peptide, this embodiment employs a secretion signal peptide of human CD33 protein when constructing an expression vector.
  • the PCR template is a human cDNA prepared by a conventional method.
  • the primer at the 5' end is
  • the first 16 nucleotide sequences are identical to the 3' end of the nucleotide sequence encoded by CD33, and are used to link the PCR fragments of fragments 1 and 2.
  • the primer sequence at the 3 ' end was TGTMCMGATTTGGGTTC CCTGAGCTCTGGTGGAAG (SEQ ID NO.: 9).
  • the PCR template is a human cDNA prepared by a conventional method.
  • the amplified DNA sequence encodes the human Y l immunoglobulin Fc amino acid sequence from 216 to 447.
  • the primer at the 5' end is GAACCCAAATCTTGTTACA (SEQ ID NO.: 10), which is complementary to the 3 'end sequence of the fragment 2 cDNA.
  • the primer sequence at the 3' end was TGGTGGTGTCTAGAGACTCATTTACCCGGAGACAGGGAGAGGC (SEQ ID N0.: 11).
  • the iiT xba I restriction site was introduced at the 3' end.
  • the purified three fragments were mixed as a template, using the primer at the 5' end of fragment 1, AGGTATAGCGGCCGCCACCATGCCGCTGC (SEQ ID NO.: 13) and the primer at the 3' end of fragment 3 (ibid., SEQ ID N0.: 11) to carry out polymerase chain reaction.
  • the amplified PCR fragment was purified and cloned into the vector pCR-Blunt I I-T0P0 (purchased from Invi trogen).
  • the recombinant plasmid was transfected into competent bacteria XL-1 (purchased from Invi trogen), positive colonies were selected, and the recombinant plasmid was purified.
  • the correctness of the recombinant gene was identified by restriction enzyme digestion and sequencing.
  • the recombinant plasmid is cloned into a mammalian cell expression vector.
  • the recombinant plasmid expression vector was digested with Notl and Xbal, and the excised recombinant gene and the double-digested expression vector were purified by gel purification, and then transfected into competent bacteria DH50 (Invi trogen) after in vitro linkage. Positive clones were selected and the TACI-Fc expression plasmid was amplified and purified.
  • CH0 DG44 cell line a stable transfection and gene amplification method was used to establish a stable and highly efficient protein expression of the CH0 DG44 cell line.
  • the cloned CH0 DG44 cells were cultured in suspension in serum-free, animal protein-free medium.
  • the original CH0 DG44 cells were obtained from Invi trogen, and the methods of cell culture and passage were referred to the company's operating manual.
  • the non-transfected cells were cultured in CD DG44 culture medium containing 8 mM Glutamine and 0.98% Fluronic F-18.
  • a large number of expression plasmids encoding fusion proteins were prepared using Qiagen's kit. In order to prevent the plasmid from being contaminated by bacteria, the plasmid was purified by alcohol precipitation.
  • CD DG44 cells Prior to gene transfection, CD DG44 cells were transfected for three generations. When the density of the cells reached l X 10 6 /ml, pipette 1 ml and transfer to a 12-well culture plate at 1 ml per well.
  • Transfection was performed using Invitrogen's transfection reagent LIPOFECTAMINE 2000 CD. In order to find the highest transfection efficiency, the ratio of transfection reagents for each plasmid using three different plasmids
  • the screening medium was a CH0 medium containing no Hypothanxine and Thymidine, containing 20 nM or ⁇ of methotrexate (MTX). Two weeks later, the cell culture medium of each well was analyzed by ELISA, and the content of the fusion protein was detected and the protein in the culture solution was analyzed by SDS-PAGE.
  • the cell line with high expression of the fusion protein was transferred to a 24-well culture plate, and after 7 days of culture, the ELISA protein content was analyzed. From each fusion protein pool, three highly expressed cells were selected for amplification and cryopreservation.
  • Fusion protein A, B cell lines as seed cells, using the initial density of 0. 5 X 10 6 cells / ml to start cultivation medium was S4 (containing 8g / L glucose, 4mmol / L glutamine and 0. 5mg / L. Insulin), pH is controlled at 6. 7-7. 1, dissolved oxygen is not less than 30%, initial temperature is 35 ° C, cell density is as long as 6. 5-7.
  • the CM cation chromatography the purity of the target protein reached 88.4%, the yield was 8.7 6%, the yield of the target protein was 87. 4%, 3% ⁇
  • the third step of Phenyl-650M hydrophobic chromatography the target protein purity of 94. 5%, the yield of 78.0%; the last step of ultrafiltration concentrated liquid change: the target protein purity reached 95. 3% , 9% ⁇ The yield was 92.9%.
  • the in vitro biological activity of the fusion protein TACI-Fc is mainly manifested by the inhibition of the growth of human myeloma cell line RPMI 8226 (purchased from ATCC) in the presence of dexamethasone and rhBAFF.
  • the role of dexamethasone is to induce apoptosis in RPMI 8226 cells, reducing the background of the experiment.
  • BAFF can restore and promote cell growth. Adding TACI-Fc and BAFF on the basis of this can attenuate or even eliminate the cell growth of BAFF. enhancement.
  • the in vitro biological activity of TACI-Fc can be detected by this method.
  • the reaction was carried out in a 96-well cell culture plate, and 10,000 RPMI 8226 cells were seeded per well in 150 ⁇ l of the culture medium, including 0.1 ⁇ M dexamethasone (Wako, C0DE #041- 18861) and 1 ⁇ g/ml rhBAFF (R&D, Cat#2149-BF/CF), the final concentration of the sample to be tested was 4-fold gradient dilution from the start of 16 ⁇ g/ml, and a set of experiments without rhBAFF was used as a control.
  • the fusion protein A/B in vitro binding assay mainly uses the ELISA principle: a certain amount of rhBAFF is immobilized on the microplate, and each well is added with a series of concentration gradient fusion protein A/B for binding reaction, and finally by HRP-conjugated anti-human Fc detects the amount of fusion protein bound to the plate rhBAFF to examine its binding to rhBAFF.
  • the soluble fragment of recombinant human BAFF was diluted to 0.5 g/ml with PBS (pH 7.4) and added to the microplate (greiner bio-one, Cat # 655001). ), 50 ⁇ l/well, 4° C coated overnight. After washing the next day with ultrapure water, it was blocked with 5% skim milk powder/PBST. The sample to be tested was then started from 3 g/ml, serially diluted 3 times, added to the plate, 50 ⁇ l/well, and incubated for 2 hours at room temperature. After washing 3 times with PBST, 50 ⁇ ⁇ 0. 8 g/ml detection antibody was added to each well.
  • the logarithm of the absorbance value of the sample to be measured is plotted, and the S-curve fit is performed by Origin software to calculate EC 5 .
  • the concentration corresponding to 50% change in absorption value represents the in vitro binding activity of the sample to be tested.
  • the relationship between the plasma concentration and the administration time was determined. Choose With normal female BALB/C mice (18-20 g) 48, 3/time points, a total of 7 time points were established, namely 1, 2, 4, 8, 24, 48, 72 h. 5 mg/kg of fusion protein A or fusion protein B was intraperitoneally injected, and the administration time of each group was accurately recorded. Then, the mice were subjected to ether anesthesia at each time point, and then the eyeballs were taken for blood collection, and serum was collected. The blood concentration of the two drugs at each time point was measured by ELISA.
  • the blood drug concentration of the two drugs was similar to the time curve, but the blood concentration of the fusion protein A at different time points was significantly higher than that of the fusion protein B, which was about 2 times.
  • the half-lives of fusion protein A and fusion protein B in mice were 14h and l lh, respectively.
  • mice Normal female BALB/C mice (18 ⁇ 20g) were used, 42 rats, 6 rats/group, divided into 7 groups, namely control (control), fusion protein A-5mg/kg, fusion protein A-2. 5mg/kg , fusion protein A-1. 25mg/kg), fusion protein B-5mg/kg, fusion protein B-2. 5mg/kg, fusion protein B-1. 25mg/kg group.
  • spleen weight/body weight was significantly reduced on day 14 after administration of different doses of fusion protein A and fusion protein B (5 mg/kg, 2. 5 mg/kg, 1. 25 mg/kg), of which fusion protein A (5mg/kg, 2.5 mg/kg, 1. 25mg/kg), fusion protein B (5mg/kg, 2. 5mg/kg) group was significantly different from the control
  • mice 44 normal female C57/B6 mice (18-20 g) were selected and divided into 7 groups, namely the control group ( 6), model group (11), fusion protein A-5mg/kg (13), fusion protein B_5mg/kg (14) were studied.
  • the model group and the experimental group mice were sensitized by intraperitoneal injection of 0VA/ALUM suspension on days 1 and 14, respectively, and PBS, different doses of fusion protein A and fusion protein B were injected intraperitoneally on the 13th day. The drug was administered every other day for a total of 12 doses.
  • mice were weighed first, and the blood was taken from the fundus venous plexus after ether anesthesia. After the sacrifice, the mouse spleens were dissected, the surrounding tissues were removed, and the cells were slightly blotted and weighed. . The alveolar lavage fluid was drawn and the total number of white blood cells was calculated. The concentration of IgE and IgM in the serum of each group of mice was detected by ELISA.
  • the serum IgM concentration of the model group on day 36 was significantly higher than that of the control. Fusion protein A-5mg/kg and fusion protein B-5mg/kg were significantly lower than the model group IgM concentration (**p ⁇ 0.01).
  • the concentration of serum IgE in the model group, fusion protein A-5mg/kg, and fusion protein B_5mg/kg on day 36 increased to different degrees compared with the control, in which fusion protein A group and model group IgE There was a significant difference in concentration comparison (**P ⁇ 0.01).
  • fusion protein A significantly reduced the concentration of IgE compared to fusion protein B, suggesting that fusion protein A is more effective in inhibiting allergic reactions.
  • fusion protein A 40 mg/ml
  • fusion protein A pharmaceutical composition including aggregation and degradation was investigated under liquid accelerated test conditions.
  • the stability of the sample was analyzed by SEC-HPLC and SDS-PAGE methods to finally determine the pharmaceutical composition of fusion protein A.
  • buffer solution 10 mM acetic acid buffer solution (pH 5.0), composition: 80 mg/mL trehalose;
  • buffer solution 10 mM succinic acid buffer solution (pH 5.0), composition: 80 mg/mL sucrose;
  • D) buffer solution 10 mM acetic acid buffer solution (pH 5.0), composition: 80 mg / mL trehalose, 0.05 mg / mL polyhydroxyl 188;
  • buffer solution 10 mM succinic acid buffer solution (pH 5.0), composition: 80 mg / mL trehalose, 0. 05mg / mL Tween 20;
  • Buffer solution 10 mM succinic acid buffer solution (pH 5.0), composition: 80 mg/mL trehalose, 0.05 mg/mL polyhydroxyl 188.
  • the six groups of pharmaceutical compositions were stored at 4 ° C, 25 ° C, and 40 ° C for 2 weeks.
  • the stability results showed that the drug composition did not aggregate and degrade at 2 ° C for 2 weeks. No degradation fragments were produced at 25 °C and 40 °C, but the aggregates increased.
  • the specific results are shown in Table 1.
  • the fusion protein A has a relatively high stability under the conditions of the pharmaceutical compositions A and D, and is stable at 4 ° C and 25 ° C, but for the 40 ° C environment, the combination Site D has a relatively higher tendency to aggregate than Composition A.
  • a particularly preferred pharmaceutical composition consists of 40 mg/mL fused egg SA, 80 mg/mL trehalose, 10 mM acetic acid buffer solution (pH 5.0). discuss
  • TNFR tumor necrosis factor receptor
  • CFD cysteine-rich domains
  • TRAIL receptor 1 CD40, 60 kDa TNFR, and 80 kDa TNFR
  • PLAD PLAD is necessary and sufficient for the formation of acceptor polymers.
  • PLAD is not directly involved in the recognition and binding of TNF ⁇ and TNF ⁇
  • deletion of PLAD or PLAD mutations results in loss of ligand-receptor affinity. Therefore, PLAD plays a crucial role in the formation of TNFR multimers and the binding of ligands in TNF-related signaling pathways.
  • the fusion protein of the present invention can effectively block the PLAD region of TACI, thereby specifically inhibiting the polymerization of TACIR, thereby terminating the formation of the TACI-BAFF complex and achieving the purpose of terminating signal transduction.
  • test results show that the TACI-FC fusion protein of the present invention prolongs the half-life of the drug, significantly lowers the concentration of IgE in the serum, and effectively inhibits the formation of the TACI-BAFF complex, thereby terminating signal transduction.

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Abstract

本发明提供了抑制TACI-BAFF复合物形成的融合蛋白及其制法和用途。具体地，本发明的融合蛋白具有封闭BAFF/APPRIL的高生物活性，可明显降低正常Balb/c小鼠血清IgM浓度，以及C57/B6哮喘小鼠血清IgM和IgE浓度。本发明的TACI-Fc融合蛋白可用于治疗自身免疫性疾病，包括哮喘、系统性红斑狼疮、类风湿性关节炎等，还可用于治疗B细胞增多相关疾病，包括多发性骨髓瘤，慢性淋巴细胞白血病，巨球蛋白血症，浆细胞性白血病等。

Description

抑制 TACI-BAFF复合物形成的融合蛋白及其制法和用途 技术领域
本发明涉及生物和药物领域， 更具体地涉及抑制 TACI-BAFF复合物形成的融合 蛋白（如 TACI-Fc融合蛋白）及其制法和用途。 背景技术
TNF及其受体超家族成员在机体的防御、炎症反应、 免疫调节等过程中起着重 要作用。 它们能通过旁分泌、 自分泌及内分泌， 以膜结合型或可溶性形式而发挥 作用， 当其与相应的受体结合后， 可引起一系列的生物效应。
TNF家族受体通常是 I型跨膜蛋白，由若干富含半胱氨酸结构域 (CRD)的胞外区 和包含 TRAF蛋白连接位点的胞内区组成， 胞内区在某些情况下还含有死亡结构。
A TACI (transmembrane activator and calcium modulator and cyclophi l in l igand -interactor)属 TNFR超家族， 是有 293个氨基酸残基的 III型膜蛋白（胞外 功能区 166个， 穿膜区 10个， 胞内功能区 117 个）， 氨基端无信号肽。 胞外有 2 个富含半胱氨酸的重复区 （S33-66和 C70-C104) , 但与其它 TNF受体超家族成员 相比缺少 TNF家族的 "死亡结构" 。 TACI主要在 B细胞和活化的 T细胞上表达， 调控 T细胞非依赖性的 B细胞抗体反应、 类型转换以及 B细胞的稳态， 可能在传 导负性信号方面具有更重要的作用。
B淋巴细胞剌激因子（B lymphocyte stimulator factor, BAFF, 或称 BLyS、
THANK, zTNF-4, TALL- 1及 TNFSF- 13B)和增殖诱导配体（A prol iferation-inducing factor, APRIL, 或称 TALL-2、 TRDL-1及 TNFSF-13)是新近发现的 TNF配体超家族 两个关系密切的成员， 与 B淋巴细胞的发育、 T细胞活化及体液免疫直接相关， 而 BAFF与 APRIL的过度表达又有可能参与自身反应性 B细胞的产生和自身免疫耐受 ^破坏， 影响免疫应答， 导致各种自身免疫性疾病和恶性肿瘤的发生。 BAFF可与 受体 BCMA (B cel l maturation antigen) ^ TACI (TNFR homology transmernrane activator and calcium modulator and cyclophi l in l igand intemtor)禾口 BAFF-R (BAFF receptor, BR3)结合， 3个受体均为 III型跨膜蛋白， 属于 TNFR超家 族， 而 BCMA与 TACI同时也是 APRIL的受体。
TACI为 BAFF和 APRIL的受体， 即能识别 BAFF, 又能作用于 APPIL。 此外，
BAFF/APRRIL异源三聚体似乎只能被 TACI所识别。
BAFF/APPRIL作为重要的免疫调节分子， 在维持 B细胞动态平衡中发挥关键作 用。
BAFF具有很强的 B细胞趋化性，能诱导活化的 B细胞分泌大量的免疫球蛋白如 IgG、 IgA、 IgM等， 增强体液免疫应答， 而且也可作为共剌激因子调节 T细胞的 活化与应答。 在体内， 它可以促进脾脏内 T1〜B细胞向 T2〜B细胞以及成熟 B细 胞的发育。 BAFF的过度表达有可能涉及自身反应性 B细胞的产生和自身免疫耐受 的破坏， 即 BAFF信号的改变会影响体液免疫应答， 导致自身免疫性疾病或肿瘤。
研究显示， 在自身免疫疾病患者体内如系统性红斑狼疮病人血清中
BAFF/APPRIL水平明显升高， 其抗 -dsDNA抗体的滴度呈正相关。 在类风湿性关节 炎病人， 血清 BAFF/APPRIL水平明显升高。
由于 BAFF/APPRIL得过量表达与自身免疫性疾病密切相关， 以其为靶分子设法 阻断其活性， 可以减少疾病的发生， 减轻发病的症状， 从而达到缓解和治疗自身 免疫疾病的目的。 封闭 BAFF/APPRIL信号的抗体或其相关受体可以特异地作用于 靶细胞， 有望成为治疗自身免疫疾病的新的生物制剂。
因此， 本领域迫切需要开发新的、 可有效抑制或封闭 BAFF/APPRIL途径的化合 物。 发明内容
本发明的目的就是提供一种新的可有效抑制或封闭 BAFF/APPRIL途径的化合 物。 在本发明的第一方面， 提供了一种融合蛋白， 所述融合蛋白包括融合在一起的 以下元件： （a) TNF受体或其活性片段； （b) BAFF受体或其活性片段， 其中所述的 BAFF受体 b包括 TACI , BCMA, BAFFR或其组合； 和（c) 抗体 Fc区域；
并且所述融合蛋白具有以下功能： 抑制 TACI-BAFF复合物的形成。
在另一优选例中， 所述的融合蛋白保留了上述元件 (a)和 (b)的生物活性。
在另一优选例中， 所述的融合蛋白还具有以下一种或多种功能：
(a) 结合于 BAFF的活性；
(b) 结合于 TALL-2/APRIL的活性；
(c) 降低 IgE的血清浓度；
(d) 降低血清免疫球蛋白的浓度如 IgM等；
(e) 降低脾脏重量；
(f) 抑制或封闭 BAFF/APPRIL途径；
(g) 减少 B细胞数量。 在另一优选例中， 所述的融合蛋白具有下式 I或 I I 所示的结构：
X-Y-Z Y-X-Z
式 I 式 I I
X为 TNF受体胞外区的第一结构域、或含第一结构域的 TNF受体胞外区（如第一 至第二结构域、 第一至第三结构域和第一至第四结构域）；
Υ为全长的 TACI、BCMA或 BAFFR胞外区，或至少含有 30-119位的 TACI胞外区； Z为无、 或任选的人抗体的 Fc区域；
-表示肽键或肽接头。
在另一优选例中， 所述的 X、 Υ、 Ζ中的任何两者以头 -头、 头 -尾、 或尾 -尾方 式相连。
在另一优选例中， 所述的 "头部"指多肽或其片段的 Ν端， 尤其是野生型多肽 的或其片段的 Ν端。
在另一优选例中， 所述的 "尾部"指多肽或其片段的 C端， 尤其是野生型多肽 的或其片段的 C端。
在另一优选例中， 所述的肽接头是 1-20个长度的肽接头。
所述的 X含有或具有 TNF-R2氨基酸序列的第 23-76位;第 23-118位；第 23-162 位； 第 23-201位； 和 /或 所述的 Y含有或具有 TACI氨基酸序列的第 30-119位； 和 /或 BAFF受体 BCMA 氨基酸序列第 1-54位；和 /或 BAFF受体 BAFFR氨基酸序列第 1-78位。
在另一优选例中， 所述的 Z含有或具有人 γΐ氨基酸序列的第 216-447位。 在另一优选例中，所述的融合蛋白具有 SEQ ID NO.： 5中第 1-398位，第 23-398 位或第 39-398位的氨基酸序列。 在本发明的第二方面， 提供了一种核酸分子， 所述的核酸分子编码本发明第一 方面中任一所述的融合蛋白。
在另一优选例中， 所述的核酸分子具有 SEQ ID NO : 4所示的核苷酸序列中第 1-1197位、 第 67-1197位或第 115-1197位的核苷酸序列。 在本发明的第三方面， 提供了一种载体， 它含有本发明第二方面所述的核酸分 子。 在本发明的第四方面， 提供了一种基因工程化的细胞， 所述的细胞含有本发明 第三方面所述的载体； 或所述的细胞基因组中整合有本发明第二方面中所述的核 酸分子。 在本发明的第五方面， 提供了一种产生本发明所述的融合蛋白的方法， 所述的 方法包括步骤：
在适合表达所述融合蛋白的条件下， 培养本发明第四方面所述的宿主细胞， 从 而表达所述的融合蛋白； 和
分离或纯化所述的融合蛋白。 在本发明的第六方面， 提供了一种药物组合物， 所述的药物组合物含有本发明 所述的融合蛋白及其药物学上可接受的载体。
在本发明的第七方面， 提供了本发明所述的融合蛋白的用途， 用于制备选自下 组的一种或多种组合物： （a) 抑制 BAFF/APPRIL信号通路组合物； （b)降低血清或 血液 IgE、 IgM浓度的组合物； （c)治疗免疫疾病的组合物； 以及（d)治疗 B细胞增 多相关疾病的组合物。
在另一优选例中， 所述的组合物为药物组合物。
在另一优选例中， 所述的免疫疾病包括： 哮喘、 系统性红斑狼疮、 类风湿性关 节炎等。
在另一优选例中， 所述的 B细胞增多相关疾病包括多发性骨髓瘤、 慢性淋巴细 胞白血病、 巨球蛋白血症和浆细胞性白血病
在另一优选例中， 所述的组合物还用于降低脾脏重量。 在本发明的第八方面，提供了一种（a)降低血清或血液 IgE和 /或 IgM浓度； （c) 治疗免疫疾病的组合物； 或（d)治疗 B细胞增多相关疾病的方法， 所述的方法包括 步骤： 给需要的对象施用本发明第一方面所述的融合蛋白。 应理解， 在本发明范围内中， 本发明的上述各技术特征和在下文 (如实施例）中 具体描述的各技术特征之间都可以互相组合， 从而构成新的或优选的技术方案。 限于篇幅， 在此不再一一累述。 附图说明
图 1显示了人的 TACI氨基酸序列（SEQ ID NO.： 1)。 斜体标记的是二个富含半 胱氨酸区域 (Cysteine-Rich Domain, CRD)。 用框标记的是穿膜序列。 下划线标记 的是主茎序列。
图 2显示了人的 TNF-R2氨基酸序列（SEQ ID NO.： 2)。 下划线的序列是第一个 富半胱氨酸区。 用框标记的是穿膜序列。
图 3显示了人的野生型 Y 1不变区 Fc氨基酸序列（SEQ ID NO.： 3)。 绞链区，
CH2和 CH3区如图所示。 由于 CH1区不是 Fc的一部分， 故没有显示在此图。
图 4A显示了融合蛋白 A和 B的结构示意图。
图 4B显示了编码融合蛋白 A前体的核苷酸序列（SEQ ID NO.： 4)和其编码的氨 基酸序列（SEQ ID NO.： 5)， 其中各元件如下：
TNF-R2元件： 核苷酸序列： 第 67-228位；氨基酸序列：第 23-76位， 其中， 富 含半胱氨酸区域 CRD1位于核苷酸序列第 115-228位;氨基酸序列位于第 39-76位。
TACI元件： 核苷酸序列： 第 229-498位；氨基酸序列：第 77-166位
Fc区元件： 核苷酸序列： 第 499-1194位；氨基酸序列：第 167-398位。
此外， 融合蛋白 A前体的第 1-22位氨基酸也来自 TNF-R2的第 1-22位氨基酸。 这一段氨基酸序列是穿膜信号肽， 在蛋白穿过细胞膜分泌到细胞外时被切掉， 所 以成熟的融合蛋白 A不含此段氨基酸序列。
图 5显示了 Western Blot 检测融合蛋白在转染细胞里的瞬间表达。 显色剂是 碱性磷酸酶标记的抗人的 Fc抗体。各泳道如下： 1， 没转染的细胞培养液； 2, l u g
DNA质粒的转染； 3， 2 g DNA质粒的转染； 4， 4 g DNA质粒的转染。
图 6显示了 SDS-PAGE分析融合蛋白的表达量。 细胞在 96孔板里生长 13天后， 取出 20 ul培养液， 进行 SDS-PAGE分离， 用 Coomassie Blue进行蛋白显色。 表 达的融合蛋白分别用箭头标记。
图 7显示了经四步分离纯化后融合蛋白 A和 B的非还原电泳图。
图 8显示了融合蛋白 A和 B对 RPMI 8226细胞增殖的影响。 其中图 A为融合蛋 白 A/B的细胞活性比较； 图 B为融合蛋白 A与 TACI-Fc (R&D)的细胞活性比较。
图 9显示了融合蛋白 A和 B与 rhBAFF的体外结合分析。 结果表明， 融合蛋白 保留了 TACI与其配体结合的活性。
图 10显示了单次给予融合蛋白 A和 B后血药浓度与时间曲线。 结果表明， 融 合蛋白的半衰期延长， 血药浓度显著提高。
图 11显示了不同剂量融合蛋白 A和 B对正常 BALB/C小鼠血清 IgM浓度的影响。 图 12显示了不同剂量融合蛋白 A和 B对正常 BALB/C小鼠脾重 /体重的影响。 图 13显示了融合蛋白 A和 B对 0VA诱导的 C57/B6哮喘小鼠血清 IgE和 IgM浓 度影响。
图 14显示了融合蛋白 A禾卩 B对 0VA诱导的 C57/B6哮喘小鼠脾重 /体重的影响。 具体实施方式
本发明人经过深入的研究，意外地发现，将 (a) TNF受体或其活性片段、 (b) TACI , BCMA, BAFFR或其活性片段、 和（c) 抗体 Fc区域相融合， 获得的融合蛋白具有极 其优异的生物活性， 并且可以非常显著地降低 IgE的血清浓度。 此外， 该融合蛋 白且稳定性好、 半衰期长， 因此有助于治疗某些自身免疫疾病。 在此基础上完成 了本发明。
具体地， 本发明人制备了优化的 TACI-Fc融合蛋白， 研究表明， 融合蛋白具有 生物活性强且明显降低正常 Balb/c小鼠血清 IgM浓度和脾脏重量。 此外， 本发明 融合蛋白可显著降低 C57/B6哮喘小鼠血清 IgE浓度， 这将为 TACI-Fc融合蛋白的 制备以及在疾病治疗方面的应用奠定基础。 如本文所用， 除非另外说明， TNF-R2 , TNFR I I， hTNFR I I可互换使用， 都指 人肿瘤坏死因子 I I型受体。
如本文所用， 除非另外说明， Fc是指人免疫球蛋白的 Fc片段。 术语 "免疫球 蛋白 Fc区"指免疫球蛋白链恒定区， 特别是免疫球蛋白重链恒定区的羧基端或其 中的一部分， 例如免疫球蛋白 Fc区可包括重链 CH1、 CH2、 CH3的两个或更多结构 域与免疫球蛋白铰链区的组合， 在优选例中， 所用的免疫球蛋白的 Fc区包括至少 一个免疫球蛋白绞链区， 一个 CH2结构域和一个 CH3结构域， 优选缺少 CH1结构 域。
已知人免疫球蛋白有多种类别，如 IgA、 IgD、 IgE、 IgM及 IgG (包括 IgGl、 IgG2、 IgG3、 IgG4四个亚类）， 从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白 Fc区是在本领域技术人员所掌握的范围之内的， 在一个优选的实例中， 免疫球蛋 白 Fc区可选择包含有人免疫球蛋白 IgG4亚类 Fc区的编码序列， 其中缺失一个免 疫球蛋白重链 1结构域 (CH1)， 但包括了铰链区以及 CH2、 CH3、 二个结构域的编码 序列。
如本文所用， 所述的 "含有" ， "具有"或 "包括"包括了 "包含" 、 "主要 由 ··· ···构成"、 "基本上由 ··· ···构成"、和 "由 ··· ···构成"； "主要由 ··· ···构成"、
"基本上由……构成"和 "由……构成"属于 "含有" 、 "具有"或 "包括" 的 下位概念。
如本文所用， 除非另外说明， 所述的融合蛋白是一种分离的蛋白， 与其它蛋白、 多肽或分子无联系， 是重组宿主细胞培养的纯化产物或作为一种纯化的提取物。
本发明提供了一种融合蛋白， 包含以下元件： （a) TNF受体或其活性片段、 （b)
BAFF受体（如 TACI， BCMA和 BAFFR)或其活性片段、 和（c) 抗体 Fc区域。 本发明 所述的融合蛋白中，所述的各元件之间（如元件 a与元件 b、元件 b或元件 c之间）， 可以含有或不含有连接序列。 所述的连接序列通常是对两个蛋白不产生影响作用 的序列。
本发明的融合蛋白， 不仅具有更长的体内半衰期， 可以更有效地抑制血清中免 疫疾病相关的抗体 (尤其是 IgE)的浓度。
根据本发明提供的氨基酸序列， 本技术领域人员可方便地用各种已知方法制得 本发明的融合蛋白。这些方法例如但不限于：重组 DNA法，人工合成，等 [参见 Murray KM, Dahl SLAnn; Pharmacother 1997 Nov; 31 (1 1) : 1335- 8]。
在得知了本发明的融合蛋白的氨基酸序列后， 本领域人员可以方便地根据所述 的氨基酸序列获得编码本发明的融合蛋白的基因序列。
作为本发明的优选方式， 本发明的融合蛋白的编码基因具有 SEQ ID NO : 4所 示的序列， 采用该序列， 特别适合于在真核细胞 (优选 CH0细胞）中高表达本发明 的融合蛋白， 其中包括其氨基酸序列如 SEQ ID NO.： 5全长（即第 1-398位)或其 活性片段， 例如第 23-398位或第 39-398位所示的多肽 (融合蛋白）。
根据本文所述的核苷酸序列， 本技术领域人员可方便地用各种已知方法制得本 发明的编码核酸。 这些方法例如但不限于： PCR， DNA人工合成等， 具体的方法可 参见 J. 萨姆布鲁克， 《分子克隆实验指南》 。 作为本发明的一种实施方式， 可通 过分段合成核苷酸序列再进行重叠延伸 PCR的方法来构建本发明的编码核酸序列。
本发明还提供了一种表达载体， 包含编码本发明的融合蛋白的序列以及与之操 作性相连的表达调控序列。 所述的 "操作性相连"或 "可操作地连于"指这样一 种状况， 即线性 DNA序列的某些部分能够调节或控制同一线性 DNA序列其它部分 的活性。 例如， 如果启动子控制序列的转录， 那么它就是可操作地连于编码序列。
表达和克隆载体可含有一个或多个筛选基因， 也称作可筛选标记。 典型的筛选 基因编码蛋白可以 (a)抵抗抗菌素等； （b)补偿营养缺陷或 (c)提供关键的在培养基 中没有的营养物质。 例如， DHFR (二氢叶酸还原酶缺陷细胞)缺陷的 DG44细胞不能 生长在不含次黄嘌呤-胸腺嘧啶的培养基里生长， 在细胞被可表达 DHFR的载体转 染后， 转染的细胞不仅可生长在不含含次黄嘌呤-胸腺嘧啶的培养基里生长， 还可 生长在含有一定量的 MTX (氨甲喋呤)培养基里生长。
表达载体和克隆载体通常都会含有一个或多个基因转录启动子， 或者被原核细 胞转录机制识别， 或者被真核细胞转录机制识别。 用于真核细胞转录的启动子有 但不限于巨细胞病毒（CMV)启动子， 反转录病毒启动子， 猴病毒 40 (SV40)前期启动 子等。
表达载体可采用市售的例如但不限于： pIRES、 pDR， pUC18等可用于真核细胞 系统表达的载体。 本领域技术人员可以根据宿主细胞来选择合适的表达载体。
根据已知空载表达载体的酶切图谱， 本领域技术人员可按照常规方法通过限 制性酶剪切与拼接， 将本发明的融合蛋白的编码序列插入合适的限制性位点， 制 得本发明的重组表达载体。
本发明还提供了表达本发明融合蛋白的宿主细胞， 其中含有本发明的融合蛋白 的编码序列。 所述的宿主细胞优选的是真核细胞， 例如但不限于 CH0， COS细胞， 293细胞， RSF细胞等。 作为本发明的优选方式， 所述的细胞是 CH0细胞， 其可良 好地表达本发明的融合蛋白， 可获得结合活性良好， 稳定性良好的融合蛋白。
本发明还提供一种用重组 DNA制备本发明融合蛋白的方法， 其步骤包括：
1) 提供编码融合蛋白的核酸序列 （如 SEQ ID NO : 4序列）；
2) 将 1)的核酸序列插入到合适的表达载体， 获得重组表达载体；
3) 将 2)的重组表达载体导入合适的宿主细胞；
4) 在适合表达的条件下培养转化宿主细胞；
5) 收集上清液， 并纯化融合蛋白产物。
将所述编码序列导入宿主细胞可采用本领域的多种已知技术， 例如但不限于： 磷酸钙沉淀， 原生质体融合， 脂质体转染， 电穿孔， 微注射， 反转录法， 噬菌体 转导法， 碱金属离子法。
有关宿主细胞的培养和表达可参见 Olander RM Dev Biol Stand 1996 : 86 : 338。 可通过离心去除悬浮液中的细胞和残渣， 收集清液。 可通过聚丙烯酰胺凝胶电泳 技术进行鉴定。 可将上述制备获得的融合蛋白纯化为基本均一的性质， 例如在 SDS-PAGE电泳 上呈单一条带。 例如， 当重组蛋白为分泌表达时， 可以采用商品化的超滤膜来分 离所述蛋白， 例如 Mi ll ipore、 Pell icon等公司产品， 首先将表达上清浓缩。 浓 缩液可采用凝胶层析的方法进一步加以纯化， 或采用离子交换层析的方法纯化。 例如阴离子交换层析 (DEAE等)或阳离子交换层析。 凝胶基质可为聚丙烯酰胺、 葡 聚糖、聚酰胺等常用于蛋白纯化的基质。 Q-或 SP-基团是较为理想的离子交换基团。 最后， 还可用羟基磷灰石吸附层析， 金属螯合层析， 疏水相互作用层析和反相高 效液相色谱 (RP-HPLC)等方法对上述纯化产物进一步精制纯化。 上述所有纯化步骤 可利用不同的组合， 最终使蛋白纯度达到基本均一。
可利用含有所述融合蛋白的特异性抗体、 受体或配体的亲和层析柱对表达的融 合蛋白进行纯化。 根据所使用的亲和柱的特性， 可利用常规的方法， 如高盐缓冲 液、 改变 pH等方法洗脱结合在亲和柱上的融合性多肽。 可选择地， 所述的融合蛋 白的氨基端或羧基端还可含有一个或多个多肽片段， 作为蛋白标签。 任何合适的 标签都可以用于本发明。 例如， 所述的标签可以是 FLAG, HA, HA1， c-Myc , 6-His 或 8-His等。 这些标签可用于对融合蛋白进行纯化。
本发明还提供了一种组合物， 它含有有效量 （如 0. 000001-90wt%; 较佳的 0. l-50wt%; 更佳的， 5-40^%)的本发明的融合蛋白， 以及药学上可接受的载体。
通常， 可将本发明的融合蛋白配制于无毒的、 惰性的和药学上可接受的水性载 体介质中， 其中 pH通常约为 5-8， 较佳地， pH约为 6-8。
如本文所用， 术语 "有效量"或 "有效剂量"是指可对人和 /或动物产生功能 或活性的且可被人和 /或动物所接受的量。
如本文所用， "药学上可接受的"的成分是适用于人和 /或哺乳动物而无过度 不良副反应 （如毒性、 剌激和变态反应） 的， 即具有合理的效益 /风险比的物质。 术语 "药学上可接受的载体"指用于治疗剂给药的载体， 包括各种赋形剂和稀释 剂。
本发明的药物组合物含有安全有效量的本发明的融合蛋白以及药学上可接受 的载体。 这类载体包括 (但并不限于）： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 及其组合。 通常药物制剂应与给药方式相匹配， 本发明的药物组合物可以被制成 针剂形式， 例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行 制备。 所述的药物组合物宜在无菌条件下制造。 活性成分的给药量是治疗有效量。 本发明的药物制剂还可制成缓释制剂。
本发明所述的融合蛋白的有效量可随给药的模式和待治疗的疾病的严重程度 等而变化。 优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定 (例如通过临床试验）。 所述的因素包括但不限于： 所述的融合蛋白的药代动力学 参数例如生物利用率、 代谢、 半衰期等； 患者所要治疗的疾病的严重程度、 患者 的体重、 患者的免疫状况、 给药的途径等。 通常， 当本发明的融合蛋白每天以约 0. 00001mg-50mg/kg动物体重（较佳的 0. OOOlmg-lOmg/kg动物体重）的剂量给予， 能得到令人满意的效果。 例如， 由治疗状况的迫切要求， 可每天给予若干次分开 的剂量， 或将剂量按比例地减少。 本发明的主要优点在于：
(a) 本发明融合蛋白半衰期长； (b) 本发明融合蛋白可显著地降低 IgE的血清浓度；
(c) 本发明融合蛋白可特异性地抑制 TACI-BAFF复合物的形成， 从而终止膜上 TACI的激活， 进而终止信号传导。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本 发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通 常按照常规条件， 例如 Sambrook等人， 分子克隆： 实验室手册 (New York : Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建议 的条件。 除非另外说明， 否则百分比和份数是重量百分比和重量份数。 实施例 1
融合蛋白 A表达质粒的构建
融合蛋白 A前体（图 4)是由下列三部分片段从 N端到 C端组成，具体氨基酸序 列如下：
片段 1. TNF-R2氨基酸序列从第 1至 76位，包含第一个富半胱氨酸区 （图 2)。 片段 2. TACI氨基酸序列从第 30至 119位， 含有二个富半胱氨酸区和部分主 茎序列（图 1)。
片段 3. 人的 Y l氨基酸序列从第 216至 447位， 含有绞链区和二个 CH区（图
3)。
片段 1的 cDNA的合成。 PCR模板是用常规方法制备的人 cDNA。 5 ' 端的引物
AAGCTTGCGGCCGCGAGCTCGGATCCACT (SEQ ID NO.： 6)是质粒载体的序列。 为了克隆 进 pT载体， 在 5 ' 端引进了 Not I 酶切位点。 3 ' 端的引物序列是 GGGGCAGGA TCTCATAGCGTCACACACGGTGTCCGAG (SEQ ID NO.： 7)， 为了和 TACI cDNA连接， 它含 有 19 个 TACI的核糖核苷酸。
片段 2和 3的 cDNA的合成。编码片段 2和 3的 cDNA已重组在编码融合蛋白 B 的载体里 (见实施例 2)， 用此载体作模板， 用片段 2的 5 ' 端引物和片段 3的 3 ' 端的引物来扩增编码片段 2和 3的重组基因。
为了把片段 1和的片段 2和 3的 cDNA连接起来，将提纯的这二个片段的 cDNA 混合作为模板， 用片段 1的 5 ' 端的引物和片段 2/3的 3 ' 端的引物来实施聚合酶 链反应。 提纯扩增的 PCR片段， 克隆到载体 pCR-Blunt II-T0P0 (购自 Invitrogen 公司）。 将重组质粒转染进感受态细菌 XL-1 (购自 Invitrogen公司）细菌， 挑选阳 性菌落， 提纯重组质粒。 用酶切和测序鉴定重组基因的正确性。
把重组质粒克隆进哺乳动物细胞表达载体。 所用的市售表达载体含人的 CMV启 动子， 并含有 DHFR表达基因， 可用来筛选融合蛋白表达的稳定细胞株和扩增表达 基因拷贝数。 用 Notl和 Xbal酶切重组质粒和表达载体， 用胶提纯法纯化切下的 重组基因和双酶切的载体，在体外链接后转染到感受态细菌 DH50 (购自 Invitrogen 公司）， 挑选阳性克隆， 扩增和提纯 TACI-Fc表达质粒。 实施例 2
融合蛋白 B表达质粒的构建
融合蛋白 B前体（图 4)是由下列三部分来源从 N端到 C端组成，具体氨基酸序 列如下： 片段 1 . 人的 CD33蛋白信号肽， 含有 N端前 16个氨基酸。
片段 2. TACI氨基酸序列从第 30至 119， 含有二个富半胱氨酸区和部分主茎 序列（图 1)。
片段 3.人的 IgGl的氨基酸序列从第 216至 447，含有绞链区和二个 CH区（图 3)。
编码融合蛋白 B的重组基因是由重叠聚合酶链反应 (PCR)构建。 PCR反应用的 模板是用常规方法制备的人 cDNA。 聚合酶链反应 (PCR)是用 Invi trogen 公司的 Plantium SuperMix, 按厂商的操作说明进行 PCR反应。 用 Qiagen公司的胶提纯 DNA片段的试剂盒来提纯各 PCR片段。 由于 TACI本身没有蛋白分泌信号肽， 所以 在构建表达载体时， 本实施例采用人 CD33蛋白的分泌信号肽。
编码 CD33蛋白分泌信号肽基因的合成。 人工合成此片段的疆，
Q ID NO: 12)， 为了克隆进哺乳动物细胞表达载体， 在 5 ' 端引进了 Not I 酶切 位点。
片段 2的 cDNA的合成。 PCR模板是常规方法制备的人 cDNA。 5 ' 端的引物是
GGCAGGGGCCCTGGCT GCTATGAGATCCTGCCCC (SEQ ID NO.： 8)， 头 16个核苷酸序列 和 CD33编码的核苷酸 3 ' 端序列相同， 是为了链接片段 1和 2的 PCR片段。 3 ' 端 的引物序列是 TGTMCMGATTTGGGTTC CCTGAGCTCTGGTGGAAG (SEQ ID NO.： 9)。
片段 3的 cDNA的合成。 PCR模板是常规方法制备的人 cDNA。 扩增出的 DNA序 列编码人的 Y l免疫球蛋白 Fc氨基酸序列， 从 216 至 447位。 5 ' 端的引物是 GAACCCAAATCTTGTTACA (SEQ ID NO.： 10)， 和片段 2 cDNA 3 ' 端序列互补。 3 ' 端 的引物序列是 TGGTGGTGTCTAGAGACTCATTTACCCGGAGACAGGGAGAGGC (SEQ ID N0.： 11)， 为了克隆进 pT载体， 在 3 ' 端引 iiT xba I 酶切位点。
为了把三个片段的 cDNA连接起来， 将提纯的三个片段混合作为模板， 用片段 1 的 5 ' 端的引物， AGGTATAGCGGCCGCCACCATGCCGCTGC (SEQ ID NO.： 13)和片段 3的 3 ' 端的引物（同上， SEQ ID N0.： 11)来实施聚合酶链反应。 提纯扩增的 PCR片段， 克隆入载体 pCR-Blunt I I-T0P0 (购自 Invi trogen公司）。 将重组质粒转染进感受 态细菌 XL-1 (购自 Invi trogen公司）， 挑选阳性菌落， 提纯重组质粒。 用酶切和测 序鉴定重组基因的正确性。
把重组质粒克隆进哺乳动物细胞表达载体。 同实施例 1， 用 Notl和 Xbal酶切 重组质粒表达载体， 用胶提纯法纯化切下的重组基因和双酶切的表达载体， 在体 外链接后转染到感受态细菌 DH50 (Invi trogen) , 挑选阳性克隆， 扩增和提纯 TACI-Fc表达质粒。 实施例 3
融合蛋白表达细胞株的建立
在本实施例中， 用稳定转染和基因扩增的方法建立稳定高效蛋白表达的 CH0 DG44细胞系。 克隆出来的 CH0 DG44 细胞悬浮培养在无血清， 无动物蛋白的培育 液里。
原始 CH0 DG44细胞来源于 Invi trogen公司， 细胞培养和传代的方法参照公司 的操作手册。 无转染细胞悬浮培养在 CD DG44 培养液里， 培养液含有 8 mM Glutamine , 0. 18% Fluronic F - 18。 用 Qiagen的试剂盒制备大量的编码融合蛋白的表达质粒。 为了质粒不被细菌 污染， 用酒精沉淀法纯化一次质粒。 在基因转染前， CD DG44 细胞传三代后再进 行转染。 当细胞的密度达到 l X 106/ml时， 吸取 lml 转到 12-孔培养板里， 每孔 lml。
转染是用 Invitrogen公司的转染试剂 LIPOFECTAMINE 2000 CD来进行的。 为 了找到最高的转染效率， 每种质粒用三种不同的质粒对转染试剂比例
(1. 5ug : 40ul，2ug : 40ul和 3ug : 40ul)进行转染。 转染 2天后， 对细胞培养液进行 Western Blot分析（图 5)， 结果显示 2ug DNA质粒转染效率最高（图 5， 第 3泳道）。
选取 2 μ g DNA质粒转染的细胞进行下一步的筛选， 用有限稀释法在 96孔培养 板里进行筛选。 筛选培养液是不含有 Hypothanxine和 Thymidine的 CH0培养液， 含有 20nM或 ΙΟΟηΜ 的氨甲碟呤 （MTX)。 二个星期后， 用 ELISA方法对每个孔的 细胞培养液进行分析， 检测融合蛋白的含量并 SDS-PAGE分析培养液里的蛋白。
结果如图 6所示。结果表明编码融合蛋白 A和 B的基因已整合进细胞的染色体， 并能表达和分泌融合蛋白 A和 B， 分子量分别为约 42和 47千道尔顿， 比理论值略 高 (约 5千道尔顿）， 与预测值相符 (在真核细胞中表达时存在蛋白糖基化现象）。
把融合蛋白高表达的细胞株转到 24孔培养板， 培养 7天后， 再进行 ELISA蛋 白含量的分析。 从每个融合蛋白库里， 再挑选 3个高表达的细胞进行扩增， 冻存。 实施例 4
融合蛋白的制备
融合蛋白 A、 B细胞株作为种子细胞， 以 0. 5 X 106细胞 /ml的初试密度开始进 行培养， 培养基为 S4 (含 8g/L葡萄糖、 4mmol/L谷氨酰胺和 0. 5mg/L的胰岛素）， pH控制在 6. 7-7. 1， 溶氧不低于 30%， 初始温度 35°C， 细胞密度长至 6. 5-7. O X 106 cells/ml时降温至 31 °C， 第二天再降至 29°C， 通过补加葡萄糖维持糖浓度， 细胞 存活率接近 50%时结束培养， 融合蛋 Θ Α、 Β的目的蛋白表达量可以达到 200mg/L。 细胞培养收获液经第一步 Protein A亲和层析： 目标蛋白纯度达到 79. 1%， 收率为 54. 4%； 第二步 CM阳离子层析： 目标蛋白纯度达到 87. 4%， 收率为 81. 6%; 第三步 Phenyl-650M疏水层析： 目标蛋白纯度达到 94. 5%， 收率为 78. 0%; 最后一步超滤 浓缩换液： 目标蛋白纯度达到 95. 3%， 收率为 92. 9%。
四步分离纯化后融合蛋白 A、 B的非还原电泳结果见图 7。 实施例 5
融合蛋白 A和融合蛋白 B对 RPMI 8226细胞的生物学活性
融合蛋白 TACI-Fc的体外生物学活性主要通过在地塞米松（dexamethasone)和 rhBAFF存在时， 对人骨髓瘤细胞 RPMI 8226 (购自 ATCC)生长的抑制作用得以体现。 地塞米松的作用是使 RPMI 8226细胞发生凋亡， 降低实验的背景， BAFF则能恢复 并促进细胞生长， 在此基础上加入 TACI-Fc中和 BAFF, 则能够减弱甚至消除 BAFF 对细胞生长的促进作用。 通过这种方法即可检测 TACI-Fc的体外生物学活性。
反应在 96孔细胞培养板中进行， 每孔接种 10000个 RPMI 8226细胞于 150 μ 1 培养液中， 其中包括 0. 1 μ Μ地塞米松（dexamethasone) (Wako, C0DE#041- 18861) 禾口 1 μ g/ml rhBAFF (R&D, Cat#2149-BF/CF)， 待测样品终浓度为从 16 μ g/ml起始 依次 4倍梯度稀释， 同时进行一组不含 rhBAFF的实验作为对照。 在 37 ° C二氧化 碳培养箱中培养 5天后， 每孔加入 l(^ lcck-8溶液（Cell Counting Kit-8, D0JIND0, cat: ck04 )进行显色反应。 37 ° C显色 6小时，用酶标仪（Bio-Rad， iMark) 以 655nm为参比， 检测 450nm的吸收值。 由于待测样品自身对细胞有抑制作用， 从实验组 0D450中减去不含 rhBAFF对照组的 0D450, 得到校正的 0D450值
(cal ibrated 0D450) , 以此对待测样品浓度的对数做图， 通过 Origin软件进行 S 型曲线拟合，计算 EC5。，即为半数有效抑制浓度，可代表待测样品的体外细胞活性。
结果见图 8， 显示了融合蛋白 A与融合蛋白 B的 RPMI 8226细胞活性的比较。 各自的浓度效应曲线显示： 当融合蛋白 TACI-Fc浓度很低时， 溶液中 rhBAFF基本 均呈游离状态， 促进 RPMI 8226细胞生长并增殖； 随着其浓度逐渐升高， 开始中 和培养液中的 rhBAFF, 游离 rhBAFF逐渐减少， 细胞的增殖也随之降低； 直至有足 够的融合蛋白与 rhBAFF结合， 可以完全抵消 rhBAFF的作用， 细胞生长速度降到 最低。 将 450nm吸收值对样品浓度对数作图， 发现三者的效应曲线呈现 S型， 经 过拟合后计算得到的半数有效抑制浓度表明： 融合蛋白 A的 RPMI 8226细胞活性 略低于融合蛋白 B的细胞活性。 实施例 6
融合蛋白 A和融合蛋白 B体外结合实验研究
融合蛋白 A/B体外结合分析检测主要利用 ELISA原理： 将一定量 rhBAFF固定 在酶标板上， 各孔加入系列浓度梯度的融合蛋白 A/B进行结合反应， 最后通过 HRP 偶联的 anti-human Fc检测结合在平板 rhBAFF上的融合蛋白量来考察其与 rhBAFF 的结合情况。
用 PBS (pH7. 4)将重组人 BAFF的可溶性片段 (rhBAFF, R&D, Cat#2149- BF/CF) 稀释成 0. 5 g/ml， 加入酶标板中（greiner bio-one, Cat # 655001)， 50 μ 1/孔， 4° C包被过夜。 第二天经超纯水洗涤后， 用 5%脱脂奶粉 /PBST进行封闭。 之后将 待测样品从 3 g/ml起始， 依次 3倍梯度稀释， 加入酶标板中， 50 μ 1/孔， 室温 孵育 2小时。 经 PBST洗涤 3次后， 每孔加入 50 μ ΐ 0. 8 g/ml检测抗体
(peroxidase- conj μ gated Goat ant i -Human IgG, Fc y Fragment Specific : Jackson ImmunoResearch LABORATORIES, INC. Code#109- 036- 098)， 室温孵育 1 小时。 PBST再次洗涤之后， 进行显色反应， 每孔加入 100 μ ΐ of 0. 4mg/ml 0PD， 避光显色 10分钟， 用 100 μ 11M H2S04终止反应。 在酶标仪（Bio-Rad， iMark)上以 655nm为参比， 检测 490nm的吸收值。 将吸收值对待测样品摩尔浓度的对数作图， 通过 Origin软件进行 S型曲线拟合， 计算出 EC5。（吸收值变化 50%对应的浓度）代 表待测样品的体外结合活性。
结果如图 9所示。 随酶标板中每孔加入融合蛋白浓度的升高， 其与固定于酶标 板上的 rhBAFF结合增加， 之后结合的检测抗体就越多， 因此最后通过显色反应读 取的 490nm吸光值就越高。 两者的结合曲线均呈现典型的 S型曲线特征， 经过软 件拟合计算得到的 EC5。表明：融合蛋白 A的 EC5。略大于融合蛋白 B，说明其与 rhBAFF 的体外结合能力略低于融合蛋白 B。 实施例 7
融合蛋白 A和融合蛋白 B对动物药代动力学研究
为了研究药物在体内的药代动力学特点， 确定血药浓度与给药时间的关系。 选 用正常雌性 BALB/C小鼠（18〜20g) 48只， 3只 /时间点， 共设立 7个时间点， 即 1、 2、 4、 8、 24、 48、 72h。 分别腹腔注射 5mg/kg的融合蛋白 A或融合蛋白 B， 准确 记录每组的给药时间， 然后于每个时间点对小鼠进行乙醚麻醉后摘取眼球取血， 收集血清。 ELISA法检测各时间点两种药物的血药浓度。
如图 10所示， 两种药物的血药浓度与时间的拟合曲线基本相似， 但是融合蛋 白 A在不同时间点的血药浓度明显高于融合蛋白 B， 基本是 2倍左右。通过软件得 出融合蛋白 A和融合蛋白 B在小鼠体内的半衰期分别为 14h和 l lh。 实施例 8
融合蛋白 A和融合蛋白 B对正常小鼠体内药效学研究
本实施例评价两种药物融合蛋白 A和融合蛋白 B对小鼠的药理作用。 选用正常 雌性 BALB/C小鼠（18〜20g) 42只， 6只 /组， 共分为 7组， 即对照（Control)、 融 合蛋白 A-5mg/kg、 融合蛋白 A-2. 5mg/kg、 融合蛋白 A-1. 25mg/kg)、 融合蛋白 B- 5mg/kg、 融合蛋白 B- 2. 5mg/kg、 融合蛋白 B- 1. 25mg/kg组。
首先于第 0天由眼底静脉丛取血， 收集血清， 然后分别腹腔给予 PBS、 不同剂 量的融合蛋白 A和融合蛋白 B (5mg/kg、 2. 5mg/kg、 1. 25mg/kg)， 隔天给药， 共 2 周。 最后一次给药后 24h， 先将小鼠称重， 乙醚麻醉后眼眦取血， 处死后解剖取出 小鼠脾脏， 去除周围组织， 用滤纸稍稍吸干后称重， 计算脾重 /体重。 ELISA法检 测给药前和给药结束后每组动物血清中 IgM的浓度。
如图 11所示， 对照组血清 IgM浓度给药前后无显著差异， 不同剂量的融合蛋 白 A和融合蛋白 B (5mg/kg、 2. 5mg/kg、 1. 25mg/kg)给药后第 14天血清 IgM浓度均 有显著下降， 与第 0天比较差异显著 (**p〈0. 01)。 融合蛋白 A组与融合蛋白 B组 相比没有显著差异。
如图 12所示， 不同剂量的融合蛋白 A和融合蛋白 B (5mg/kg、 2. 5mg/kg、 1. 25mg/kg)给药后第 14天脾重 /体重显著降低，其中融合蛋白 A (5mg/kg、2. 5mg/kg、 1. 25mg/kg)、 融合蛋白 B (5mg/kg、 2. 5mg/kg)组与对照比较具有显著差异
(**p〈0. 01, *p〈0. 05)。 融合蛋白 B (l. 25mg/kg)组与对照比较无统计学意义。 结果 可见， 融合蛋白 A的降低小鼠脾重 /体重的作用明显优于融合蛋白 B。 实施例 8
融合蛋白 A和融合蛋白 B对哮喘模型小鼠体内药效学研究
为了评价两种药物融合蛋白 A和融合蛋白 B对哮喘模型小鼠的药理作用， 选用 正常雌性 C57/B6小鼠（18〜20g) 44只， 共分为 7组， 即对照（Control)组（6只）、 模型组（11只）、 融合蛋白 A-5mg/kg (13只）、 融合蛋白 B_5mg/kg (14只）进行研究。 首先， 将模型组和实验组小鼠分别于第 1和 14天用 0VA/ALUM混悬液腹腔注射致 敏， 于第 13天开始分别腹腔注射 PBS、 不同剂量的融合蛋白 A和融合蛋白 B， 隔 天给药， 共给药 12次。 其次， 第 28天、 30天、 32天接受 0VA蛋白溶液的鼻滴入 攻击。 最后， 给药全部结束后 24h， 即第 36天， 先将小鼠称重， 乙醚麻醉后眼底 静脉丛取血， 处死后解剖取出小鼠脾脏， 去除周围组织， 用滤纸稍稍吸干后称重。 抽取其肺泡灌洗液，计算其中总的白细胞数量。 ELISA法检测每组小鼠血清中 IgE、 IgM浓度。
如图 13 (A)所示， 模型组第 36天血清 IgM浓度与对照相比明显升高。融合蛋白 A-5mg/kg、 融合蛋白 B-5mg/kg与模型组 IgM浓度比较显著降低（**p〈0. 01)。 如图 13 (B)所示， 模型组、 融合蛋白 A-5mg/kg、 融合蛋白 B_5mg/kg第 36天血清 IgE浓 度与对照相比均不同程度升高， 其中融合蛋白 A组与模型组 IgE浓度比较具有显 著差异（**P〈0. 01)。
出乎意料的是， 与融合蛋白 B相比， 融合蛋白 A可显著降低 IgE的浓度， 提示 融合蛋白 A可更有效地抑制变态反应。
如图 14所示， 模型组第 36天脾重 /体重与对照相比明显升高。 融合蛋白
A-5mg/kg、 融合蛋白 B-5mg/kg与模型组脾重 /体重比较显著降低（**p〈0. 01)。 实施例 9
药物组合物
根据融合蛋白 A和融合蛋白 B药物特点， 以融合蛋白 A (40mg/ml)为例， 设计了 几组药物组合物， 并在液体加速试验条件下考察了融合蛋白 A药物组合物包括聚 集与降解的稳定性， 通过 SEC-HPLC和 SDS-PAGE方法对样品的聚集与降解进行分 析， 最终确定融合蛋白 A的药物组合物。
A) 缓冲溶液： 10mM乙酸缓冲溶液（pH5. 0)， 组成： 80 mg/mL海藻糖；
B) 缓冲溶液： 10mM琥珀酸缓冲溶液（pH5. 0)， 组成： 80mg/mL蔗糖；
C) 缓冲溶液： 10mM乙酸缓冲溶液（pH5. 0)，组成： 80mg/mL海藻糖， 0. 05mg/mL 吐温 20；
D) 缓冲溶液： 10mM乙酸缓冲溶液（pH5. 0)，组成： 80mg/mL海藻糖， 0. 05mg/mL 聚羟亚烃 188 ;
E) 缓冲溶液： 10mM琥珀酸缓冲溶液（pH5. 0)，组成： 80mg/mL海藻糖， 0. 05mg/mL 吐温 20;
F) 缓冲溶液： 10mM琥珀酸缓冲溶液（pH5. 0)，组成： 80mg/mL海藻糖， 0. 05mg/mL 聚羟亚烃 188。
六组药物组合物在 4°C、 25 °C、 40°C条件下储存 2周， 稳定性结果可见， 4°C 条件下储存 2周， 药物组合物均未发生聚集与降解。 25 °C、 40°C条件下未产生降 解片段， 但聚集体有增加， 具体结果见表 1。
表 1
Figure imgf000014_0001
由表 1结果可见， 药物组合物 A和 D条件下， 融合蛋白 A具有相对较高的稳 定性， 在 4°C和 25 °C环境下是稳定的， 但对于 40°C环境而言， 组合物 D比组合物 A有相对较高的聚集趋势。 因此， 对于融合蛋白 A而言， 一种特别优选的药物组 合物组成为 40 mg/mL融合蛋 S A、 80 mg/mL海藻糖、 10mM乙酸缓冲溶液（pH5. 0)。 讨论
肿瘤坏死因子受体 (TNFR)超家族的胞外区通常含有 1至 6个富含半胱氨酸的结 构域 (CRD)主要负责与配体的识别和结合。 这些受体通常以三聚体的形式结合配体 发挥受体功能。 而上述功能则由含有至少 1个富含半胱氨酸的前配体装配域 (Pre-l igand binding assembly domain, PLAD)所介导。 更具体的说， TNFR超家 族成员， 包括 TRAIL受体 1、 CD40、 60kDa的 TNFR和 80kDa的 TNFR, 均表现出这 种同型缔合功能。 研究发现， 在缺少配体的情况下， PLAD对受体聚合体的形成是 必须且充分的。虽然 PLAD不直接参与对 TNF α和 TNF β的识别与结合，但删除 PLAD 或者 PLAD发生突变， 配体与受体的亲和能力会丧失。 因此， PLAD对于 TNFR多聚 体的形成以及配基的结合相关， 在 TNF相关信号传导途径中起着至关重要的作用。
本发明的融合蛋白可有效封闭 TACI的 PLAD区， 从而特异性地抑制 TACIR的聚 合， 从而终止 TACI-BAFF复合物的形成， 达到终止信号传导的目的。
试验结果还表明， 本发明的 TACI-FC融合蛋白具有延长药物的半衰期， 显著降 低血清中 IgE的浓度， 并可有效抑制 TACI-BAFF复合物的形成， 进而终止信号传 导。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单 独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领 域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所附 权利要求书所限定的范围。

Claims

权 利 要 求
1. 一种融合蛋白， 其特征在于， 所述融合蛋白包括融合在一起的以下元件： (a) TNF受体或其活性片段； （b) BAFF受体或其活性片段， 其中所述的 BAFF受体 b 包括 TACI , BCMA, BAFFR或其组合； 和（c) 抗体 Fc区域；
并且所述融合蛋白具有以下功能： 抑制 TACI-BAFF复合物的形成。
2. 如权利要求 1所述的融合蛋白， 其特征在于， 所述的融合蛋白还具有以下 一种或多种功能：
(a) 结合于 BAFF的活性；
(b) 结合于 TALL-2/APRIL的活性；
(c) 降低 IgE的血清浓度；
(d) 降低血清免疫球蛋白的浓度如 IgM等；
(e) 降低脾脏重量；
(f) 抑制或封闭 BAFF/APPRIL途径；
(g) 减少 B细胞数量。
3. 如权利要求 1所述的融合蛋白， 其特征在于， 所述的融合蛋白具有下式 I 或 II 所示的结构：
X-Y-Z Y-X-Z
式 I 式 II
式中，
X为 TNF受体胞外区的第一结构域、 或含第一结构域的 TNF受体胞外区；
Y为全长的 TACI、BCMA或 BAFFR胞外区，或至少含有 30-119位的 TACI胞外区； Z为无、 或任选的人抗体的 Fc区域；
-表示肽键或肽接头。
4. 如权利要求 1所述的融合蛋白， 其特征在于， 所述的 X、 Y、 Ζ中的任何两 者以头 -头、 头 -尾、 或尾-尾方式相连； 和 /或
所述的 X含有或具有 TNF-R2氨基酸序列的第 23-76位；第 23-118位；第 23-162 位； 第 23-201位； 和 /或
所述的 Υ含有或具有 TACI氨基酸序列的第 30-119位； 和 /或 BAFF受体 BCMA 氨基酸序列第 1-54位；和 /或 BAFF受体 BAFFR氨基酸序列第 1-78位； 和 /或
所述的 Z含有或具有人 γΐ氨基酸序列的第 216-447位； 和 /或 所述的融合蛋白具有 SEQ ID NO.： 5中第 1-398位， 第 23-398位或第 39-398 位的氨基酸序列。
5. 一种核酸分子， 其特征在于， 所述的核酸分子编码权利要求 1-4任一所述 的融合蛋白。
6. 一种载体， 其特征在于， 它含有权利要求 5所述的核酸分子。
7. 一种基因工程化的细胞， 其特征在于， 所述的细胞含有权利要求 6所述的 载体； 或所述的细胞基因组中整合有权利要求 5所述的核酸分子。
8. —种产生权利要求 1所述的融合蛋白的方法， 其特征在于， 所述的方法包 括步骤：
在适合表达所述融合蛋白的条件下， 培养权利要求 7所述的宿主细胞， 从而表 达所述的融合蛋白； 和
分离或纯化所述的融合蛋白。
9. 一种药物组合物， 其特征在于， 所述的药物组合物含有权利要求 1所述的 融合蛋白及其药物学上可接受的载体。
10. 如权利要求 1所述的融合蛋白的用途， 其特征在于， 用于制备选自下组的 一种或多种组合物： （a) 抑制 BAFF/APPRIL信号通路组合物； （b)降低血清或血液 IgE、 IgM浓度的组合物； （c)治疗免疫疾病的组合物， 较佳地所述免疫疾病包括哮 喘、 哮喘、 系统性红斑狼疮和类风湿性关节炎； 以及（d)治疗 B细胞增多相关 疾病的组合物， 较佳地所述的 B细胞增多相关疾病包括多发性骨髓瘤、 慢性淋 巴细胞白血病、 巨球蛋白血症和浆细胞性白血病。
11.一种（a)降低血清或血液 IgE和 /或 IgM浓度； （c)治疗免疫疾病的组合物； 或 (d)治疗 B细胞增多相关疾病的方法， 其特征在于， 所述的方法包括步骤： 给需 要的对象施用权利要求 1所述的融合蛋白。
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