WO2022188883A1 - Tnfr2与april/baff受体的融合蛋白 - Google Patents

Tnfr2与april/baff受体的融合蛋白 Download PDF

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WO2022188883A1
WO2022188883A1 PCT/CN2022/080467 CN2022080467W WO2022188883A1 WO 2022188883 A1 WO2022188883 A1 WO 2022188883A1 CN 2022080467 W CN2022080467 W CN 2022080467W WO 2022188883 A1 WO2022188883 A1 WO 2022188883A1
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fusion protein
tnfr2
baff
bcma
april
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PCT/CN2022/080467
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English (en)
French (fr)
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蔡则玲
陈羿
李鹏
张利强
张博阳
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上海赛金生物医药有限公司
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Priority to US18/550,055 priority Critical patent/US20240150432A1/en
Priority to CN202280007564.0A priority patent/CN116802301A/zh
Priority to EP22766405.9A priority patent/EP4306545A1/en
Publication of WO2022188883A1 publication Critical patent/WO2022188883A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the field of biotechnology. Specifically, it relates to a fusion protein of TNFR2 and APRIL/BAFF receptor.
  • autoimmune diseases are a type of diseases in which one's own cells and body fluids react to one's own body.
  • Common autoimmune diseases include rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis (PS), systemic lupus erythematosus (SLE), etc. big impact.
  • TNF ⁇ targeted inhibitors have effectively controlled the disease process by antagonizing TNF ⁇ and inhibiting the up-regulation of its downstream cytokines such as IL-2, IL-6, IFN- ⁇ , and at the same time inhibiting overactivated T cells. It is widely used in a variety of autoimmune diseases.
  • TNF ⁇ inhibitors have achieved good curative effect, there are still 50% of patients with unsatisfactory treatment effects, so new combinations are urgently needed to achieve more effective control of autoimmune diseases.
  • the present invention provides a safer, more effective and more precise therapeutic fusion protein for autoimmune diseases.
  • the purpose of the present invention is to provide a safer, more effective and more precise therapeutic fusion protein for autoimmune diseases.
  • a fusion protein comprising the following elements fused together:
  • TNF receptors or active fragments thereof (a) TNF receptors or active fragments thereof;
  • APRIL/BAFF receptor or an active fragment thereof wherein the APRIL/BAFF receptor comprises TACI, BCMA, BAFFR or a combination thereof;
  • the fusion protein retains the biological activities of the above-mentioned elements (a) and (b).
  • the TNF receptor is selected from the group consisting of TNFR2, TNFR1, or a combination thereof.
  • the TNF receptors are derived from human or non-human mammals, more preferably from rodents (eg, mice, rats), primates and humans.
  • the TNF receptor includes wild type and mutant type.
  • the TNF receptor includes a full-length, mature form of TNF receptor, or an active fragment thereof.
  • the TNF receptor also includes derivatives of TNF receptor.
  • the derivatives of TNF receptors include modified TNF receptors, protein molecules whose amino acid sequences are homologous to natural TNF receptors and have natural TNF receptor activity, and TNF receptor dimers Or multimer, fusion protein containing the amino acid sequence of TNF receptor.
  • the modified TNF receptor is a PEGylated TNF receptor.
  • the "protein molecule whose amino acid sequence is homologous to the natural TNF receptor and has the activity of the natural TNF receptor” means that its amino acid sequence has ⁇ 85% homology compared with the TNF receptor, Preferably > 90% homology, more preferably > 95% homology, most preferably > 98% homology; and a protein molecule with TNF receptor activity.
  • the TNF receptor includes a first domain, a second domain, a third domain, and/or a fourth domain.
  • first domain, the second domain, the third domain, and/or the fourth domain are each independently a cysteine-rich region (CRD).
  • CCD cysteine-rich region
  • the TNF receptor contains or has positions 1-235, or 17-179, or 17-140 of the amino acid sequence of TNFR2 (SEQ ID NO.: 13), or 55-179th, or 55-140th.
  • the APRIL/BAFF receptors are derived from human or non-human mammals, more preferably from rodents (eg, mice, rats), primates and humans.
  • the APRIL/BAFF receptor includes wild type and mutant type.
  • the APRIL/BAFF receptor further includes derivatives of APRIL/BAFF receptor.
  • the derivatives of the APRIL/BAFF receptors include modified APRIL/BAFF receptors, protein molecules whose amino acid sequences are homologous to the natural APRIL/BAFF receptors and have the activity of the natural APRIL/BAFF receptors , APRIL/BAFF receptor dimer or multimer, fusion protein containing APRIL/BAFF receptor amino acid sequence.
  • the "protein molecule whose amino acid sequence is homologous to the natural APRIL/BAFF receptor and has the activity of the natural APRIL/BAFF receptor” means that its amino acid sequence has ⁇ 85 compared with that of the APRIL/BAFF receptor. % homology, preferably ⁇ 90% homology, more preferably ⁇ 95% homology, most preferably ⁇ 98% homology; and a protein molecule with APRIL/BAFF receptor activity.
  • the APRIL/BAFF receptor is selected from the group consisting of TACI, BCMA, BAFFR, or a combination thereof.
  • the TACI contains or has positions 1-109, or 33-109, or 68-109 of the amino acid sequence of TACI (SEQ ID NO.: 14).
  • the BCMA contains or has positions 1-54, or positions 6-54 of the BCMA amino acid sequence (SEQ ID NO.: 15).
  • the BAFFR contains or has positions 1-78, or positions 12-78, or 12-46 of the amino acid sequence of BAFFR (SEQ ID NO.: 16).
  • the Fc fragment is derived from human or non-human mammals, more preferably from rodents (eg, mice, rats), primates and humans.
  • the Fc fragment is the Fc fragment of immunoglobulin IgG, preferably the Fc part of IgG1.
  • the Fc fragments include natural Fc fragments and Fc mutants.
  • the Fc fragment contains or has positions 99-329 of the amino acid sequence of human IgG1 (accession number: UniProtKB-P01857).
  • amino acid sequence of the Fc fragment is shown in SEQ ID NO.: 17.
  • the fusion protein also has one or more of the following functions:
  • the fusion protein has the dimer structure shown in the following formula I or II:
  • X is the extracellular segment of the TNF receptor
  • Y is the extracellular segment of TACI, BCMA or BAFFR;
  • Z is none, or optionally, the Fc region of a human antibody
  • the fusion protein is a homodimer.
  • amino acid sequence of the fusion protein is shown in SEQ ID NO.: 18 or 19 or 20.
  • any two of the X, Y, and Z are connected in a head-to-head, head-to-tail, or tail-to-tail manner.
  • the "head” refers to the N-terminus of a polypeptide or a fragment thereof, especially the N-terminus of a wild-type polypeptide or a fragment thereof.
  • the "tail” refers to the C-terminus of a polypeptide or a fragment thereof, especially the C-terminus of a wild-type polypeptide or a fragment thereof.
  • the peptide linker is a peptide linker with a length of 1-20, preferably, 1-10 amino acids.
  • the X contains or has positions 1-235, or 17-179, or 17-140, or 55- 179, or 55-140; and/or
  • Said Y contains or has:
  • the Z contains or has positions 99-329 of the amino acid sequence of human IgG1 (accession number: UniProtKB-P01857).
  • nucleic acid molecule encoding the fusion protein of the first aspect of the present invention.
  • the nucleic acid molecule additionally contains auxiliary elements selected from the group consisting of signal peptide, secretory peptide, tag sequence (such as 6His), or its flank on the ORF of the mutein or fusion protein combination.
  • auxiliary elements selected from the group consisting of signal peptide, secretory peptide, tag sequence (such as 6His), or its flank on the ORF of the mutein or fusion protein combination.
  • the nucleic acid molecule is selected from the group consisting of DNA sequence, RNA sequence, or a combination thereof.
  • a vector comprising the nucleic acid molecule described in the second aspect of the present invention.
  • the vector comprises one or more promoters operably associated with the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker , nucleic acid restriction sites, and/or homologous recombination sites.
  • the vectors include plasmids and viral vectors.
  • the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.
  • AAV adeno-associated virus
  • adenovirus adenovirus
  • lentivirus lentivirus
  • retrovirus lentivirus
  • herpes virus SV40
  • poxvirus poxvirus
  • the vector includes an expression vector, a shuttle vector, and an integration vector.
  • a genetically engineered cell in the fourth aspect of the present invention, the cell contains the vector of the third aspect of the present invention; or the cell genome is integrated with the vector of the second aspect of the present invention described nucleic acid molecules.
  • the genetically engineered cells are eukaryotic cells, such as yeast cells, plant cells or mammalian cells (including human and non-human mammals).
  • the genetically engineered cells are prokaryotic cells, such as Escherichia coli.
  • the genetically engineered cells are selected from the group consisting of: Escherichia coli, wheat germ cells, insect cells, SF9, SP2/0, Hela, HEK293, CHO (such as CHOKS), yeast cells, or their combination.
  • a method for producing the fusion protein of the first aspect of the present invention comprising the steps of:
  • the genetically engineered cell of the fourth aspect of the present invention is cultured, thereby expressing the fusion protein
  • the fusion protein is isolated or purified.
  • a pharmaceutical composition comprising the fusion protein of the first aspect of the present invention and a pharmaceutically acceptable carrier thereof.
  • the pharmaceutical composition further includes other drugs for treating immune diseases.
  • other drugs for treating immune diseases are selected from the group consisting of hormone drugs, immunosuppressive drugs, small molecule targeted drugs, biological agents, or combinations thereof.
  • the hormonal drug is selected from the group consisting of cortisone, hydrocortisone, prednisone, prednisone, prednisolone, dexamethasone, betamethasone, or a combination thereof .
  • the immunosuppressive drug is selected from the group consisting of methotrexate, cyclophosphamide, azathioprine, cyclosporine, mycophenolate mofetil, tacrolimus, sirolimus, Flunomide, sulfasalazine, hydroxychloroquine, or a combination thereof.
  • the small molecule targeted drug is selected from the group consisting of tofacitinib, baricitinib, or a combination thereof.
  • the biological agent is selected from the group consisting of etanercept, certolizumab, adalimumab, golimumab, infliximab, tocilizumab, Kinumab, ustekinumab, canakinumab, anakinra, linacept, abatacept, rituximab, belimumab, or a combination thereof.
  • a fusion protein according to the first aspect of the present invention a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, and the fourth aspect of the present invention.
  • compositions that inhibit the BAFF/APPRIL signaling pathway (a) compositions that inhibit the BAFF/APPRIL signaling pathway
  • compositions for the treatment of immune diseases comprising
  • composition for treating a disease associated with B cell hyperplasia (c) A composition for treating a disease associated with B cell hyperplasia.
  • the composition is a pharmaceutical composition.
  • the immune disease is selected from the group consisting of rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis (PS), psoriatic arthritis (PsA), juvenile Idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), Behcet's disease (BD), multiple sclerosis (MS), Sjögren's syndrome (SS), Graves' disease, Crohn's disease ( CD), ulcerative colitis (UC), primary glomerulonephritis, IgA nephropathy, autoimmune vasculitis, polymyositis (PM), noninfectious uveitis, autoimmune hemolytic anemia ( AIHA), autoimmune purpura (ATTP), N-methyl-d-aspartate receptor (NMDAR) encephalitis, myasthenia gravis, hidradenitis suppurativa (HS), myelin-oligodendrocyte
  • the immune diseases include autoimmune diseases, such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis (PS), psoriatic arthritis (PsA) , Juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), autoimmune purpura (ATTP), Behçet's disease, IgA nephropathy.
  • autoimmune diseases such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis (PS), psoriatic arthritis (PsA) , Juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (SLE), autoimmune purpura (ATTP), Behçet's disease, IgA nephropathy.
  • the B cell hyperplasia-related diseases include multiple myeloma, chronic lymphocytic leukemia, macroglobulinemia and plasma cell leukemia.
  • the eighth aspect of the present invention there is provided a method for treating an immune disease by administering the fusion protein according to the first aspect of the present invention to a patient in need.
  • the fusion protein is administered in the form of monomers and/or dimers.
  • the immune diseases include rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis (PS), psoriatic arthritis (PsA), juvenile idiopathic joint inflammation (JIA), systemic lupus erythematosus (SLE), Behçet's disease (BD), multiple sclerosis (MS), Sjögren's syndrome (SS), Graves' disease, Crohn's disease (CD), ulcerative colitis ( UC), primary glomerulonephritis, autoimmune vasculitis, polymyositis (PM), noninfectious uveitis, autoimmune hemolytic anemia (AIHA), autoimmune purpura (ATTP), N-methyl-d-aspartate receptor (NMDAR) encephalitis, myasthenia gravis, hidradenitis suppurativa (HS), myelin-oligodendrocyte glycoprotein profile disorder (MOGSD) and neurode rhe
  • Figure 1 shows the schematic structure of the fusion protein (1A and 1B),
  • Figure 1C shows the amino acid sequence of the TNFR2-BCMA-Fc fusion protein (SEQ ID NO.: 19), the 1-235th position is the TNFR2 active fragment, bold Labeled (positions 236-284) is the BCMA active fragment and underlined (positions 285-515) is the IgG Fc fragment;
  • Figure 1D shows the amino acid sequence of the TNFR2-BAFFR-Fc fusion protein (SEQ ID NO.:20 ), the 1-235th position is the active fragment of TNFR2, the BAFFR active fragment marked in bold (positions 236-270), and the IgG Fc fragment marked by underline (positions 271-501);
  • Figure 1E shows the TNFR2-TACI -The amino acid sequence of the Fc fusion protein (SEQ ID NO.: 18), the 1-235th position is the TNFR2 active fragment, and the bold mark (the 236th-277th position) is
  • Figure 2 shows the results of reducing and non-reducing SDS-PAGE electrophoresis analysis of fusion proteins (TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc)
  • Figure 3 shows the in vitro binding activity of (3A) TNFR2-BAFFR-Fc, (3B) TNFR2-BCMA-Fc fusion proteins to human TNF[alpha].
  • Figure 4 shows the in vitro binding activity of (4A) TNFR2-BCMA-Fc, (4B) TNFR2-TACI-Fc fusion proteins to human BAFF.
  • Figure 5 shows the in vitro binding activity of (5A) TNFR2-BCMA-Fc, (5B) TNFR2-TACI-Fc fusion proteins to APRIL.
  • Figure 6 shows that in vitro binding of TNFR2-BCMA-Fc to TNFa was not affected by BAFF (6A) or APRIL (6B).
  • Figure 7 shows the activity of TNFR2-BCMA-Fc (7A), TNFR2-TACI-Fc (7B) fusion proteins in inhibiting TNF ⁇ -induced apoptosis of L929 cells.
  • Figure 8 shows that TNFR2-BCMA-Fc fusion protein inhibits the function of BAFF ( Figure 8A) or APRIL (8B) to protect RPMI8226 cells from being killed by DEX, where control is a control.
  • Figure 9 shows the function of TNFR2-BCMA-Fc fusion protein to inhibit LPS-induced death of mice from septic shock.
  • Figure 10 shows that TNFR2-BCMA-Fc significantly reduces the number of B cells in the blood (10A) and spleen (10B) of mice with etanercept being etanercept.
  • Figure 11 shows the inhibitory function of TNFR2-BCMA-Fc on inflammation in CIA mice.
  • (11A) shows the dose-response relationship of TNFR2-BCMA-Fc inhibiting the degree of inflammation in CIA mice;
  • (11B) shows the inhibitory effect of TNFR2-BCMA-Fc and etanercept (Enbrel) at a dose of 5 mg/kg
  • (11C) shows the inhibitory function of TNFR2-BCMA-Fc on inflammation in CIA mice.
  • Figure 12 shows the results of a toe histopathological study of the inhibitory effect of TNFR2-BCMA-Fc on inflammation in CIA mice.
  • (12A) shows the inhibitory effect of TNFR2-BCMA-Fc on joint tissue synovitis
  • (12B) shows the inhibitory effect of TNFR2-BCMA-Fc on joint tissue pannus
  • (12C) shows the inhibitory effect of TNFR2-BCMA-Fc on joint tissue pannus Inhibitory effect of bone erosion in joint tissue.
  • Figure 13 shows the effect of TNFR2-BCMA-Fc on the spleen of CIA mice.
  • 13A shows the function of TNFR2-BCMA-Fc to reduce the percentage of B lymphocytes in the spleen;
  • 13B shows the function of TNFR2-BCMA-Fc to inhibit spleen enlargement due to inflammation.
  • Figure 14 shows that TNFR2-BCMA-Fc inhibits the production of anti-collagen antibodies in CIA mice by CIA.
  • the present inventors discovered unexpectedly for the first time that by fusing (a) TNF receptor or its active fragment, (b) TACI, BCMA, BAFFR or its active fragment, and (c) the Fc region of an antibody to obtain
  • the fusion protein has excellent biological activity. While inhibiting TNF ⁇ -induced inflammation, the fusion protein of the present invention can efficiently recognize key molecules (such as BAFF or APRIL) in immune response, inhibit the activation of B cells, play a synergistic effect with TNFR2, and help treat some autoimmune diseases sexually transmitted diseases.
  • the TNFR2-BCMA-Fc fusion protein of the present invention can significantly reduce the content of B lymphocytes in the blood and spleen of mice, and the functions of inhibiting inflammation and spleen enlargement in CIA mice are significantly better than those of etanercept .
  • the present invention has been completed on this basis.
  • the inventors prepared optimized fusion proteins TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc. Studies have shown that the fusion proteins have strong biological activity and can block the binding of BAFF or APRIL to their cell membrane receptors .
  • the fusion protein of the present invention can significantly reduce the content of B lymphocytes in the spleen of C57 mice, inhibit TNFa-induced apoptosis, protect mice from shock death induced by LPS, and relieve osteoarticular inflammation in CIA and AIA rat models.
  • the fusion protein constructed by the present invention is a new combination of APRIL/BAFF related receptor and TNFR2, and the fusion protein has the advantages of precise identification, synergistic effect and controllable toxicity. Blocking the binding of BAFF-R, BCMA, TACI to ligands BAFF and APRIL on B cells by deceiving receptors while inhibiting TNF ⁇ and its downstream pathways to induce autoimmune responses. The fusion protein will further improve the therapeutic effect on autoimmune diseases by effectively regulating B cells, T cells and a series of cytokines.
  • Fc refers to the Fc fragment of a human immunoglobulin.
  • immunoglobulin Fc region refers to the constant region of an immunoglobulin chain, particularly the carboxy-terminus or a part thereof, of the constant region of an immunoglobulin heavy chain, for example, an immunoglobulin Fc region may include both heavy chains CH1, CH2, CH3.
  • the combination of one or more domains and the immunoglobulin hinge region, in a preferred embodiment, the Fc region of the immunoglobulin used includes at least one immunoglobulin hinge region, one CH2 domain and one CH3 domain, preferably lacking CH1 domain.
  • the Fc region of a globulin is within the purview of those skilled in the art, and in a preferred example, the Fc region of an immunoglobulin can be selected to comprise a coding sequence comprising an Fc region of a human immunoglobulin IgG4 subclass, in which an immunoglobulin Fc region is deleted.
  • the globulin heavy chain 1 domain (CH1) but includes the hinge region and the coding sequences for the CH2, CH3, and two domains.
  • a Proliferation Inducing Ligand APRIL
  • APRIL is a ligand of the TNF superfamily, which is secreted into the extracellular intercellular substance after intracellular synthesis.
  • APRIL exists in the form of homotrimers and can also form biologically active heterotrimers with BAFF.
  • APRIL mainly exerts its physiological functions by binding to the receptors BCMA and TACI.
  • APRIL enhances the proliferation and survival of plasma cells and also enhances T cell-dependent humoral immunity.
  • BAFF B cell stimulator factor
  • BAFF is a ligand of the TNF superfamily and its structure is a type II membrane-bound protein. BAFF is an essential factor for B cell survival and maturation. BAFF exerts its physiological effects mainly by binding to its receptors BAFF-R, BCMA and TACI. In a lupus mouse model, it was found that the content of soluble BAFF protein in serum was significantly positively correlated with the severity of the disease, which was mainly related to the increase of autoreactive B cells in peripheral blood, abnormal activation of B cells and the production of autoantibodies. In addition, the study found that transgenic mice overexpressing BAFF were prone to spontaneous SLE.
  • BAFF-R (BAFF receptor) is the receptor of BAFF, which can specifically bind to it, and its structure is a type III transmembrane protein.
  • BAFF-R plays a crucial role in B cell maturation. It promotes B cell proliferation and differentiates B cells into plasma cells by combining with soluble BAFF. It is an important receptor in the early stage of B cell development.
  • the combination of BAFF and BAFF-R activates a series of downstream pathways of protein synthesis and energy metabolism, thereby prolonging the half-life of immature B cells, transitional B cells, and mature B cells. After BAFF binds to BAFF-R, it can also continuously induce the activation of B cells through the NF- ⁇ B pathway and the PI3K pathway.
  • BAFF-R three receptors
  • BCMA B Cell Maturation Antigen
  • BCMA is a member of the tumor necrosis factor receptor superfamily. BCMA and its ligands play an important role in regulating the immune system, especially in regulating B cell proliferation, differentiation and apoptosis. BCMA is mainly expressed on mature B cells and plays an important role in maintaining B cell homeostasis, tolerance and terminal differentiation. The main function of BCMA is to mediate the long-term survival of plasma cells and maintain long-term humoral immunity. . BCMA is known to be closely related to the occurrence and development of multiple myeloma and autoimmune diseases.
  • Proliferation-inducing ligand (APRIL, or TALL-2, TRDL-1 and TNFSF-13) is a member of the TNF ligand superfamily, which is directly related to the development of B lymphocytes, T cell activation and humoral immunity. related.
  • Calmodulin ligand interactor transmembrane activator and calmodulin ligand interactor, TACI
  • TACI is the third receptor for BAFF in addition to BAFF-R and BCMA. It is currently believed that TACI negatively regulates the process of B cell maturation. In addition to binding to BAFF, TACI, like BCMA, can also bind to APRIL with high affinity. The combination of BCMA/TACI with APRIL or BAFF induces the activation of NF- ⁇ B and initiates multiple downstream signal transduction pathways, resulting in an over-activated immune response and then autoimmune diseases. TACI fusion protein has been confirmed to bind free BAFF and APRIL with high affinity in vivo and in vitro, and competitively inhibit its binding to receptors on lymphocytes (TACI, BCMA, BAFF-R), thereby reducing mature B cells and APRIL in circulation. amount of immunoglobulin.
  • fusion protein of the present invention refers to the fusion protein of the first aspect of the present invention.
  • the fusion protein is an isolated protein, not associated with other proteins, polypeptides or molecules, either as a purified product of recombinant host cell culture or as a purified extract.
  • the present invention provides a fusion protein comprising the following elements: (a) TNF receptor or active fragment thereof, (b) APRIL/BAFF receptor (eg TACI, BCMA and BAFFR) or active fragment thereof, and (c) antibody Fc region.
  • the elements eg, between element a and element b, element b or element c
  • the linker sequence is usually a sequence that has no effect on the two proteins.
  • the fusion protein of the present invention not only has a longer half-life in vivo, but also can more effectively inhibit the concentration of immune disease-related antibodies (especially IgE) in serum.
  • the amino acid sequence provided by the present invention those skilled in the art can easily prepare the fusion protein of the present invention by various known methods. These methods include, but are not limited to, recombinant DNA methods, artificial synthesis, and the like.
  • telomeres As a preferred mode of the present invention, it is particularly suitable for highly expressing the fusion protein of the present invention in eukaryotic cells (preferably CHO cells), including the TNFR2-BCMA-Fc fusion protein whose amino acid sequence is shown in SEQ ID NO.: 19. Full-length (ie, positions 1-515) or active fragments thereof, eg, the polypeptides shown at positions 55-515 (fusion proteins).
  • the fusion protein of the present invention comprises the following elements:
  • APRIL/BAFF receptor or its active fragment (a) TNF receptor or its active fragment; (b) APRIL/BAFF receptor or its active fragment, wherein said APRIL/BAFF receptor (APRIL and/or BAFF receptor) includes TACI, BCMA, BAFFR or its and optional (c) antibody Fc region; in the fusion protein of the present invention, between the elements (such as element a and element b, element b or element c), may contain or not Contains linker sequences.
  • the linker sequence is usually a sequence that has no effect on the two proteins.
  • fusion protein also includes fusion proteins having the above-mentioned activities (eg, TNFR2-TACI-Fc (amino acid sequence shown in SEQ ID NO.: 18), TNFR2-BCMA-Fc (amino acid sequence shown in SEQ ID NO.: 18), TNFR2-BCMA-Fc (amino acid sequence shown in SEQ ID NO. .: 19), a variant form of TNFR2-BAFFR-Fc (amino acid sequence shown in SEQ ID NO.: 20)).
  • TNFR2-TACI-Fc amino acid sequence shown in SEQ ID NO.: 18
  • TNFR2-BCMA-Fc amino acid sequence shown in SEQ ID NO.: 18
  • TNFR2-BCMA-Fc amino acid sequence shown in SEQ ID NO. .: 19
  • a variant form of TNFR2-BAFFR-Fc amino acid sequence shown in SEQ ID NO.: 20
  • variants include (but are not limited to): deletions, insertions and/or substitutions of 1-3 (usually 1-2, more preferably 1) amino acids, and C-terminal and/or N-terminal additions or One or several (usually within 3, preferably within 2, more preferably within 1) amino acids are deleted.
  • substitution with amino acids of similar or similar properties generally does not alter the function of the protein.
  • addition or deletion of one or several amino acids at the C-terminus and/or N-terminus usually does not alter the structure and function of the protein.
  • the term also includes monomeric and multimeric forms of the polypeptides of the invention.
  • the term also includes linear as well as nonlinear polypeptides (eg, cyclic peptides).
  • the present invention also includes active fragments, derivatives and analogs of the above fusion proteins.
  • fragment refers to polypeptides that substantially retain the function or activity of the fusion proteins of the present invention.
  • polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) in one or more A polypeptide with a substituent group in each amino acid residue, or (iii) a polypeptide formed by fusion of an antigenic peptide with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide formed by fusing this polypeptide sequence (a fusion protein formed by fusing with a leader sequence, a secretory sequence or a tag sequence such as 6 ⁇ His).
  • a class of preferred active derivatives refers to that compared with the amino acid sequence of formula I or formula II, there are at most 3, preferably at most 2, more preferably at most 1 amino acid replaced by amino acids with similar or similar properties. peptide. These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • the present invention also provides analogs of the fusion proteins of the present invention.
  • the differences between these analogs and the polypeptides shown in SEQ ID NO.: 18, 19 or SEQ ID NO.: 20 can be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both Of.
  • Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
  • Modified (generally without altering the primary structure) forms include chemically derivatized forms such as acetylation or carboxylation of the polypeptide in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modifications can be accomplished by exposing the polypeptide to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize their solubility properties.
  • the present invention also provides an expression vector comprising a sequence encoding the fusion protein of the present invention and an expression control sequence operably linked thereto.
  • the expression “operably linked” or “operably linked” refers to the condition that some parts of a linear DNA sequence are capable of modulating or controlling the activity of other parts of the same linear DNA sequence.
  • a promoter is operably linked to a coding sequence if it controls transcription of the sequence.
  • Expression and cloning vectors may contain one or more selectable genes, also known as selectable markers.
  • Typical screening genes encode proteins that can (a) resist antibiotics, etc.; (b) compensate for auxotrophic deficiencies or (c) provide critical nutrients that are not present in the medium.
  • DG44 cells deficient in DHFR dihydrofolate reductase deficient cells
  • hypoxanthine-thymine-free medium After cells are transfected with a DHFR-expressing vector, the transfected cells not only It can be grown in a medium without hypoxanthine-thymine, or in a medium containing a certain amount of MTX (methotrexate).
  • Expression vectors and cloning vectors usually contain one or more gene transcription promoters, either recognized by prokaryotic cell transcription machinery or eukaryotic cell transcription machinery. Promoters used for transcription in eukaryotic cells include, but are not limited to, cytomegalovirus (CMV) promoter, retrovirus promoter, simian virus 40 (SV40) prophase promoter, and the like.
  • CMV cytomegalovirus
  • SV40 simian virus 40 prophase promoter
  • expression vector commercially available vectors such as but not limited to: pIRES, pDR, pUC18 and the like can be used for expression in eukaryotic cell systems. Those skilled in the art can select a suitable expression vector according to the host cell.
  • those skilled in the art can insert the coding sequence of the fusion protein of the present invention into a suitable restriction site through restriction enzyme cutting and splicing according to conventional methods to prepare the present invention recombinant expression vector.
  • the present invention also provides a host cell expressing the fusion protein of the present invention, which contains the coding sequence of the fusion protein of the present invention.
  • the host cells are preferably eukaryotic cells, such as but not limited to CHO cells, COS cells, 293 cells, RSF cells and the like.
  • the cell is a CHO cell, which can well express the fusion protein of the present invention, and can obtain a fusion protein with good binding activity and good stability.
  • the present invention also provides a method for preparing the fusion protein of the present invention with recombinant DNA, the steps comprising:
  • the coding sequences can be introduced into host cells using a variety of 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 fusion protein prepared above can be purified to a substantially homogeneous nature, eg, as a single band on SDS-PAGE electrophoresis.
  • a commercial ultrafiltration membrane can be used to separate the protein, such as products from companies such as Millipore and Pellicon, and the expression supernatant is first concentrated.
  • the concentrate can be further purified by gel chromatography, or purified by ion exchange chromatography. For example, anion exchange chromatography (DEAE etc.) or cation exchange chromatography.
  • the gel matrix can be polyacrylamide, dextran, polyamide, etc., which are commonly used in protein purification. Q- or SP-groups are ideal ion exchange groups.
  • the above-mentioned purified product can be further refined and purified by methods such as hydroxyapatite adsorption chromatography, metal chelation chromatography, hydrophobic interaction chromatography and reversed-phase high performance liquid chromatography (RP-HPLC).
  • RP-HPLC reversed-phase high performance liquid chromatography
  • the expressed fusion protein can be purified using an affinity chromatography column containing specific antibodies, receptors or ligands for 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 other methods.
  • the amino terminus or carboxyl terminus of the fusion protein may also contain one or more polypeptide fragments as protein tags. Any suitable label can be used in the present invention.
  • the tag can be FLAG, HA, HA1, c-Myc, 6-His or 8-His and the like. These tags can be used to purify fusion proteins.
  • the present invention provides a fusion protein, which may optionally contain a peptide linker.
  • Peptide linker size and complexity may affect protein activity.
  • the peptide linker should be of sufficient length and flexibility to ensure that the two proteins being joined have sufficient freedom in space to perform their function. At the same time, the influence of the formation of ⁇ helix or ⁇ sheet in the peptide linker on the stability of the fusion protein is avoided.
  • the length of the linker peptide is generally 0-20 amino acids, preferably 0-10 amino acids.
  • the present invention also provides a pharmaceutical composition, which contains an effective amount (eg, 0.000001-90 wt %; preferably 0.1-50 wt %; more preferably, 5-40 wt %) of the fusion protein of the present invention, and pharmaceutically acceptable accepted vector.
  • an effective amount eg, 0.000001-90 wt %; preferably 0.1-50 wt %; more preferably, 5-40 wt % of the fusion protein of the present invention, and pharmaceutically acceptable accepted vector.
  • the fusion proteins of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably, the pH is about 6-8.
  • the term “effective amount” or “effective dose” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by using normal saline or an aqueous solution containing glucose and other adjuvants by conventional methods.
  • the pharmaceutical compositions are 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 made into a sustained-release preparation.
  • the effective amount of the fusion protein of the present invention may vary with the mode of administration and the severity of the disease to be treated. Selection of the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, and the administration. way etc. Generally, when the fusion protein of the present invention is administered at a dose of about 0.00001 mg-50 mg/kg animal body weight (preferably 0.0001 mg-10 mg/kg animal body weight) per day, satisfactory effects can be obtained. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.
  • the fusion protein of the present invention has the advantages of precise identification, synergistic effect and controllable toxicity.
  • the fusion protein of the present invention can efficiently recognize key molecules (such as TNFa, BAFF or APRIL) in immune response.
  • the fusion protein of the present invention can simultaneously bind to TNFa, and BAFF or APRIL, and simultaneously inhibit the biological functions of TNFa, and BAFF or APRIL.
  • the ribonucleotide sequence encoding the amino acid sequence of human TNFR2 (Met1-Asp257) and the ribonucleotide sequence encoding the amino acid sequence of human IgGFc were synthesized by GenScript (USA). The nucleotide sequence was synthesized by Genewiz (China).
  • PCR polymerase chain reaction
  • 5'-end primer KDP068 (SEQ ID NO: 1): 5'-CTTTGGCAAAGAATTGGG-3', located on the vector 5' of the gene.
  • the 3'-end primer KDP422 (SEQ ID NO: 2): 5'-GTCGCCAGTGCTCCCTTCAG-3' is a specific primer for the TNFR2 gene.
  • the first 20 nucleotide sequences of the primer KDP410 are complementary to the nucleotide sequence of the primer KDP422, and the first 19 nucleotide sequences of the primer KDP411 are complementary to the nucleotide sequence of the primer KDP134, so that in the overlap extension PCR of the second step During the process, the three PCR fragments can be ligated together.
  • the digested PCR fragment was then cloned into the same digested mammalian cell expression vector.
  • This mammalian cell expression vector is an improved pcDNA3.1 (Invitrogen).
  • the anti-neomycin (neomycin) gene in pcDNA3.1 is replaced by the DHFR (dihydrofolate reductase) gene.
  • the improved vector is suitable for screening for stable transfection. Mammalian cells with high protein expression.
  • the recombinant plasmid was transfected into DH5a competent bacteria, positive colonies containing the correct recombinant plasmid were identified by colony PCR, and the recombinant plasmid was purified.
  • amino acid sequence (SEQ ID NO.: 19) of the TNFR2-BCMA-Fc fusion protein is shown in Figure 1C, and the specific amino acids are as follows:
  • Fragment 1 Positions 1-235 of the TNFR2 amino acid sequence (SEQ ID NO.: 13);
  • Fragment 3 Positions 99-329 (SEQ ID NO.: 17) of the human IgGl amino acid sequence (Accession No. UniProtKB-P01857).
  • the TNFR2-BAFFR-Fc fusion protein gene was constructed by the method of Example 1.
  • the ribonucleotide sequence encoding the amino acid sequence of human BAFFR (Asp12-Ala46) was synthesized by Genewiz (China).
  • the first step is to amplify the gene of human TNFR2 (Met1-Asp257) and human IgG1 Fc (Glu99-Gly329) by PCR method (high-fidelity polymerase Pfx, Invitrogen) (the method is the same as that in Example 1).
  • the first 20 nucleotide sequences of the primer KDP408 are complementary to the nucleotide sequence of the primer KDP422, and the first 19 nucleotide sequences of the primer KDP409 are complementary to the nucleotide sequence of the primer KDP134, so that in the overlap extension PCR of the second step During the process, the three PCR fragments can be ligated together.
  • amino acid sequence (SEQ ID NO.: 20) of the TNFR2-BAFFR-Fc fusion protein is shown in Figure 1D, and the specific amino acids are as follows:
  • Fragment 1 Positions 1-235 of the TNFR2 amino acid sequence (SEQ ID NO.: 13);
  • Fragment 3 Positions 99-329 (SEQ ID NO.: 17) of the human IgGl amino acid sequence (Accession No. UniProtKB-P01857).
  • the TNFR2-TACI-Fc fusion protein gene was constructed by the method of Example 1.
  • the ribonucleotide sequence encoding the amino acid sequence of human TACI (Ser68-Arg109) was synthesized by Genewiz (China).
  • the first step is to amplify the gene of human TNFR2 (Met1-Asp257) and human IgG1 Fc (Glu99-Gly329) by PCR method (high-fidelity polymerase Pfx, Invitrogen) (the method is the same as that in Example 1).
  • the first 20 nucleotide sequences of the primer KDP412 are complementary to the nucleotide sequence of the primer KDP422, and the first 19 nucleotide sequences of the primer KDP412 are complementary to the nucleotide sequence of the primer KDP134, so that in the overlap extension PCR of the second step During the process, the three PCR fragments can be ligated together.
  • amino acid sequence (SEQ ID NO.: 18) of the TNFR2-TACI-Fc fusion protein is shown in Figure 1E, and the specific amino acids are as follows:
  • Fragment 1 Positions 1-235 of the TNFR2 amino acid sequence (SEQ ID NO.: 13);
  • Fragment 3 Positions 99-329 (SEQ ID NO.: 17) of the human IgGl amino acid sequence (Accession No. UniProtKB-P01857).
  • CHO-KS Chinese hamster ovary cells
  • FBS fetal bovine serum
  • OptiCHO medium Invitrogen
  • the transfected cells were cultured for 24-48 hours, and the transfected cells were screened and cultured on a 96-well culture plate by limiting dilution method.
  • the selection medium was OptiCHO, 5 ⁇ g/ml recombinant human insulin and 10 ⁇ M sulfoxide methionine (MSX). Cells were cultured in a 37 °C, 8% CO 2 incubator. After 3 weeks, the cell culture medium of each well with a cell population was analyzed by ELISA method (alkaline phosphatase-conjugated goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab), and cells positive for fusion protein expression were identified. The population is further expanded, detected by ELISA, and then amplified, and finally a stable cell line expressing the fusion protein is obtained.
  • ELISA method alkaline phosphatase-conjugated goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab
  • the cell lines stably expressing each fusion protein obtained in Example 4 were cultured and expanded.
  • the cell culture medium was centrifuged, the supernatant was collected, and the fusion protein was purified from the supernatant using a Protein-A affinity chromatography column.
  • the fusion protein of the present invention is a homologous covalent dimer, the molecular weight of TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc is not much different, and the dimer is about 112kDa (Fig. 2, lane 1, 3, 7, non-reducing SDS-PAGE), the monomer is about 56kDa ( Figure 2, lanes 2, 4, 8, reducing SDS-PAGE), all slightly larger than TNFR2-Fc (etanercept), its dimer and the molecular weight of the monomer were 102 kDa and 51 kDa, respectively (Fig. 2, lanes 5, 6).
  • Example 6 In vitro binding studies of TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc with recombinant human TNFa
  • the in vitro binding activity of the fusion protein to recombinant human TNFa was studied by ELISA.
  • Recombinant human TNFa (Novoprotein) was dissolved in PBS (pH 7.4) to a final concentration of 0.8 ⁇ g/mL, and 50 ⁇ L/well of recombinant protein was added to a 96-well ELISA plate, and was kept in a refrigerator at 4°C overnight. The next day, the ELISA plate was washed three times with PBST (PBS containing 0.05% Tween-20), and 100 ⁇ L/well of PBST containing 3% BSA blocking solution was added. The ELISA plate was placed in a 37°C incubator for 1 hour. Three-fold serial dilutions were prepared by separately diluting the fusion proteins in PBST containing 1% BSA binding solution.
  • Figure 3 shows that both fusion proteins TNFR2-BAFFR-Fc and TNFR2-BCMA-Fc can specifically bind to human TNF ⁇ .
  • TNFR2-BAFFR-Fc and TNFR2-BCMA-Fc bound to TNFa with affinity EC50 of 189 ng/mL and 46 ng/mL, respectively ( Figures 3A and 3B).
  • Example 7 Binding study of TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc to recombinant human BAFF
  • the binding activity of the fusion protein to recombinant human BAFF was studied by ELISA.
  • Recombinant human BAFF was dissolved with 50 mM NaHCO 3 (pH 9.6) to a final concentration of 1.0 ⁇ g/mL, and 50 ⁇ L/well of recombinant protein was added to a 96-well ELISA plate, and kept in a refrigerator at 4°C overnight. The next day, the ELISA plate was washed three times with PBST (PBS containing 0.05% Tween-20), and 100 ⁇ L/well of PBST containing 3% BSA blocking solution was added. The ELISA plate was placed in a 37°C incubator for 1 hour. 3-fold serial dilutions were prepared by separately diluting each fusion protein in PBST containing 1% BSA binding solution.
  • Figure 4 shows that both fusion proteins TNFR2-BCMA-Fc and TNFR2-TACI-Fc can specifically bind to recombinant human BAFF in vitro.
  • Example 8 Binding study of TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc to recombinant human APRIL
  • the binding activity of the fusion protein to recombinant human APRIL was studied by ELISA.
  • Recombinant human APRIL was dissolved with 50 mM NaHCO 3 (pH 9.6) to a final concentration of 1.0 ⁇ g/mL, and 50 ⁇ L/well of recombinant protein was added to a 96-well ELISA plate, and kept in a refrigerator at 4°C overnight. The next day, the ELISA plate was washed three times with PBST (PBS containing 0.05% Tween-20), and 100 ⁇ L/well of PBST containing 3% BSA blocking solution was added. The ELISA plate was placed in a 37°C incubator for 1 hour. 3-fold serial dilutions were prepared by separately diluting each fusion protein in PBST containing 1% BSA binding solution.
  • Figure 5 shows that both fusion proteins TNFR2-BCMA-Fc and TNFR2-TACI-Fc can specifically bind to recombinant human APRIL in vitro.
  • TNFR2-BCMA-Fc and TNFR2-TACI-Fc bound APRIL with affinities of 23 ng/mL and 104 ng/mL, respectively ( Figures 5A and 5B).
  • TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc fusion proteins simultaneously bind TNFa and BAFF or APRIL without affecting each other
  • the fusion protein of the present invention binds TNFa and BAFF or APRIL with high affinity.
  • Recombinant human TNFa (Novoprotein) is dissolved in PBS (pH 7.4) to a final concentration of 0.8 ⁇ g/mL, and 50 ⁇ L/well of rhTNFa is added to a 96-well ELISA plate at 4° C. overnight in the refrigerator. The next day, the ELISA plate was washed three times with PBST (PBS containing 0.05% Tween-20), and 100 ⁇ L/well of PBST containing 3% BSA blocking solution was added. The ELISA plate was placed in a 37°C incubator for 1 hour.
  • Fusion proteins were serially diluted 3-fold in binding solution (PBST with 1% BSA) with or without BAFF or APRIL at 1 ⁇ g/mL. Pour off the blocking solution, add serially diluted fusion proteins, 50 ⁇ L/well, and react in a 37°C incubator for 1 hour. The solution was poured off, the ELISA plate was washed three times with PBST, 50 ⁇ L/well of secondary antibody (alkaline phosphatase-conjugated goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab) was added, and the reaction was carried out in a 37°C incubator for 1 hour.
  • secondary antibody alkaline phosphatase-conjugated goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab
  • TNFR2-BCMA-Fc bound TNFa identically in solutions with and without BAFF (Fig. 6A), with no significant difference in binding to TNFa in solutions with and without APRIL (Fig. 6B).
  • Example 10 Study on the biological activity of TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc fusion proteins inhibiting TNFa-induced apoptosis
  • the TNFR2 biological activity study method of the fusion protein uses in vitro neutralization of the biological activity of TNFa.
  • TNFa biological activity was detected by mouse fibroblast L929 cytotoxicity. 5ng/mL of rhTNFa was mixed with serial dilutions of different concentrations of each fusion protein, and then added to L929 cells cultured in a 96-well culture plate. The viability of L929 cells was detected by crystal sub-staining method after culturing for 20 hours.
  • Figure 7 shows the results of the study that the fusion protein inhibits TNFa-induced apoptosis.
  • the EC50 of TNFR2-BCMA-Fc and TNFR2-TACI-Fc inhibition of TNFa-induced apoptosis in L929 cells was 27 ng/mL and 23 ng/mL, respectively ( Figures 7A and 7B).
  • TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc fusion proteins inhibit the growth of RPMI8226 cells stimulated by BAFF and APRIL in vitro
  • RPMI8226 is a human multiple myeloma cell.
  • Dexamethasone (DEX) can induce RPMI8226 cell death.
  • BAFF or APRIL can inhibit DEX-induced RPMI8226 cell death.
  • RPMI8226 cells were cultured in 96-well plate RPMI1660 medium (containing 10% FBS), each reagent shown in Figure 8 was added, and cultured in a cell incubator for 5 days, and then the number of viable cells was detected with CCK-8.
  • TNFR2-BCMA-Fc inhibited the function of BAFF, reducing the cell viability of RPMI8226 to 42%, with a significant inhibitory effect on BAFF activity (p ⁇ 0.0001) (Fig. 8A).
  • TNFR2-BCMA-Fc also inhibited the function of APRIL to protect RPMI8226 cells, reducing the survival rate of RPMI8226 cells from 63% to 46% (p ⁇ 0.0001) (Fig. 8B).
  • the results of this experiment prove that the fusion protein of the present invention has the function of inhibiting BAFF or APRIL to protect the survival of RPMI8226 cells.
  • TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc fusion proteins reduce LPS-induced mortality in mice with septic shock
  • mice To study the effect of the fusion protein on LPS-induced septic shock death in mice, and to evaluate the biological activity of the fusion protein to neutralize TNFa in vivo. Sixteen 7-8 week old Balb/c male mice were divided into 2 groups with 8 mice in each group. Each mouse was intraperitoneally injected with 1 mg of LPS, and then the two groups of mice were injected with PBS and 6 mg/kg of TNFR2-BCMA-Fc intravenously, respectively. The state of the mice was observed in the following 80 hours, and the time of death of the mice was recorded.
  • Figure 9 shows that within 24 hours after administration of LPS, 2 mice in the PBS group died, while no mice in the TNFR2-BCMA-Fc group died. Within 41 hours, another 5 mice in the PBS group died, with a mortality rate of 88%, and all the mice in the PBS group died within 48 hours. In the TNFR2-BCMA-Fc group, 5 mice died within 41 hours, with a mortality rate of 63%, and 1 mouse died within 72 hours. A total of 6 mice died within 80 hours, with a mortality rate of 75%.
  • TNFR2-BCMA-Fc could reduce the mortality of mice induced by LPS in shock.
  • Example 13 Study on TNFR2-BAFFR-Fc, TNFR2-BCMA-Fc and TNFR2-TACI-Fc fusion proteins reducing the content of B lymphocytes in mice
  • the fusion protein was injected into the mice twice, the content of B lymphocytes in the spleen of the mice was detected, and the effect of the fusion protein on B lymphocytes was evaluated.
  • mice Male C57BL/6 mice aged 6-7 weeks were randomly divided into groups according to body weight, 5 mice in each group, and the corresponding drugs were administered in the tail veins of D1 and D5.
  • D9 collect 100 ⁇ L of mouse fundus blood, add 100 ⁇ L of PBS containing anticoagulant to each, add 1 ⁇ L of anti-mouse B220(CD45R)-APC and anti-mouse CD3-FITC, after staining on ice for 30min, add 6 times the volume of Flow hemolysin was lysed, shaken and allowed to stand for 10 minutes, centrifuged at 800G for 15 minutes, and 200 ⁇ L of FACS working solution was added to resuspend.
  • the proportion of B220+ cell population and CD3+ cell population in the samples was detected by flow cytometry. On D10, all mice were sacrificed by decapitation, blood was collected, spleen was dissected, and spleen lymphocytes were isolated. The proportion of B220+ cell population and CD3+ cell population in spleen lymphocytes was detected by the same method as above.
  • the membrane protein B220 is a biomarker of mouse B lymphocytes, and all B cells express B220.
  • Figure 10 shows that TNFR2-BCMA-Fc can significantly reduce the content of B lymphocytes in mouse blood (Figure 10A) and spleen ( Figure 10B), 7.5mg/kg and 15mg/kg doses of TNFR2-BCMA-Fc decreased respectively.
  • the content of B lymphocytes in the blood was 37% and 43%, and the content of B lymphocytes in the spleen was decreased by 44% and 56%.
  • Etanercept did not have any effect on the content of B lymphocytes in the blood, and had a reduced effect on the content of B lymphocytes in the spleen (23% reduction), but it was significantly weaker than the same dose of TNFR2-BCMA-Fc. effect (44% reduction).
  • Example 14 Inflammatory effects of TNFR2-BCMA-Fc fusion protein on collagen-induced arthritis (CIA) mouse model
  • mice were divided into 6 groups of 9 mice each. Mice were immunized with Collagen Emulsion A (Bovine type II collagen + Complete Freund's Adjuvant) on DO and Collagen Emulsion B (Bovine type II collagen + Incomplete Freund's Adjuvant) on D21. On D21, 3 doses of TNFR2-BCMA-Fc (1.25, 5 and 20 mg/kg) and 2 doses of etanercept (Enbrel) were intraperitoneally injected into 5 groups of mice, respectively, and mice in group 6 were intraperitoneally administered with PBS. In addition, set 1 group including 4 normal mice. Dosing 3 times a week for a total of 4 weeks. The trial ends on D49. The degree of inflammation in mice was assessed by clinical joint score and measurement of hindpaw thickness three times a week.
  • the entire left hind paw was taken, fixed with 4% paraformaldehyde, and then soaked in 75% ethanol for preservation.
  • HE staining and histopathological analysis were performed on the ankle tarsal toe joint (Wuhan Sewell Biotechnology Co., Ltd.). In a quantitative manner, the severity of synovial inflammation, pannus formation, and bone erosion and cartilage damage was assessed.
  • the spleens of all mice were harvested, the spleen was weighed, the spleen lymphocytes were separated, the cells were stained with anti-CD3 antibody and anti-B220 antibody, and the numbers of T lymphocytes and B lymphocytes were detected by flow cytometry.
  • the spleen weight and the changes of B lymphocytes and T lymphocytes were compared in each group.
  • the titers of anti-collagen antibodies in mouse serum at the end of the experiment were measured to evaluate the effect of TNFR2-BCMA-Fc on the production of anti-collagen antibodies.
  • TNFR2-BCMA-FC The pathological study (hematoxylin-eosin staining) of the dorsal joint tissue of the mouse toes showed that 3 doses of TNFR2-BCMA-FC could inhibit the synovitis of the joint tissue (Fig. 12A) and pannus (Fig. 12B). and the extent of bone erosion (Fig. 12C).
  • the dorsal tissue of the toes of all mice in the 5 mg/kg and 20 mg/kg dose groups of TNFR2-BCMA-FC was the same as the normal tissue. 1.25mg/kg of TNFR2-BCMA-FC also had a partial protective effect on tissues, which was better than 5mg/kg of Enbrel.
  • the pathological results of joint tissue also proved that TNFR2-BCMA-FC inhibited inflammation in CIA mice significantly better than etanercept.
  • TNFR2-BCMA-Fc By analyzing the content of B lymphocytes in the spleen of mice, 5 and 20 mg/kg doses of TNFR2-BCMA-Fc were shown to reduce the percentage of B lymphocytes in the spleen (Fig. 13A). The same dose of Enbrel also reduced B lymphocytes in the spleen. Cell percentage, but the effect was weaker than that of TNFR2-BCMA-Fc, especially at the dose of 20 mg/kg, TNFR2-BCMA-Fc reduced the percentage of B lymphocytes significantly stronger than Enbrel (p ⁇ 0.0001). Inflammation caused spleen enlargement compared to normal mice (FIG. 13B, PBS group). The 5 and 20 mg/kg doses of TNFR2-BCMA-Fc inhibited spleen enlargement (FIG. 13B), and had a stronger inhibitory effect than the same dose of Enbrel.
  • TNFR2-BCMA-Fc was able to inhibit the titers of anti-collagen antibodies ( Figure 14). Compared with the PBS group, the 20 mg/kg dose of TNFR2-BCMA-Fc significantly reduced the titer of anti-collagen antibodies in serum, while the same dose of Enbrel had no effect on antibody production.

Abstract

本发明提供了一类TNFR2与APRIL/BAFF受体蛋白融合的制法和应用。具体地，本发明提供了一类融合蛋白，所述融合蛋白包括TNFR2胞外段和BAFFR或BCMA或TACI的胞外段。本发明的融合蛋白在抑制TNFα诱导的炎症的同时，通过结合BAFF或APRIL，抑制B细胞的激活，起到与TNFR2协同作用，增强对各类自身免疫性疾病治疗的效果。

Description

TNFR2与APRIL/BAFF受体的融合蛋白 技术领域
本发明涉及生物技术领域。具体地，涉及一种TNFR2与APRIL/BAFF受体的融合蛋白。
背景技术
自身免疫性疾病是自身细胞和体液对自身机体产生反应的一类疾病，目前全球范围内自身免疫性疾病的发病率和患病率都呈逐年上升状态。常见的自身免疫疾病包括类风湿关节炎(RA)、强直性脊柱炎(AS)、银屑病(PS)、系统性红斑狼疮(SLE)等，给患者的工作能力和生活质量带来了很大的影响。近年来，TNFα靶向抑制剂通过拮抗TNFα，并抑制其下游IL-2、IL-6、IFN-γ等细胞因子的上调，同时抑制被过度激活的T细胞，有效的控制了疾病的进程，被广泛应用于多种自身免疫性疾病。虽然TNFα抑制剂取得了不错的疗效，但是仍有50％患者治疗效果不甚理想，因此亟需新的组合来实现对自身免疫性疾病更有效的控制。
本发明提供了一种更安全、有效、精准用于自身免疫性疾病的治疗性融合蛋白。
发明内容
本发明的目的在于提供一种更安全、有效、精准用于自身免疫性疾病的治疗性融合蛋白。
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)APRIL/BAFF受体或其活性片段，其中所述的APRIL/BAFF受体包括TACI，BCMA，BAFFR或其组合；
和任选的(c)抗体Fc区域；
其中，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TNF受体选自下组：TNFR2、TNFR1、或其组合。
在另一优选例中，所述TNF受体来源于人或非人哺乳动物，更佳地来源于啮齿动物(如小鼠、大鼠)、灵长动物和人。
在另一优选例中，所述TNF受体包括野生型和突变型。
在另一优选例中，所述TNF受体包括全长的、成熟形式的TNF受体，或其活性片段。
在另一优选例中，所述TNF受体还包括TNF受体的衍生物。
在另一优选例中，所述TNF受体的衍生物包括经修饰的TNF受体、氨基酸序列与天然TNF受体同源且具有天然TNF受体活性的蛋白分子、TNF受体的二聚体或多聚体、含有TNF受体氨基酸序列的融合蛋白。
在另一优选例中，所述经修饰的TNF受体是PEG化的TNF受体。
在另一优选例中，所述“氨基酸序列与天然TNF受体同源且具有天然TNF受体活性的蛋白分子”是指其氨基酸序列与TNF受体相比具有≥85％的同源性，较佳地≥90％的同源性，更佳地≥95％的同源性，最佳地≥98％同源性；并且具有TNF受体活性的蛋白分子。
在另一优选例中，所述TNF受体包括第一结构域、第二结构域、第三结构域、和/或第四结构域。
在另一优选例中，所述第一结构域、第二结构域、第三结构域、和/或第四结构域各自独立地为半胱氨酸富集区(CRD)。
在另一优选例中，所述的TNF受体为含有或具有TNFR2氨基酸序列(SEQ ID NO.:13)的第1-235位，或第17-179位，或第17-140位，或第55-179位，或第55-140位。
在另一优选例中，所述APRIL/BAFF受体来源于人或非人哺乳动物，更佳地来源于啮齿动物(如小鼠、大鼠)、灵长动物和人。
在另一优选例中，所述APRIL/BAFF受体包括野生型和突变型。
在另一优选例中，所述APRIL/BAFF受体还包括APRIL/BAFF受体的衍生物。
在另一优选例中，所述APRIL/BAFF受体的衍生物包括经修饰的APRIL/BAFF受体、氨基酸序列与天然APRIL/BAFF受体同源且具有天然APRIL/BAFF受体活性的蛋白分子、APRIL/BAFF受体的二聚体或多聚体、含有APRIL/BAFF受体氨基酸序列的融合蛋白。
在另一优选例中，所述“氨基酸序列与天然APRIL/BAFF受体同源且具有天然APRIL/BAFF受体活性的蛋白分子”是指其氨基酸序列与APRIL/BAFF受体相比具有≥85％的同源性，较佳地≥90％的同源性，更佳地≥95％的同源性，最佳地≥98％同源性；并且具有APRIL/BAFF受体活性的蛋白分子。
在另一优选例中，所述APRIL/BAFF受体选自下组：TACI、BCMA、BAFFR，或其组合。
在另一优选例中，所述TACI含有或具有TACI氨基酸序列(SEQ ID NO.:14)的第1-109位，或第33-109位，或第68-109位。
在另一优选例中，所述BCMA含有或具有BCMA氨基酸序列(SEQ ID NO.:15)的第1-54位，或第6-54位。
在另一优选例中，所述BAFFR含有或具有BAFFR氨基酸序列(SEQ ID NO.:16)的第1-78位，或第12-78位，或第12-46位。
在另一优选例中，所述Fc片段来源于人或非人哺乳动物，更佳地来源于啮齿动物(如小鼠、大鼠)、灵长动物和人。
在另一优选例中，所述Fc片段为免疫球蛋白IgG的Fc片段，较佳地为IgG1的Fc部分。
在另一优选例中，所述Fc片段包括天然的Fc片段及Fc突变体。
在另一优选例中，所述的Fc片段含有或具有人IgG1氨基酸序列(登录号为UniProtKB-P01857)的第99-329位。
在另一优选例中，所述Fc片段的氨基酸序列如SEQ ID NO.:17所示。
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TNFα的活性；
(b)抑制TNFα诱导的炎症；
(c)结合BAFF或APRIL的活性；
(d)抑制或封闭BAFF/APPRIL途径；
(e)降低体内B细胞的数量；
(f)降低脾脏或血液中的B淋巴细胞含量；
(g)抑制脾脏增大；
(h)抑制APRIL/BAFF诱导的疾病。
在另一优选例中，所述融合蛋白具有下式I或II所示的二聚体结构：
X-Y-Z  (I)
Y-X-Z  (II)；
式中，
X为TNF受体的胞外段；
Y为TACI，BCMA或BAFFR的胞外段；
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，所述融合蛋白为同源二聚体。
在另一优选例中，所述融合蛋白的氨基酸序列如SEQ ID NO.:18或19或20所示。
在另一优选例中，所述的X、Y、Z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N端，尤其是野生型多肽的或其片段的N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，较佳地，1-10个氨基酸。
在另一优选例中，所述的X含有或具有TNFR2氨基酸序列(SEQ ID NO.:13)的第1-235位，或第17-179位，或第17-140位，或第55-179位，或第55-140位；和/或
所述的Y含有或具有：
(a)TACI氨基酸序列(SEQ ID NO.:14)的第1-109位，或第33-109位，或第68-109位；
(b)BCMA氨基酸序列(SEQ ID NO.:15)的第1-54位，或第6-54位；或
(c)BAFFR氨基酸序列(SEQ ID NO.:16)的第1-78位，或第12-78位，或第12-46位。
在另一优选例中，所述的Z含有或具有人IgG1氨基酸序列(登录号为UniProtKB-P01857)的第99-329位。
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的第一方面所述的融合蛋白。
在另一优选例中，所述的核酸分子在所述突变蛋白或融合蛋白的ORF的侧翼还额外含有选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His)、或其组合。
在另一优选例中，所述的核酸分子选自下组：DNA序列、RNA序列、或其组合。
在本发明的第三方面，提供了一种载体，它含有在本发明的第二方面所述 的核酸分子。
在另一优选例中，所述载体包含一个或多个启动子，所述启动子可操作地与所述核酸序列、增强子、转录终止信号、多腺苷酸化序列、复制起点、选择性标记、核酸限制性位点、和/或同源重组位点连接。
在另一优选例中，所述载体包括质粒、病毒载体。
在另一优选例中，所述的病毒载体选自下组：腺相关病毒(AAV)、腺病毒、慢病毒、逆转录病毒、疱疹病毒、SV40、痘病毒、或其组合。
在另一优选例中，所述载体包括表达载体、穿梭载体、整合载体。
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的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的第二方面所述的核酸分子。
在另一优选例中，所述基因工程化的细胞为真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。
在另一优选例中，所述基因工程化的细胞为原核细胞，如大肠杆菌。
在另一优选例中，所述基因工程化的细胞选自下组：大肠杆菌、麦胚细胞，昆虫细胞，SF9、SP2/0、Hela、HEK293、CHO(比如CHOKS)、酵母细胞、或其组合。
在本发明的第五方面，提供了一种产生本发明第一方面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，培养本发明的第四方面所述的基因工程化的细胞，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第一方面所述的融合蛋白及其药物学上可接受的载体。
在另一优选例中，所述药物组合物还包括其他用于治疗免疫性疾病的药物。
在另一优选例中，其他用于治疗免疫性疾病的药物选自下组：激素类药物、免疫抑制药物、小分子靶向药物、生物制剂、或其组合。
在另一优选例中，所述激素类药物选自下组：可的松、氢化可的松、强的松、 波尼松、波尼松龙、地塞米松、倍他米松、或其组合。
在另一优选例中，所述免疫抑制药物选自下组：甲氨蝶呤、环磷酰胺、咪唑硫嘌呤、环孢素、霉酚酸酯、他克莫司、西罗莫司、来氟米特、柳氮磺砒啶、羟氯喹、或其组合。
在另一优选例中，所述小分子靶向药物选自下组：托法替布、巴瑞替尼、或其组合。
在另一优选例中，所述生物制剂选自下组：依那西普、赛妥珠单抗、阿达木单抗、戈利木单抗、英夫利昔单抗、托珠单抗、苏金单抗、优特克单抗、卡纳单抗、阿那白滞素、利纳西普、阿巴西普、利妥昔单抗、贝利尤单抗、或其组合。
在本发明的第七方面，提供了一种如本发明第一方面所述的融合蛋白、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、本发明第四方面所述的基因工程化的细胞的用途，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)治疗免疫疾病的组合物；和/或
(c)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，所述免疫性疾病选自下组：类风湿关节炎(RA)、强直性脊柱炎(AS)、银屑病(PS)、银屑病关节炎(PsA)、幼年特发性关节炎(JIA)、系统性红斑狼疮(SLE)、白塞病(behcet’s disease,BD)、多发性硬化症(MS)、干燥综合征(SS)、Graves病、克罗恩病(CD)、溃疡性结肠炎(UC)、原发性肾小球肾炎、IgA肾病、自身免疫性血管炎、多发性肌炎(PM)、非感染性葡萄膜炎、自身免疫性溶血性贫血(AIHA)、自身免疫性紫癜(ATTP)、N-甲基-d-天冬氨酸受体(NMDAR)脑炎、重症肌无力、化脓性汗腺炎(HS)、髓鞘-少突胶质细胞糖蛋白谱紊乱(MOGSD)和视神经脊髓炎频谱障碍(NMOSD)、或其组合。
在另一优选例中，所述免疫性疾病包括自身免疫性疾病，例如类风湿关节炎(RA)、强直性脊柱炎(AS)、银屑病(PS)、银屑病关节炎(PsA)、幼年特发性关节炎(JIA)、系统性红斑狼疮(SLE)、自身免疫性紫癜(ATTP)、白塞病、IgA肾病。
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。
在本发明的第八方面，提供了一种治疗免疫性疾病的方法，向有需要的患者施用如本发明第一方面所述的融合蛋白。
在另一优选例中，所述的融合蛋白以单体和/或二聚体形式施用。
在另一优选例中，所述免疫性疾病包括类风湿关节炎(RA)、强直性脊柱炎(AS)、银屑病(PS)、银屑病关节炎(PsA)、幼年特发性关节炎(JIA)、系统性红斑狼疮(SLE)、白塞病(BD)、多发性硬化症(MS)、干燥综合征(SS)、Graves病、克罗恩病(CD)溃疡性结肠炎(UC)、原发性肾小球肾炎、自身免疫性血管炎、多发性肌炎(PM)、非感染性葡萄膜炎、自身免疫性溶血性贫血(AIHA)、自身免疫性紫癜(ATTP)、N-甲基-d-天冬氨酸受体(NMDAR)脑炎、重症肌无力、化脓性汗腺炎(HS)、髓鞘-少突胶质细胞糖蛋白谱紊乱(MOGSD)和视神经脊髓炎频谱障碍(NMOSD)。
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显示了融合蛋白的结构示意图(1A和1B)，图1C显示了TNFR2-BCMA-Fc融合蛋白的氨基酸序列(SEQ ID NO.:19)，第1-235位为TNFR2活性片段，加粗标记(第236-284位)的是BCMA活性片段，下划线标记(第285-515位)的是IgG Fc片段；图1D显示了TNFR2-BAFFR-Fc融合蛋白的氨基酸序列(SEQ ID NO.:20)，第1-235位为TNFR2活性片段，加粗标记(第236-270位)的是BAFFR活性片段，下划线标记(第271-501位)的是IgG Fc片段；图1E显示了TNFR2-TACI-Fc融合蛋白的氨基酸序列(SEQ ID NO.:18)，第1-235位为TNFR2活性片段，其中加粗标记(第236-277位)的是TACI活性片段，下划线标记(第278-508位)的是IgG Fc片段。
图2显示了融合蛋白(TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc)的还原和非还原SDS-PAGE电泳分析结果
图3显示了(3A)TNFR2-BAFFR-Fc、(3B)TNFR2-BCMA-Fc融合蛋白体外结合人TNFα的活性。
图4显示了(4A)TNFR2-BCMA-Fc、(4B)TNFR2-TACI-Fc融合蛋白体外结合人BAFF的活性。
图5显示了(5A)TNFR2-BCMA-Fc、(5B)TNFR2-TACI-Fc融合蛋白体外结合APRIL的活性。
图6显示了TNFR2-BCMA-Fc与TNFa的体外结合不被BAFF(6A)或APRIL(6B)影响。
图7显示了TNFR2-BCMA-Fc(7A)、TNFR2-TACI-Fc(7B)融合蛋白抑制TNFα诱导L929细胞凋亡的活性。
图8显示了TNFR2-BCMA-Fc融合蛋白抑制BAFF(图8A)或APRIL(8B)保护RPMI8226细胞被DEX杀伤的功能，其中control为对照。
图9显示了TNFR2-BCMA-Fc融合蛋白抑制LPS诱导的小鼠感染性休克死亡的功能。
图10显示了TNFR2-BCMA-Fc显著降低小鼠血液(10A)和脾脏(10B)中B细胞的数量，其中etanercept为依那西普。
图11显示了TNFR2-BCMA-Fc对CIA小鼠炎症的抑制功能。(11A)显示了TNFR2-BCMA-Fc抑制CIA小鼠炎症发生程度的量效关系；(11B)显示了在5mg/kg的剂量下TNFR2-BCMA-Fc与依那西普(Enbrel)抑制炎症效果的比较；(11C)显示了在20mg/kg的剂量下TNFR2-BCMA-Fc与Enbrel抑制炎症效果的比较。
图12显示了TNFR2-BCMA-Fc对CIA小鼠炎症抑制效果的脚趾组织病理研究结果。(12A)显示了TNFR2-BCMA-Fc对关节组织滑膜炎的抑制效果；(12B)显示了TNFR2-BCMA-Fc对关节组织血管翳的抑制效果；(12C)显示了TNFR2-BCMA-Fc对关节组织骨侵蚀的抑制效果。
图13显示了TNFR2-BCMA-Fc对CIA小鼠脾脏的影响。(13A)显示了TNFR2-BCMA-Fc降低脾脏中B淋巴细胞百分比的功能；(13B)显示了TNFR2-BCMA-Fc抑制因炎症引起的脾脏增大的功能。
图14显示了TNFR2-BCMA-Fc对CIA抑制CIA小鼠的抗胶原抗体的产生。
具体实施方式
本发明人经过广泛而深入地研究，首次意外发现，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优异的生物活性。本发明的融合蛋白在抑制TNFα诱导的炎症的同时，能够高效识别免疫反应中关键分子(如BAFF或APRIL)，抑制B细胞的激活，起到与TNFR2协同作用，有助于治疗某些自身免疫性疾病。特别地，本发明中的TNFR2-BCMA-Fc融合蛋白能够显著降低小鼠血液中和脾脏中的B淋巴细胞含量，抑制CIA小鼠炎症效果、抑制脾脏增大的功能显著优于依那西普。在此基础上完成了本发明。
具体地，本发明人制备了优化的融合蛋白TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc，研究表明，融合蛋白生物活性强，能够阻断BAFF或APRIL与其细胞膜受体结合。此外，本发明的融合蛋白能够明显降低C57小鼠脾脏B淋巴细胞的含量，抑制TNFa诱导细胞凋亡，保护LPS诱导小鼠休克死亡，缓解CIA和AIA大鼠模型骨关节炎症。
本发明构建的APRIL/BAFF相关受体与TNFR2的全新组合的融合蛋白，该融合蛋白具有精确识别、协同作用、毒性可控的优势。在抑制TNFα及其下游通路引起自身免疫反应的同时，通过欺骗受体阻断B细胞上BAFF-R、BCMA、TACI与配体BAFF和APRIL的结合。该融合蛋白将通过对B细胞、T细胞以及一系列细胞因子有效的调控，进一步提高对自身免疫性疾病的疗效。
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IgG1、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。
增殖诱导配体(A Proliferation Inducing Ligand，APRIL)
APRIL是TNF超级家族的配体，在细胞内合成后被分泌到细胞外的细胞间素。APRIL以同源三聚体形式存在，也能与BAFF形成具有生物活性的异源三聚体。APRIL主要通过结合受体BCMA和TACI发挥生理功能。APRIL增强浆细胞的增殖和存活，也能加强T细胞依赖性的体液免疫。
B细胞活化因子(B lymphocyte stimulator factor,BAFF)
BAFF是TNF超级家族的配体，其结构是一种II型膜结合蛋白。BAFF是B细胞存活与成熟的必需因子。BAFF主要通过与其受体BAFF-R、BCMA和TACI结合发挥生理作用。在狼疮小鼠模型中发现，血清中可溶性BAFF蛋白含量与疾病严重程度间呈显著正相关，主要与外周血自身反应性B细胞增加、B细胞异常活化以及自身抗体产生有关。另外，研究发现BAFF过表达的转基因小鼠易自发SLE。
BAFF-R(BAFF receptor)是BAFF的受体，可特异性与其结合，其结构是一种III型跨膜蛋白。BAFF-R在B细胞成熟阶段起到至关重要的作用，其通过与可溶性BAFF结合后促进B细胞增殖，并使B细胞向浆细胞分化，是B细胞发育早期阶段的重要受体。BAFF与BAFF-R结合后激活了一系列蛋白质合成和能量代谢的下游途径，进而延长了未成熟B细胞、过渡B细胞以及成熟B细胞的半衰期。BAFF与BAFF-R结合后，还可以通过NF-κB途径以及PI3K途径持续诱导B细胞的活化。研究显示，在系统性红斑狼疮患者中同样存在着BAFF的异常表达增高，而给予抗BAFF抗体后可有效的抑制体内可溶性的BAFF与其三种受体(BAFF-R、BCMA、TACI)的结合，进而抑制B细胞的分化和存活，达到治疗效果。
B细胞成熟抗原(B Cell Maturation Antigen，BCMA)
BCMA是肿瘤坏死因子受体超家族的成员，BCMA及其配体在调节免疫系统，尤其是调控B细胞增殖、分化和凋亡中起着重要的作用。BCMA主要在成熟的B细胞上表达，与维持B细胞的稳态、耐受性和终末分化中起着重要的作用，BCMA主要功能是介导浆细胞的长期存活并维持长效的体液免疫。BCMA已知与多发性骨髓瘤以及自身免疫性疾病的发生发展都有着紧密的联系。增殖诱导 配体(A proliferation-inducing ligand,APRIL，或称TALL-2、TRDL-1及TNFSF-13)是TNF配体超家族的成员，与B淋巴细胞的发育、T细胞活化及体液免疫直接相关。
既往研究表明，B细胞上表达的BCMA的调控作用与其配体BAFF和APRIL有着紧密的联系。APRIL/BAFF与BCMA结合在体内和体外都被证明是B细胞激活和抗体产生的共刺激分子。在狼疮模型小鼠中进行干预阻断BAFF和APRIL后，可以发现外周血B细胞和浆细胞数量都显著降低。研究显示，BCMA与其配体BAFF或APRIL的结合会启动浆细胞抗凋亡基因的转录，延长细胞的存活时间。一项研究显示，在类风湿关节炎患者中发现了APRIL显著增加并加剧了疾病的进展，而在使用BCMA-Fc融合蛋白进行干预后，发现BCMA-Fc融合蛋白在体内可以作为欺骗受体高亲和力的结合APRIL，进而抑制APRIL的生物活性，控制自身免疫性疾病的进展。
钙调亲环素配体相互作用分子(transmembrane activator and calmodulin ligand interactor，TACI)
TACI是除BAFF-R和BCMA外，BAFF的第三种受体。目前认为TACI在B细胞成熟的过程中产生负性调节作用。除了与BAFF结合外，TACI同BCMA一样，还可与APRIL高亲和力的结合。BCMA/TACI与APRIL或BAFF结合后诱导NF-κB激活，并启动下游多个信号转导途径，导致过度激活的免疫反应进而造成自身免疫性疾病。TACI融合蛋白在体内和体外都被证实可以高亲和力的结合游离BAFF和APRIL，并竞争性抑制其与淋巴细胞上受体(TACI、BCMA、BAFF-R)结合，从而降低循环中成熟B细胞和免疫球蛋白的量。
融合蛋白
如本文所用，“本发明的融合蛋白”、或“多肽”均指本发明第一方面所述的融合蛋白。
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)APRIL/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。
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，特别适合于在真核细胞(优选CHO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.:19所示的TNFR2-BCMA-Fc融合蛋白的全长(即第1-515位)或其活性片段，例如第55-515位所示的多肽(融合蛋白)。
在一优选实施方式中，本发明的融合蛋白包含以下元件：
(a)TNF受体或其活性片段；(b)APRIL/BAFF受体或其活性片段，其中所述的APRIL/BAFF受体(APRIL和/或BAFF受体)包括TACI，BCMA，BAFFR或其组合；和任选的(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。
如本文所用，术语“融合蛋白”还包括具有上述活性的融合蛋白(例如TNFR2-TACI-Fc(氨基酸序列如SEQ ID NO.:18所示)、TNFR2-BCMA-Fc(氨基酸序列如SEQ ID NO.:19所示)、TNFR2-BAFFR-Fc(氨基酸序列如SEQ ID NO.:20所示))的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述融合蛋白的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明融合蛋白的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有 取代基团的多肽，或(iii)抗原肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6×His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I或式II的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供本发明融合蛋白的类似物。这些类似物与SEQ ID NO.:18、19或SEQ ID NO.:20所示的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的 酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
表达载体和宿主细胞、制备方法
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CHO，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CHO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；
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Millipore、Pellicon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。
肽接头
本发明提供了一种融合蛋白，它可任选地含有肽接头。肽接头大小和复杂性可能会影响蛋白的活性。通常，肽接头应当具有足够的长度和柔韧性，以保证连接的两个蛋白在空间上有足够的自由度以发挥其功能。同时避免肽接头中形成α螺旋或β折叠等对融合蛋白的稳定性的影响。
连接肽的长度一般为0-20个氨基酸，较佳地0-10个氨基酸。
药物组合物
本发明还提供了一种药物组合物，它含有有效量(如0.000001-90wt％；较佳的0.1-50wt％；更佳的，5-40wt％)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、刺激和变态反应)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0.0001mg-10mg/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。
本发明的主要优点包括
(1)本发明融合蛋白具有精确识别、协同作用、毒性可控的优势。
(2)本发明融合蛋白能够高效识别免疫反应中关键分子(如TNFa、BAFF 或APRIL)。
(3)本发明融合蛋白能同时结合TNFa、和BAFF或APRIL，同时抑制TNFa、和BAFF或APRIL的生物学功能。
下面结合具体实施例，进一步陈述本发明。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详细条件的实验方法，通常按照常规条件如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，否则百分比和份数按重量计算。
引物
本发明涉及的引物如下表1所示：
表1引物序列
Figure PCTCN2022080467-appb-000001
实施例1.TNFR2-BCMA-Fc融合蛋白表达质粒的构建
编码人TNFR2氨基酸序列(Met1-Asp257)的核糖核苷酸序列和编码人IgGFc氨基酸序列的核糖核苷酸序列均由GenScript(USA)公司合成，编码人BCMA氨基酸序列(Gly6-Ala54)的核糖核苷酸序列由Genewiz(中国)公司合成。
编码TNFR2-BCMA-Fc融合蛋白的重组基因用两步聚合酶链式反应技术 (PCR)方法连接起来。第一步，用PCR方法(高保真聚合酶Pfx，Invitrogen)扩增人TNFR2(Met1-Asp257)基因：
5’端引物KDP068(SEQ ID NO:1)：5'-CTTTGGCAAAGAATTGGG-3'，位于基因5’的载体上。
3’端引物KDP422(SEQ ID NO:2)：5'-GTCGCCAGTGCTCCCTTCAG-3'，是TNFR2基因专一引物。
同样，用PCR方法扩增BCMA(Gly6-Ala54)的基因：
5’端引物KDP410(SEQ ID NO:3)：
5'-CTGAAGGGAGCACTGGCGACGGGCAGTGCTCCCAAAATG-3'；
3’端引物KDP411(SEQ ID NO:4)：
5'-TATCACAGCTCTTGGGCTCCGCATTCGTTCCTTTCACTG-3'。
同样，用PCR方法扩增人IgG1 Fc(Glu99-Gly329)的基因：
5’端引物KDP134(SEQ ID NO:5)：
5'-GAGCCCAAGAGCTGTGATA-3'；
3’端引物BGHR(SEQ ID NO:6)：
5'-AACTAGAAGGCACAGTCGAGGC-3'，位于基因3’端的载体上。
其中引物KDP410的头20个核苷酸序列与引物KDP422的核苷酸序列互补，引物KDP411的头19个核苷酸序列与引物KDP134的核苷酸序列互补，这样在第二步的重叠延伸PCR过程中，可以把这3个PCR片段连接起来。
上面3个PCR片段经DNA胶纯化后(天根生化科技有限公司，北京)，进行第二步重叠PCR。
5’端引物KDP066(SEQ ID NO:7)，5'-CGAACATCGATTGAATTCC-3'。
3’端引物KDP093(SEQ ID NO:8)，5'-TCTAGCATTTAGGTGACAC-3'。
TNFR2基因转录起始位点前有Not I的酶切位点，IgG1 Fc终止密码子的3’端有Xba I酶切位点，在胶纯化重叠延伸PCR反应获得到的DNA片段后，进行Not I/Xba I双酶切(Takara)。然后把酶切的PCR片段克隆到同样酶切的哺乳动物细胞表达载体上。此哺乳动物细胞表达载体是改进的pcDNA3.1(Invitrogen)，pcDNA3.1里的抗neomycin(新霉素)基因被DHFR(二氢叶酸还原酶)基因取代，改进后的载体适用于筛选稳定转染蛋白高表达的哺乳动物细胞。将重组质粒转染进DH5a感受态细菌，用菌落PCR方法鉴定含有正确重组质粒的阳性菌落，提纯重组质粒。
TNFR2-BCMA-Fc融合蛋白的氨基酸序列(SEQ ID NO.:19)如图1C所示，具体氨基酸如下：
片段1.TNFR2氨基酸序列(SEQ ID NO.:13)的第1-235位；
片段2.BCMA氨基酸序列(SEQ ID NO.:16)的第6-54位；
片段3.人IgG1氨基酸序列(登录号为UniProtKB-P01857)的第99-329位(SEQ ID NO.:17)。
实施例2.TNFR2-BAFFR-Fc融合蛋白表达质粒的构建
用实施例1的方法构建TNFR2-BAFFR-Fc融合蛋白基因。编码人BAFFR氨基酸序列(Asp12-Ala46)的核糖核苷酸序列由Genewiz(中国)公司合成。
第一步，用PCR方法(高保真聚合酶Pfx，Invitrogen)扩增人TNFR2(Met1-Asp257)基因和人IgG1 Fc(Glu99-Gly329)的基因(方法同实施例1)。
同样，用PCR方法扩增BAFFR(Asp12-Ala46)的基因：
5’端引物KDP408(SEQ ID NO:9)：
5'-CTGAAGGGAGCACTGGCGACGACGCGCCAGCCCCCACGC-3'；
3’端引物KDP409(SEQ ID NO:10)：
5'-TATCACAGCTCTTGGGCTCGGCCGGTTTCGGCCGCGG-3'。
其中引物KDP408的头20个核苷酸序列与引物KDP422的核苷酸序列互补，引物KDP409的头19个核苷酸序列与引物KDP134的核苷酸序列互补，这样在第二步的重叠延伸PCR过程中，可以把这3个PCR片段连接起来。
上面3个PCR片段经DNA胶纯化后(天根生化科技有限公司，北京)，进行第二步重叠PCR(方法同实施例1)，把上述3个PCR片段按照相应的顺序连接，构建成TNFR2-BAFFR-Fc融合蛋白编码基因。再用与实施例1中相同的方法把构建好的重组基因克隆到哺乳细胞表达载体中。
TNFR2-BAFFR-Fc融合蛋白的氨基酸序列(SEQ ID NO.:20)如图1D所示，具体氨基酸如下：
片段1.TNFR2氨基酸序列(SEQ ID NO.:13)的第1-235位；
片段2.BAFFR氨基酸序列(SEQ ID NO.:16)的第12-46位；
片段3.人IgG1氨基酸序列(登录号为UniProtKB-P01857)的第99-329位(SEQ ID NO.:17)。
实施例3.TNFR2-TACI-Fc融合蛋白表达质粒的构建
用实施例1的方法构建TNFR2-TACI-Fc融合蛋白基因。编码人TACI氨基酸序列(Ser68-Arg109)的核糖核苷酸序列由Genewiz(中国)公司合成。
第一步，用PCR方法(高保真聚合酶Pfx，Invitrogen)扩增人TNFR2(Met1-Asp257)基因和人IgG1 Fc(Glu99-Gly329)的基因(方法同实施例1)。
同样，用PCR方法扩增TACI(Ser70-Arg109)的基因：
5’端引物KDP412(SEQ ID NO:11)：
5'-CTGAAGGGAGCACTGGCGACTCACTCAGCTGCCGCAAGGAG-3'；
3’端引物KDP413(SEQ ID NO:12)：
5'-TATCACAGCTCTTGGGCTCCCTGAGCTTGTTCTCACAGAAG-3'。
其中引物KDP412的头20个核苷酸序列与引物KDP422的核苷酸序列互补，引物KDP412的头19个核苷酸序列与引物KDP134的核苷酸序列互补，这样在第二步的重叠延伸PCR过程中，可以把这3个PCR片段连接起来。
上面3个PCR片段经DNA胶纯化后(天根生化科技有限公司，北京)，进行第二步重叠PCR(方法同实施例1)，把上述3个PCR片段按照相应的顺序连接，构建成TNFR2-TACI-Fc融合蛋白编码基因。再用与实施例1中相同的方法把构建好的重组基因克隆到哺乳细胞表达载体中。
TNFR2-TACI-Fc融合蛋白的氨基酸序列(SEQ ID NO.:18)如图1E所示，具体氨基酸如下：
片段1.TNFR2氨基酸序列(SEQ ID NO.:13)的第1-235位；
片段2.TACI氨基酸序列(SEQ ID NO.:16)的第68-109位；
片段3.人IgG1氨基酸序列(登录号为UniProtKB-P01857)的第99-329位(SEQ ID NO.:17)。
实施例4.建立融合蛋白稳定表达细胞株
用于稳定表达这些融合蛋白的宿主细胞为中国仓鼠卵巢细胞CHO-KS。CHO-KS是生长在含胎牛血清(FBS)培养基里的CHO-K1细胞经过逐渐降低培养基中FBS含量的培养直至无FBS培养基培养，最终驯化成在不含FBS的OptiCHO培养基(Invitrogen)中悬浮生长的细胞。含有融合蛋白基因的pcDNA3.1载体中的抗新霉素基因用大鼠谷氨酰胺合成酶基因取代，采用电转染(Bio-Rad，Gene Pulser Xcell)的方法把融合蛋白表达表达质粒转染进CHO-KS细胞，转染 的细胞在培养24-48个小时后，用有限稀释法在96孔培养板上对转染的细胞进行筛选培养。筛选培养基是OptiCHO，5μg/ml重组人胰岛素和10μM氨基亚砜蛋氨酸(MSX)。在37℃，8％CO 2的培养箱里培养细胞。3个星期后，用ELISA方法(碱性磷酸酶偶联的羊抗人IgG Fc抗体，Jackson ImmunoResearch Lab)对每个长有细胞群的孔的细胞培养液进行分析，把融合蛋白表达阳性的细胞群进一步扩增，再ELISA检测，再扩增，最后得到融合蛋白表达稳定细胞株。
实施例5.融合蛋白的制备、纯化和鉴定
将实施例4所得的各融合蛋白稳定表达细胞株，培养扩增细胞。离心细胞培养液，收集上清，用Protein-A亲和层析柱从上清中纯化融合蛋白。
结果与分析
本发明的融合蛋白为同源共价二聚体，TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc的分子量差别不大，二聚体约为112kDa(图2，泳道1、3、7，非还原SDS-PAGE)，单体约为56kDa(图2，泳道2、4、8，还原SDS-PAGE)，都略为大于TNFR2-Fc(依那西普)，其二聚体和单体的分子量分别为102kDa和51kDa(图2，泳道5、6)。
实施例6.TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc与重组人TNFa体外结合研究
用ELISA方法研究融合蛋白与重组人TNFa的体外结合活性。
用PBS(pH7.4)溶解重组人TNFa(Novoprotein)至终浓度0.8μg/mL，并加入50μL/孔重组蛋白于96-孔ELISA板，4℃冰箱里过夜。第二天，用PBST(PBS含0.05％Tween-20)洗ELISA板3次，加入100μL/孔PBST含3％BSA的封闭溶液。ELISA板于37℃恒温箱里放置1小时。分别将融合蛋白稀释在PBST含1％BSA的结合溶液里，制备3倍系列稀释的溶液。倒掉封闭液，分别加入稀释的蛋白，50μL/孔，在37℃恒温箱里反应1小时。倒掉溶液，ELISA板用PBST清洗3次，加入50μL/孔的二抗(碱性磷酸酶偶联的羊抗人IgG Fc抗体，Jackson ImmunoResearch Lab)，在37℃恒温箱里反应1小时。倒掉显色抗体，向ELISA板上加200μL/孔PBST清洗溶液，ELISA板于水平摇床上放置5分钟，转速100转/分钟，倒掉清洗溶液，再重复清洗4次。加50μL/孔抗体显色液(PNPP)后ELISA板置于37℃恒温箱里进行显色。用酶标仪在波长405nm/490nm下读板。
结果与分析
图3显示了融合蛋白TNFR2-BAFFR-Fc和TNFR2-BCMA-Fc均能特异性结合人TNFα。TNFR2-BAFFR-Fc和TNFR2-BCMA-Fc与TNFa结合的亲和力EC 50分别为189ng/mL和46ng/mL(图3A和3B)。
实施例7.TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc与重组人BAFF结合研究
用ELISA方法研究融合蛋白与重组人BAFF的结合活性。
用50mM NaHCO 3(pH9.6)溶解重组人BAFF至终浓度1.0μg/mL，并加入50μL/孔重组蛋白于96-孔ELISA板，4℃冰箱里过夜。第二天，用PBST(PBS含0.05％Tween-20)洗ELISA板3次，加入100μL/孔PBST含3％BSA的封闭溶液。ELISA板于37℃恒温箱里放置1小时。分别将各融合蛋白稀释在PBST含1％BSA的结合溶液里，制备3倍系列稀释的溶液。倒掉封闭液，分别加入稀释的蛋白，50μL/孔，在37℃恒温箱里反应1小时。倒掉溶液，ELISA板用PBST清洗3次，加入50μL/孔的二抗(碱性磷酸酶偶联的羊抗人IgG Fc抗体，Jackson ImmunoResearch Lab)，在37℃恒温箱里反应1小时。倒掉显色抗体，向ELISA板上加200μL/孔PBST清洗溶液，ELISA板于水平摇床上放置5分钟，转速100转/分钟，倒掉清洗溶液，再重复清洗4次。加50μL/孔抗体显色液(PNPP)后ELISA板置于37℃恒温箱里进行显色。用酶标仪在波长405nm/655nm下读板。
结果与分析
图4显示了融合蛋白TNFR2-BCMA-Fc和TNFR2-TACI-Fc均能特异性体外结合重组人BAFF。TNFR2-BCMA-Fc和TNFR2-TACI-Fc融合蛋白与BAFF结合的亲和力分别为71ng/mL和50ng/mL(图4A和4B)。
实施例8.TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc与重组人APRIL结合研究
用ELISA方法研究融合蛋白与重组人APRIL的结合活性。
用50mM NaHCO 3(pH9.6)溶解重组人APRIL至终浓度1.0μg/mL，并加入50μL/孔重组蛋白于96-孔ELISA板，4℃冰箱里过夜。第二天，用PBST(PBS含0.05％Tween-20)洗ELISA板3次，加入100μL/孔PBST含3％BSA的封闭溶液。ELISA板于37℃恒温箱里放置1小时。分别将各融合蛋白稀释在PBST含1％BSA 的结合溶液里，制备3倍系列稀释的溶液。倒掉封闭液，分别加入稀释的蛋白，50μL/孔，在37℃恒温箱里反应1小时。倒掉溶液，ELISA板用PBST清洗3次，加入50μL/孔的二抗(碱性磷酸酶偶联的羊抗人IgG Fc抗体，Jackson ImmunoResearch Lab)，在37℃恒温箱里反应1小时。倒掉显色抗体，向ELISA板上加200μL/孔PBST清洗溶液，ELISA板于水平摇床上放置5分钟，转速100转/分钟，倒掉清洗溶液，再重复清洗4次。加50μL/孔抗体显色液(PNPP)后ELISA板置于37℃恒温箱里进行显色。用酶标仪在波长405nm/655nm下读板。
结果与分析
图5显示了融合蛋白TNFR2-BCMA-Fc和TNFR2-TACI-Fc均能特异性体外结合重组人APRIL。TNFR2-BCMA-Fc和TNFR2-TACI-Fc与APRIL结合的亲和力分别为23ng/mL和104ng/mL(图5A和5B)。
实施例9.TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc融合蛋白同时结合TNFa和BAFF或APRIL且互不影响
本发明的融合蛋白高亲和力结合TNFa和BAFF或APRIL，重组人TNFa(Novoprotein)溶解于PBS(pH7.4)至终浓度0.8μg/mL，加入rhTNFa 50μL/孔于96-孔ELISA板，4℃冰箱里过夜。第二天，用PBST(PBS含0.05％Tween-20)洗ELISA板3次，加入100μL/孔PBST含3％BSA的封闭溶液。ELISA板于37℃恒温箱里放置1小时。融合蛋白用结合溶液(PBST含1％BSA)含或不含1μg/mL的BAFF或APRIL进行3倍系列稀释。倒掉封闭液，分别加入系列稀释的融合蛋白，50μL/孔，在37℃恒温箱里反应1小时。倒掉溶液，ELISA板用PBST清洗3次，加入50μL/孔的二抗(碱性磷酸酶偶联的羊抗人IgG Fc抗体，Jackson ImmunoResearch Lab)，在37℃恒温箱里反应1小时。倒掉显色抗体，向ELISA板上加200μL/孔PBST清洗溶液，ELISA板于水平摇床上放置5分钟，转速100转/分钟，倒掉清洗溶液，再重复清洗4次。加50μL/孔抗体显色液(PNPP)后ELISA板置于37℃恒温箱里进行显色。用酶标仪在波长405nm/490nm下读板。
结果与分析
TNFR2-BCMA-Fc在含BAFF溶液里和不含BAFF溶液里与TNFa的结合完全一致(图6A)，在含APRIL溶液里和不含APRIL溶液里与TNFa的结合没有显著差别(图6B)。
实验结果证明BAFF或APRIL不影响TNFR2-BCMA-Fc与TNFa的结合， TNFR2-BCMA-Fc能同时并互不影响地结合TNFa和BAFF或APRIL。
实施例10.TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc融合蛋白抑制TNFa诱导细胞凋亡的生物学活性研究
融合蛋白的TNFR2生物学活性研究方法采用在体外中和TNFa的生物学活性。TNFa生物活性用小鼠成纤维细胞L929细胞毒性检测。5ng/mL的rhTNFa与系列稀释不同浓度的各融合蛋白混合，然后加入到培养在96-well培养板里的L929细胞中，培养液中加入Actinomycin D(终浓度20μg/mL)，在细胞培养箱里培养20小时后用结晶子染色方法检测L929细胞活率。
结果与分析
图7显示了融合蛋白抑制TNFa诱导细胞凋亡的研究结果。TNFR2-BCMA-Fc和TNFR2-TACI-Fc抑制TNFa诱导L929细胞凋亡的活性EC 50分别为27ng/mL和23ng/mL(图7A和7B)。
实施例11.TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc融合蛋白抑制BAFF和APRIL体外刺激RPMI8226细胞的生长
RPMI8226是人多发性骨髓瘤细胞，地塞米松(DEX)能诱导RPMI8226细胞死亡，BAFF或APRIL能抑制DEX诱导RPMI8226细胞死亡的作用。我们对本发明的融合蛋白对BAFF和APRIL的上述抑制作用的影响进行了研究。
RPMI8226细胞培养在96-well板RPMI1660培养基中(含10％FBS)，加入图8中所示的各试剂，在细胞培养箱中培养5天，然后用CCK-8检测活细胞数量。
结果与分析
与阴性对照细胞相比(只培养在RPMI1660+10％FBS培养基中)，RPMI8226细胞在0.1μM浓度的DEX中细胞存活率为37％，0.1μg/mL的BAFF抑制DEX诱导的细胞死亡，增加RPMI8226细胞的活率达到55％，10μg/mL的TNFR2-BCMA-Fc抑制BAFF的功能，使RPMI8226的细胞存活率降到42％，具有显著的抑制BAFF活性的效果(p<0.0001)(图8A)。TNFR2-BCMA-Fc同样能抑制APRIL保护RPMI8226细胞的功能，使RPMI8226细胞存活率从63％降低到46％(p<0.0001)(图8B)。
本实验的结果证明了本发明的融合蛋白具有抑制BAFF或APRIL保护RPMI8226细胞存活的功能。
实施例12.TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc融合蛋白降低LPS诱导的小鼠感染性休克死亡率
研究融合蛋白对LPS诱导的小鼠感染性休克死亡的影响，评估融合蛋白体内中和TNFa的生物学活性。16只7-8周龄Balb/c雄鼠分成2组，每组8只。每只小鼠腹腔注射1mg的LPS，随即给2组小鼠分别静脉注射PBS和6mg/kg的TNFR2-BCMA-Fc，在随后的80小时内观察小鼠状态，记录小鼠死亡的时间。
结果与分析
图9显示，在给予LPS后24小时内，PBS组有2只小鼠死亡，而TNFR2-BCMA-Fc组没有小鼠死亡。在41小时内，PBS组又有5只小鼠死亡，死亡率88％，在48小时内PBS组小鼠全部死亡。而TNFR2-BCMA-Fc组在41小时内死亡5只小鼠，死亡率63％，在72小时内又死亡1只，在80小时内共死亡6只，死亡率75％。
实验结果表明，TNFR2-BCMA-Fc能降低LPS诱导的小鼠休克死亡率。
实施例13.TNFR2-BAFFR-Fc、TNFR2-BCMA-Fc和TNFR2-TACI-Fc融合蛋白降低小鼠B淋巴细胞含量的研究
给小鼠注射融合蛋白2次，检测小鼠脾脏B淋巴细胞的含量，评估融合蛋白对B淋巴细胞的影响。
6-7周龄的雄性C57BL/6小鼠按体重随机分组，每组5只，在D1、D5尾静脉给予相应的药物。D9分别采集小鼠眼底血100μL，各加入100μL含抗凝剂的PBS，加入anti-mouse B220(CD45R)-APC 1μL和anti-mouse CD3-FITC，冰上染色30min后，再加入6倍体积的流式溶血素裂解，震荡后并静置10分钟，再800G离心15分钟，加入FACS工作液200μL重悬。用流式细胞仪检测样本中B220+细胞群和CD3+细胞群所占比例。D10脱颈处死全部小鼠，取血、解剖取脾脏，分离脾脏淋巴细胞。用上述同样的方法检测脾脏淋巴细胞中B220+细胞群和CD3+细胞群所占比例。
结果与分析
膜蛋白B220是小鼠B淋巴细胞的生物标志物，所有B细胞都表达B220。图10显示了TNFR2-BCMA-Fc能显著降低小鼠血液中(图10A)和脾脏中(图10B)B淋巴细胞的含量，7.5mg/kg和15mg/kg剂量的TNFR2-BCMA-Fc分别降低血液 中B淋巴细胞的含量37％和43％，降低脾脏中B淋巴细胞的含量44％和56％。依那西普(etanercept)对血液中B淋巴细胞的含量没有任何影响，对脾脏中B淋巴细胞的含量有降低的作用(降低23％)，但显著弱于同等剂量的TNFR2-BCMA-Fc的作用(降低44％)。
实施例14.TNFR2-BCMA-Fc融合蛋白对胶原诱导型关节炎(CIA)小鼠模型的炎症影响
CIA小鼠模型建立方法参考文献Schett等,Arthritis Rheum.52:1604-1611,2005。DBA/1小鼠(7周龄)分成6组，每组9只小鼠。在D0用胶原乳剂A(Bovine type II collagen+Complete Freund’s Adjuvant)免疫小鼠，在D21用胶原乳剂B(Bovine type II collagen+Incomplete Freund’s Adjuvant)免疫小鼠。在D21，对其中5组小鼠分别腹腔注射3个剂量的TNFR2-BCMA-Fc(1.25、5和20mg/kg)和2个剂量的依那西普(Enbrel)，第6组小鼠腹腔给与PBS。另外设置1组包括4个正常小鼠。每周给药3次，共给药4周。在D49结束试验。通过临床关节评分和对后爪厚度测量评估小鼠炎症发生的程度，临床评分每周三次。
在实验终点采取整个左后爪，用4％多聚甲醛固定，然后75％乙醇浸泡保存，对踝跗趾关节进行HE染色和组织病理分析(武汉塞维尔生物科技有限公司)，病理分析采用半定量的方式，对滑膜炎症、血管翳生成以及骨侵蚀和软骨损伤的严重程度进行评估。在实验结束后，摘取全部小鼠的脾脏，称量脾重，分离脾脏淋巴细胞，用抗CD3抗体和抗B220抗体染色细胞，流式细胞仪技术检测T淋巴细胞和B淋巴细胞的数量以及含量，比较了各组动物的脾脏重量以及B淋巴细胞和T淋巴细胞的变化。对试验结束时小鼠血清中抗胶原抗体的滴度进行检测，评估TNFR2-BCMA-Fc对抗胶原抗体产生的影响。
结果与分析
研究结果显示，与对照组相比，3个剂量的TNFR2-BCMA-FC均能抑制小鼠炎症发生的程度(评估小鼠脚爪肿胀程度)，抑制效果和剂量成正比关系(图11A)。2个剂量的依那西普也能抑制小鼠炎症发生的程度，但与同等剂量的TNFR2-BCMA-FC相比，抑制效果明显低于TNFR2-BCMA-FC(图11B和11C)。其中，5mg/kg的依那西普抑制炎症的效果与1.25mg/kg的TNFR2-BCMA-FC效果相当，显著低于5mg/kg的TNFR2-BCMA-FC效果，差异具有统计学意义(p<0.01)(图11B)。
对小鼠脚趾背关节组织进行病理研究(苏木精-伊红染色)，结果显示3个剂量的TNFR2-BCMA-FC均能抑制关节组织滑膜炎(图12A)、血管翳(图12B)和骨侵蚀(图12C)的发生程度。TNFR2-BCMA-FC 5mg/kg和20mg/kg剂量组的全部小鼠的脚趾背部组织与正常组织一样。1.25mg/kg的TNFR2-BCMA-FC对组织也有部分保护作用，且优于5mg/kg的Enbrel。同样的，关节组织的病理研究结果也证明了TNFR2-BCMA-FC抑制CIA小鼠炎症效果显著优于依那西普。
通过对小鼠脾脏B淋巴细胞的含量分析，5和20mg/kg剂量的TNFR2-BCMA-Fc显示出能降低脾脏中B淋巴细胞的百分比(图13A)，同等剂量的Enbrel也能降低脾脏B淋巴细胞百分比，但作用效果弱于TNFR2-BCMA-Fc，尤其是在20mg/kg剂量下，TNFR2-BCMA-Fc降低B淋巴细胞百分比显著强于Enbrel(p<0.0001)。与正常小鼠相比，炎症能引起脾脏增大(图13B，PBS组)。5和20mg/kg剂量的TNFR2-BCMA-Fc具有抑制脾脏增大的功能(图13B)，并且比同等剂量的Enbrel具有更强的抑制效果。
TNFR2-BCMA-Fc能抑制抗胶原抗体的滴度(图14)。与PBS组相比，20mg/kg剂量的TNFR2-BCMA-Fc显著降低血清中抗胶原抗体的滴度，而同等剂量的Enbrel对抗体的产生没有任何影响。
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 (12)

  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)APRIL/BAFF受体或其活性片段，其中所述的APRIL/BAFF受体包括TACI，BCMA，BAFFR或其组合；
    和任选的(c)抗体Fc区域；
    其中，所述的融合蛋白保留了上述元件(a)和(b)的生物活性。
  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TNFα的活性；
    (b)抑制TNFα诱导的炎症；
    (c)结合BAFF或APRIL的活性；
    (d)抑制或封闭BAFF/APPRIL途径；
    (e)降低体内B细胞的数量；
    (f)降低脾脏或血液中的B淋巴细胞含量；
    (g)抑制脾脏增大；
    (h)抑制APRIL/BAFF诱导的疾病。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述融合蛋白具有下式I或II所示的二聚体结构：
    X-Y-Z  (I)
    Y-X-Z  (II)；
    式中，
    X为TNF受体的胞外段；
    Y为TACI，BCMA或BAFFR的胞外段；
    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，所述融合蛋白的氨基酸序列如SEQ ID NO.:18或19或20所示。
  5. 如权利要求3所述的融合蛋白，其特征在于，所述的X含有或具有TNFR2氨基酸序列(SEQ ID NO.:13)的第1-235位，或第17-179位，或第17-140位，或第55-179位，或第55-140位；和/或
    所述的Y含有或具有：
    (a)TACI氨基酸序列(SEQ ID NO.:14)的第1-109位，或第33-109位，或第68-109位；
    (b)BCMA氨基酸序列(SEQ ID NO.:15)的第1-54位，或第6-54位；或
    (c)BAFFR氨基酸序列(SEQ ID NO.:16)的第1-78位，或第12-78位，或第12-46位；和/或
    所述的Z含有或具有人IgG1氨基酸序列(登录号为UniProtKB-P01857)的第99-329位。
  6. 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所述的融合蛋白。
  7. 一种载体，其特征在于，它含有权利要求6所述的核酸分子。
  8. 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7所述的载体；或所述的细胞基因组中整合有权利要求6所述的核酸分子。
  9. 一种产生权利要求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8所述的基因工程化的细胞，从而表达所述的融合蛋白；和
    分离或纯化所述的融合蛋白。
  10. 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。
  11. 如权利要求1所述的融合蛋白、权利要求6所述的核酸分子、权利要求7所述的载体、权利要求8所述的基因工程化的细胞的用途，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)治疗免疫疾病的组合物，较佳地，所述免疫性疾病选自下组：类风湿关节炎(RA)、强直性脊柱炎(AS)、银屑病(PS)、银屑病关节炎(PsA)、幼年特发性关节炎(JIA)、系统性红斑狼疮(SLE)、白塞病(behcet’s disease,BD)、多发性硬化症(MS)、干燥综合征(SS)、Graves病、克罗恩病(CD)、溃疡性结肠炎(UC)、原发性肾小球肾炎、IgA肾病、自身免疫性血管炎、多发性肌炎(PM)、非感染性葡萄膜炎、自身免疫性溶血性贫血(AIHA)、自身免疫性紫癜(ATTP)、N-甲基-d-天冬氨酸受体(NMDAR)脑炎、重症肌无力、化脓性汗腺炎(HS)、髓鞘-少突胶质细胞糖蛋白谱紊乱(MOGSD)和视神经脊髓炎频谱障 碍(NMOSD)、或其组合；和/或
    (c)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。
  12. 一种(a)治疗免疫疾病的组合物；或(b)治疗B细胞增多相关疾病的病的方法，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。
PCT/CN2022/080467 2021-03-12 2022-03-11 Tnfr2与april/baff受体的融合蛋白 WO2022188883A1 (zh)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993019777A1 (en) * 1992-03-30 1993-10-14 Immunex Corporation Fusion proteins comprising tumor necrosis factor receptor
US20030082736A1 (en) * 1989-09-05 2003-05-01 Immunex Corporation Fusion proteins comprising tumor necrosis factor receptor
CN1752108A (zh) * 2005-10-20 2006-03-29 高基民 可溶性肿瘤坏死因子受体ⅱ－“脂联素”球部融合蛋白
CN101061231A (zh) * 2004-08-27 2007-10-24 惠氏研究爱尔兰有限公司 TNFR－Ig融合蛋白的生产
CN101323643A (zh) * 2007-06-15 2008-12-17 烟台荣昌生物工程有限公司 优化的TACI-Fc融合蛋白
CN101535335A (zh) * 2006-08-28 2009-09-16 阿雷斯贸易股份有限公司 含Fc-蛋白的纯化方法
WO2010003766A2 (en) * 2008-06-17 2010-01-14 Apogenix Gmbh Multimeric tnf receptors
CN102171247A (zh) * 2008-07-02 2011-08-31 特鲁比昂药品公司 TNF-α拮抗剂多靶点结合蛋白
US20200369739A1 (en) * 2017-06-06 2020-11-26 Relinia, Inc. Single-chain tnf receptor 2 agonist fusion proteins

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030082736A1 (en) * 1989-09-05 2003-05-01 Immunex Corporation Fusion proteins comprising tumor necrosis factor receptor
WO1993019777A1 (en) * 1992-03-30 1993-10-14 Immunex Corporation Fusion proteins comprising tumor necrosis factor receptor
CN101061231A (zh) * 2004-08-27 2007-10-24 惠氏研究爱尔兰有限公司 TNFR－Ig融合蛋白的生产
CN1752108A (zh) * 2005-10-20 2006-03-29 高基民 可溶性肿瘤坏死因子受体ⅱ－“脂联素”球部融合蛋白
CN101535335A (zh) * 2006-08-28 2009-09-16 阿雷斯贸易股份有限公司 含Fc-蛋白的纯化方法
CN101323643A (zh) * 2007-06-15 2008-12-17 烟台荣昌生物工程有限公司 优化的TACI-Fc融合蛋白
WO2010003766A2 (en) * 2008-06-17 2010-01-14 Apogenix Gmbh Multimeric tnf receptors
CN102171247A (zh) * 2008-07-02 2011-08-31 特鲁比昂药品公司 TNF-α拮抗剂多靶点结合蛋白
US20200369739A1 (en) * 2017-06-06 2020-11-26 Relinia, Inc. Single-chain tnf receptor 2 agonist fusion proteins

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHETT ET AL., ARTHRITIS RHEUM, vol. 52, 2005, pages 1604 - 1611

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