WO2022194067A1 - 一种抗baffr抗体及其应用 - Google Patents

一种抗baffr抗体及其应用 Download PDF

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WO2022194067A1
WO2022194067A1 PCT/CN2022/080548 CN2022080548W WO2022194067A1 WO 2022194067 A1 WO2022194067 A1 WO 2022194067A1 CN 2022080548 W CN2022080548 W CN 2022080548W WO 2022194067 A1 WO2022194067 A1 WO 2022194067A1
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seq
amino acid
antibody
variable region
chain variable
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PCT/CN2022/080548
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French (fr)
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秦春铃
姜晓玲
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盛禾(中国)生物制药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to an anti-BAFFR antibody and application thereof.
  • BAFFR is encoded by the TNFRSF13C gene, and other abbreviations are: TNFRSF13C, BLyS-3, CD268, etc.
  • BAFFR belongs to the transmembrane protein of the TNFR family. It consists of 184 amino acids and is divided into an extracellular part, a transmembrane region and an intracellular part. The extracellular part is suitable for the development of antibody drugs.
  • BAFFR is a specific and highly expressed marker of B lymphocytes at different stages. Its expression pattern in B cells is similar to that of CD20. It is expressed in cells other than plasma cells (plasma), pro B cells (pro B) and pre B cells (pre B). Other developmental stages of B cells, but more abundantly expressed than CD20. BAFFR is not expressed in bone marrow stem cells, the origin cells of B cells. After the excessively proliferated B cells are eliminated, the B cells will be replenished again after drug withdrawal, which does not affect the normal needs of the human body and is safe.
  • the indications for BAFFR target blockers include lymphoma types other than ALL (acute lymphoblastic leukemia) and MM (multiple myeloma), such as MCL (mantle cell lymphoma), DLBCL (diffuse large B lymphoma, chronic lymphoma, follicular lymphoma), etc.
  • BAFFR The only ligand of BAFFR is BAFF.
  • BAFFR and BAFF act together to activate the NF-kB signaling pathway in B cells and promote the proliferation and activation of B cells.
  • the BAFF-BAFFR signaling pathway and the BCR pathway are the key pathways for the maturation and activation of Immature B cells. Blockade of both pathways can completely eliminate peripheral B lymphocytes and inhibit the development and proliferation of B lymphocytes.
  • the BAFFR-BAFF signaling pathway is located downstream of the BCR pathway and is an essential survival channel for cells, which can further inhibit the growth of B cells downstream.
  • an anti-BAFFR antibody with good performance which can specifically recognize/bind to BAFFR, block the BAFFR-BAFF signaling pathway, remove excessively proliferated B lymphocytes, inhibit the The growth of B cells; effectively solve the problem of drug resistance of existing drugs for the treatment of B cell lymphoma.
  • the first object of the present invention is to provide an anti-BAFFR antibody or an antigen-binding fragment thereof.
  • the second object of the present invention is to provide a gene encoding the above-mentioned anti-BAFFR antibody or antigen-binding fragment thereof.
  • the third object of the present invention is to provide applications of the above-mentioned anti-BAFFR antibodies or antigen-binding fragments thereof.
  • the fourth object of the present invention is to provide a pharmaceutical composition.
  • An anti-BAFFR monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain complementarity determining regions CDR1, CDR2 and CDR3, and the light chain variable region comprises a light chain Complementarity determining regions CDR1, CDR2 and CDR3, wherein,
  • CDR1 of heavy chain variable region selected from any amino acid sequence of SEQ ID NO: 33-40, or having at least 80%, 85%, 90% with any amino acid sequence of SEQ ID NO: 33-40 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared to any of the amino acid sequences of SEQ ID NOs: 33-40
  • CDR2 of the heavy chain variable region selected from any amino acid sequence of SEQ ID NOs: 41-48, or having at least 80%, 85%, 90% with any amino acid sequence of SEQ ID NOs: 41-48 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared to any of the amino acid sequences of SEQ ID NOs: 41-48
  • CDR3 of the heavy chain variable region selected from any amino acid sequence of SEQ ID NO: 49-56, or having at least 80%, 85%, 90% with any amino acid sequence of SEQ ID NO: 49-56 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared to any of the amino acid sequences of SEQ ID NOs: 49-56
  • CDR1 of the light chain variable region selected from any amino acid sequence of SEQ ID NO: 57-64, or having at least 80%, 85%, 90% with any amino acid sequence of SEQ ID NO: 57-64 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared to any of the amino acid sequences of SEQ ID NOs: 57-64
  • CDR2 of the light chain variable region selected from any amino acid sequence of SEQ ID NO: 65-72, or having at least 80%, 85%, 90% with any amino acid sequence of SEQ ID NO: 65-72 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared to any of the amino acid sequences of SEQ ID NOs: 65-72
  • CDR3 of the light chain variable region selected from any of the amino acid sequences of SEQ ID NOs: 73-80; the antibody binds siglec15, or has at least 80% of the amino acid sequence of any of SEQ ID NOs: 73-80 , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or to SEQ ID NOs: 73-80
  • the heavy chain and light chain comprise a heavy chain variable region and a light chain variable region
  • the heavy chain variable region includes a framework region FR and CDR1, CDR2 and CDR3 of the heavy chain variable region
  • the The described framework area FR includes:
  • (a) is selected from any amino acid sequence of SEQ ID NOs: 81-88,
  • (b) is selected from any amino acid sequence of SEQ ID NOs: 89-96,
  • (c) is selected from any amino acid sequence of SEQ ID NO:97-104,
  • (d) is selected from any amino acid sequence of SEQ ID NO: 105-112,
  • the heavy chain and the light chain comprise a heavy chain variable region and a light chain variable region
  • the light chain variable region includes a framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region
  • the The framework area FR includes:
  • (e) is selected from any amino acid sequence of SEQ ID NO: 113-120,
  • (f) is selected from any amino acid sequence of SEQ ID NO: 121-128,
  • (g) is selected from any amino acid sequence of SEQ ID NO: 129-136,
  • (h) is selected from any amino acid sequence of SEQ ID NO: 137-144,
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:33, SEQ ID NO:41 and SEQ ID NO:49, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 57, SEQ ID NO: 65 and SEQ ID NO: 73, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 91 , FR2 shown in SEQ ID NO: 101, FR3 shown in SEQ ID NO: 111, and FR4 shown in SEQ ID NO: 121.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 131 , FR2 shown in SEQ ID NO: 139, FR3 shown in SEQ ID NO: 147, and FR4 shown in SEQ ID NO: 155.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO: 38, SEQ ID NO: 48 and SEQ ID NO: 58, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 68, SEQ ID NO: 76 and SEQ ID NO: 84, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 92 , FR2 shown in SEQ ID NO: 102, FR3 shown in SEQ ID NO: 112, and FR4 shown in SEQ ID NO: 122.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 132 , FR2 shown in SEQ ID NO: 140, FR3 shown in SEQ ID NO: 148, and FR4 shown in SEQ ID NO: 156.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:39, SEQ ID NO:49 and SEQ ID NO:59, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 69, SEQ ID NO: 77 and SEQ ID NO: 85, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 93 , FR2 shown in SEQ ID NO: 103, FR3 shown in SEQ ID NO: 113, and FR4 shown in SEQ ID NO: 123.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 133 , FR2 shown in SEQ ID NO: 141, FR3 shown in SEQ ID NO: 149, and FR4 shown in SEQ ID NO: 157.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of the amino acid sequences SEQ ID NO: 40, SEQ ID NO: 50 and SEQ ID NO: 60, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 70, SEQ ID NO: 78 and SEQ ID NO: 86, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 84 , FR2 shown in SEQ ID NO: 94, FR3 shown in SEQ ID NO: 100, and FR4 shown in SEQ ID NO: 108.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 116 , FR2 shown in SEQ ID NO: 124, FR3 shown in SEQ ID NO: 132, and FR4 shown in SEQ ID NO: 140.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:37, SEQ ID NO:45 and SEQ ID NO:53, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 61, SEQ ID NO: 69 and SEQ ID NO: 77, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 85 , FR2 shown in SEQ ID NO: 93, FR3 shown in SEQ ID NO: 101, and FR4 shown in SEQ ID NO: 109.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 117 , FR2 shown in SEQ ID NO: 125, FR3 shown in SEQ ID NO: 133, and FR4 shown in SEQ ID NO: 141.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of the amino acid sequences SEQ ID NO:38, SEQ ID NO:46 and SEQ ID NO:54, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 62, SEQ ID NO: 70 and SEQ ID NO: 78, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 86 , FR2 shown in SEQ ID NO: 94, FR3 shown in SEQ ID NO: 102, and FR4 shown in SEQ ID NO: 110.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 118 , FR2 shown in SEQ ID NO: 126, FR3 shown in SEQ ID NO: 134, and FR4 shown in SEQ ID NO: 142.
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO:39, SEQ ID NO:47 and SEQ ID NO:55, and CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 63, SEQ ID NO: 71 and SEQ ID NO: 79, respectively;
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 87 , FR2 shown in SEQ ID NO: 95, FR3 shown in SEQ ID NO: 103, and FR4 shown in SEQ ID NO: 111.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 119 , FR2 shown in SEQ ID NO: 127, FR3 shown in SEQ ID NO: 135, and FR4 shown in SEQ ID NO: 143.
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively composed of amino acid sequences SEQ ID NO: 40, SEQ ID NO: 48 and SEQ ID NO: 56, and CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO:64, SEQ ID NO:72 and SEQ ID NO:80, respectively.
  • the heavy chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned heavy chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 88 , FR2 shown in SEQ ID NO: 96, FR3 shown in SEQ ID NO: 104, and FR4 shown in SEQ ID NO: 112.
  • the light chain variable region includes framework region FR and CDR1, CDR2 and CDR3 of the above-mentioned light chain variable region; more preferably, the framework region FR includes: FR1 shown in SEQ ID NO: 120 , FR2 shown in SEQ ID NO: 128, FR3 shown in SEQ ID NO: 136, and FR4 shown in SEQ ID NO: 144.
  • An anti-BAFFR monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; it is characterized in that, wherein:
  • the heavy chain variable region has any of the amino acid sequences given in SEQ ID NOs: 1-8,
  • the light chain variable region has any of the amino acid sequences given in SEQ ID NOs: 17-24;
  • the heavy chain variable region and the light chain variable region are selected from any one of the following amino acid sequences (1)-(8):
  • the antibody is a murine antibody, a single domain antibody, a chimeric antibody, a fully human antibody or a humanized antibody.
  • the antibody or antigen-binding fragment thereof is a monospecific antibody, bispecific antibody, multispecific antibody, antibody conjugate or car molecule for cell therapy.
  • the cytotoxic drug of the antibody conjugate is a cytotoxic molecule, a radioisotope, a drug or a cytokine.
  • the bispecific antibody includes any of the above-mentioned antibodies or antigen-binding fragments thereof, as well as antibodies or antigen-binding fragments directed against other antigens and/or other antigenic epitopes.
  • the antibody is a monoclonal antibody.
  • the antibody or antigen-binding fragment thereof binds to a BAFFR protein.
  • the BAFFR protein is a human monkey BAFFR protein.
  • the BAFFR protein forms part of a cell.
  • the BAFFR protein is expressed on the surface of the cell.
  • the cells are lymphocytes.
  • the cells are B cells.
  • the cells are cancer cells.
  • the cancer cells are lymphoma cells.
  • the present invention also provides a gene encoding any of the aforementioned anti-BAFFR antibodies or antigen-binding fragments thereof.
  • the gene is selected from any one of the following (1)-(8):
  • the present invention provides a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the gene.
  • the present invention provides an application of the antibody or antigen-binding fragment thereof described in any one of the above in the following (a) or (b) or (c):
  • the present invention provides a multifunctional fusion protein comprising the antibody or antigen-binding fragment thereof or antibody conjugated drug described in any one of the above.
  • it also comprises one or more secondary antibodies or antigen-binding portions thereof that specifically bind to other antigens.
  • the antigen that binds to the second antibody or antigen-binding portion thereof is selected from tumor-associated antigens (TAAs) or immune checkpoints.
  • TAAs tumor-associated antigens
  • immune checkpoints immune checkpoints
  • the immune checkpoints are CD3, 4-1BB, CD40, OX40, and ICOS.
  • the multifunctional fusion protein further comprises cytokines.
  • the cytokine is selected from IL8, IL10, IL15, IL18, TGF, VEGF, IFN ⁇ , IFN ⁇ or GM-CSF.
  • the present invention also provides the use of the antibody or antigen-binding fragment thereof described in any of the above, and the multifunctional fusion protein of any of the above, in the preparation of a medicament for treating cancer or autoimmune disease.
  • the cancer is lymphoma, leukemia or myeloma.
  • the lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, marginal zone lymphoma or Burkitt's lymphoma.
  • the leukemia is lymphoblastic leukemia, chronic lymphocytic leukemia or hairy cell leukemia.
  • the myeloma is multiple myeloma.
  • the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, idiopathic thrombocytopenic purpura, ulcerative colitis, Sjögren's syndrome.
  • the drug is used in combination with other drugs or therapies.
  • the other therapy is selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, gene therapy, cell therapy, and stem cell therapy.
  • the present invention provides a method for modulating cell function, comprising delivering a polynucleotide sequence encoding the multifunctional fusion protein of any of the above-mentioned antibodies or antigen-binding fragments thereof into the cells.
  • the method is used for gene therapy, CAR-T therapy or CRISPR therapy.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of the above and an acceptable carrier, diluent or excipient.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the multifunctional fusion protein described in any of the above and an acceptable carrier, diluent or excipient.
  • the present invention provides a method for inhibiting cell proliferation, the method comprising:
  • the cells are lymphocytes.
  • the cells are B cells.
  • the cells are cancer cells.
  • the cells are lymphoma cells.
  • mAb monoclonal antibody
  • VH variable region of antibody heavy chain
  • VL variable region of antibody light chain
  • CDR complementarity determining region in immunoglobulin variable region
  • FR antibody framework region, i.e. in the variable region of antibody Amino acid residues other than CDR residues
  • IgG immunoglobulin G
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescence-activated cell sorting
  • PCR polymerase chain reaction.
  • antibody refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis.
  • Antibodies can be isolated from natural resources such as plasma or serum in which the antibodies are naturally present, or from culture supernatants of antibody-producing hybridoma cells, from animal immune sera, or from phage library screening. Alternatively, it may be partially or completely synthesized by techniques using genetic recombination or the like.
  • Preferred antibodies include, for example, antibodies of immunoglobulin isotypes or subclasses of these isotypes.
  • Human immunoglobulins are known to include 9 classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM.
  • the antibodies of the invention may include IgGl, IgG2, IgG3, IgG4.
  • antigen-binding fragment refers to a polypeptide fragment of an antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or compete with the full-length antibody for specific binding to the antigen, also known as "antigen binding”. part".
  • Antigen-binding fragments of antibodies can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabodies , linear antibodies, single domain antibodies, domain antibodies, and polypeptides comprising at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which they are introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked, and are referred to herein as "expression vectors.”
  • pharmaceutically acceptable carrier includes any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water and emulsions, as well as various types of wetting agents.
  • Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence after aligning the sequences and introducing gaps where necessary to obtain maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be performed in a variety of ways that are within the skill in the art, eg, using publicly available computer software, such as BLAST software or the FASTA package.
  • Specific means that one of the molecules involved in specific binding does not exhibit any significant binding to molecules other than one or more of the binding partner molecules.
  • the term is also used when the antibody variable region-containing domain is specific for a particular epitope of multiple epitopes in an antigen.
  • an antigen-binding molecule comprising the antibody variable region-containing domain can bind to various antigens having the epitope.
  • Cytotoxicity is a purely cell-killing event caused by a cell or chemical, and does not rely on apoptosis or necrosis as a cell death mechanism. Testing for the cytotoxicity of specific substances, such as drug screening, is sometimes required. Cytotoxicity detection is mainly based on changes in cell membrane permeability, and the following methods are commonly used: MTT, XTT method; LDH method; other enzymatic methods: such as detection of alkaline phosphatase and acid phosphatase in the supernatant. activity, etc.
  • Epitope means an antigenic determinant in an antigen, and refers to an antigenic site to which a domain of an antigen-binding molecule comprising an antibody variable region disclosed in the present specification binds.
  • epitopes can be defined according to their structure.
  • the epitope can also be defined based on the antigen-binding activity in the antigen-binding molecule that recognizes the epitope.
  • the antigen is a peptide or polypeptide
  • the epitope can be specified by the amino acid residues forming the epitope; when the epitope is a sugar chain, the epitope can be identified by its specific sugar chain structure.
  • BMK is the abbreviation of Benchmark, which refers to a positive control antibody that can bind to the target protein or native or engineered cells that express the target protein.
  • BR can be assessed based on the principles of ELISA or fluorescence-activated cell sorting (FACS) using BAFFR-expressing cells as antigens.
  • the antibody variable region-containing domains in the antigen-binding molecules of the present invention can bind to the same epitope.
  • the same epitope may be present in a protein comprising the amino acid sequence of SEQ ID NO: 145.
  • the antibody variable region-containing domains in the antigen-binding molecules of the present invention may bind to different epitopes, respectively.
  • different epitopes may be present in a protein comprising the amino acid sequence of SEQ ID NO:145.
  • the present application is an anti-BAFFR antibody or an antigen-binding fragment thereof, the antibody binds to an epitope, and the epitope is contained in whole or in part in the amino acid sequence described in SEQ ID NO: 145.
  • Anti-BAFFR antibodies can be obtained as polyclonal or monoclonal antibodies using known methods.
  • a mammalian-derived monoclonal antibody can be preferably prepared.
  • These mammalian-derived monoclonal antibodies include antibodies produced by hybridomas, antibodies screened by phage surface display techniques, or antibodies produced by host cells transformed with expression vectors carrying antibody genes by genetic engineering techniques.
  • BAFFR protein was purchased from a conventional biochemical reagent store.
  • the BAFFR protein is used as a sensitizing antigen for immunization in mammals.
  • Partial peptides of BAFFR can also be used as sensitizing antigens.
  • the partial peptide can also be obtained by chemical synthesis from the amino acid sequence of human BAFFR.
  • it can also be obtained by integrating a part of the BAFFR gene into an expression vector and expressing it.
  • the region and size of the BAFFR peptide used as the partial peptide are not particularly limited to specific embodiments.
  • the number of amino acids constituting the peptide used as the sensitizing antigen is at least five or more, or preferably, for example, six or more, or seven or more. More specifically, a peptide consisting of 8 to 50 residues or preferably 10 to 30 residues can be used as the sensitizing antigen.
  • a fusion protein in which a desired partial polypeptide or peptide of the BAFFR protein is fused with a different polypeptide can be used as a sensitizing antigen.
  • a fusion protein to be used as a sensitizing antigen for example, an Fc fragment of an antibody, a peptide tag, and the like can be preferably used.
  • a vector for expressing a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in frame, and inserting the fused gene into an expression vector as described above.
  • the mammal to be immunized with the sensitizing antigen is not limited to a specific animal. However, the selection is preferably made in consideration of suitability with the parent cell for cell fusion. Generally rodents such as mice, rats and hamsters, alpacas, rabbits and monkeys are preferably used.
  • DNA expressing BAFFR protein is administered to the immunized animal.
  • DNA encoding BAFFR can be synthesized by a known method such as PCR.
  • the resulting BAFFR is inserted into an appropriate expression vector and administered to immunized animals.
  • the expression vector a commercially available expression vector such as PCDNA3.1 can be suitably used.
  • a method of administering the carrier into a living body a commonly used method can be used.
  • DNA immunization is carried out by introducing a gold particle to which an expression vector is adsorbed into the cells of an immunized animal individual with a gene gun.
  • immune cells are collected from the mammal, followed by cell fusion.
  • splenocytes can be used in particular.
  • Mammalian myeloma cells can be used as the cells to be fused with the above-mentioned immune cells.
  • Myeloma cells are preferably provided with appropriate selectable markers for screening.
  • a selectable marker refers to a property that enables (or does not) survive under specific culture conditions.
  • Well-known selectable markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency).
  • HAT sensitivity hypoxanthine-aminopterin-thymidine sensitivity
  • Cells lacking HGPRT or lacking TK can be selected in a medium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA will die. At the same time, cells lacking these enzymes that do not take up these pyrimidine analogs can survive in selective culture.
  • a selectable marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (a gentamicin analog) through a neomycin resistance gene.
  • Various myeloma cells suitable for cell fusion are known.
  • myeloma cells including the following cells can be preferably used: P3 (P3x63Ag8.653) (J. Immunol. (1979) 123(4), 1548-1550); NS-1 (C. Eur. J. Immunol. ( 1976) 6(7), 511-519); SP2/0 (Nature (1978) 276 (5685), 269-270); R210 (Nature (1979) 277 (5692), 131-133), etc.
  • Cell fusion of the above-mentioned immune cells and myeloma cells is basically carried out according to known methods, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).
  • the above-mentioned cell fusion can be carried out in a conventional nutrient medium in the presence of a cell fusion promoter.
  • Fusion promoters include, for example, polyethylene glycol (PEG) and Sendai virus (HVJ).
  • Adjuvants such as dimethyl sulfoxide can be added as needed to further improve the fusion efficiency.
  • the use ratio of immune cells to myeloma cells can be arbitrarily set. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells.
  • the culture medium for the above-mentioned cell fusion for example, RPMI1640 medium suitable for the growth of the above-mentioned myeloma cell line, MEM medium, and conventional culture medium used for cell culture of this species can be used.
  • serum supplements such as fetal calf serum (FCS) can be suitably added.
  • predetermined amounts of the above-mentioned immune cells and myeloma cells can be sufficiently mixed in the above-mentioned culture medium. Then, the PEG solution preheated to about 37°C is added at a concentration of usually 30% to 60% (w/v). The desired fused cells (hybridomas) can be formed by slowly mixing the mixture. Next, the appropriate medium exemplified above was gradually added to the cells, and centrifugation was repeated to remove the supernatant. Cell fusion agents, etc., which are not conducive to the growth of hybridomas, can be removed.
  • FACS fluorescence-activated cell sorting
  • hybridomas producing the monoclonal antibodies of the invention are first prepared.
  • Preferred cells for screening are mammalian cells that are forced to express BAFFR.
  • untransformed mammalian cells can be used as host cells to selectively detect the binding activity of an antibody to BAFFR on the cell surface. That is, hybridomas producing BAFFR monoclonal antibodies can be obtained by selecting hybridomas that produce antibodies that do not bind to host cells but that bind to cells forcibly expressing BAFFR.
  • the binding activity of the antibody to immobilized BAFFR-expressing cells can be assessed based on the principles of ELISA.
  • cells expressing BAFFR are immobilized in the wells of an ELISA plate.
  • the antibody bound to the immobilized cells is detected by contacting the culture supernatant of the hybridoma with the immobilized cells in the well.
  • the monoclonal antibody is of mouse origin, the antibody bound to the cells can be detected by an anti-mouse immunoglobulin antibody.
  • Hybridomas having antigen-binding ability and producing the desired antibody selected by the above screening can be cloned by a limiting dilution method or the like.
  • the hybridoma is cultured according to a conventional method, and the desired monoclonal antibody can be obtained from the culture supernatant.
  • monoclonal antibodies can be obtained from the ascites fluid of the hybridoma by administering it to an adapted mammal and multiplying it.
  • the former method is suitable for obtaining high-purity antibodies.
  • Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. By inserting the cloned antibody gene into an appropriate vector and introducing it into a host, the antibody encoded by the gene is expressed. Methods for isolation of antibody genes, gene insertion into vectors, and transformation of host cells, and methods for producing recombinant antibodies are also known.
  • cDNA encoding the variable region (V region) of the anti-BAFFR antibody can be prepared from hybridoma cells expressing the anti-BAFFR antibody.
  • total RNA is usually first extracted from the hybridoma.
  • a guanidine ultracentrifugation method and an AGPC method can be used as a method for extracting mRNA from cells.
  • the extracted mRNA can be purified using an mRNA purification kit (GE Healthcare Bioscience) or the like. Alternatively, kits for the direct extraction of total mRNA from cells such as the QuickPrep mRNA purification kit (GE Healthcare Bioscience) are also commercially available. mRNA can be obtained from hybridomas using such kits.
  • the cDNA encoding the antibody V region can be synthesized from the prepared mRNA using reverse transcriptase. cDNA can be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Biochemical Industry Co., Ltd.) or the like.
  • SMART RACE cDNA Amplification Kit and PCR-based 5'-RACE method can be appropriately used.
  • appropriate restriction enzyme sites described later may be introduced into both ends of the cDNA.
  • the resulting PCR product was purified to the target cDNA fragment, followed by ligation with vector DNA.
  • a recombinant vector is produced and introduced into Escherichia coli or the like.
  • the desired recombinant vector can be prepared from the E. coli forming the colony.
  • whether or not the recombinant vector has the target cDNA nucleotide sequence can be confirmed by a known method, for example, the dideoxynucleotide chain termination method or the like.
  • cDNA was synthesized using RNA extracted from hybridoma cells as a template to obtain a 5'-RACE cDNA library.
  • the 5'-RACE cDNA library can be synthesized using a commercially available kit such as the SMART RACE cDNA Amplification Kit as appropriate.
  • the antibody gene was amplified by PCR method.
  • Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences.
  • the nucleotide sequences of these primers vary depending on the immunoglobulin subclass. Therefore, the subclass is preferably determined in advance using a commercially available kit such as the Iso Strip Mouse Monoclonal Antibody Isotype Kit (Roche Diagnostics).
  • the gene encoding mouse IgG can be isolated using primers capable of amplifying genes encoding Y1, Y2a, Y2b, Y3 as heavy chains, and kappa and beta chains as light chains.
  • primers capable of amplifying genes encoding Y1, Y2a, Y2b, Y3 as heavy chains, and kappa and beta chains as light chains In order to amplify the variable region gene of IgG, a primer annealing to a constant region site close to the variable region can usually be used as a 3'-side primer.
  • the primers attached to the 5'-RACE cDNA library construction kit were used as 5' side primers.
  • immunoglobulins composed of combinations of heavy and light chains can be reconstituted. Desired antibodies can be screened using the BAFFR-binding activity of the reconstituted immunoglobulin as an indicator. For example, for the purpose of isolating an antibody against BAFFR, it is more preferable that the binding of the antibody to BAFFR is specific. Antibodies that bind to BAFFR can be screened, for example, by the following steps:
  • binding of antibodies to cells expressing BAFFR are well known. Specifically, the binding of the antibody to BAFFR-expressing cells can be detected by techniques such as FACS as described above. In order to evaluate the binding activity of the antibody, a fixed sample of BAFFR-expressing cells can be appropriately used.
  • Screening methods for antibodies using binding activity as an index also include panning methods using phage vectors. Screening methods utilizing phage vectors are advantageous in cases where antibody genes are isolated from heavy and light chain subclass libraries of cell populations expressing polyclonal antibodies.
  • the genes encoding the variable regions of the heavy and light chains can be ligated with appropriate linker sequences to form single-chain Fvs (scFvs).
  • Phage with scFv presented on the surface can be obtained by inserting a gene encoding scFv into a phage vector.
  • the phage is contacted with the antigen of interest. By collecting phages that bind to the antigen, the DNA encoding the scFv with target-binding activity can be isolated. Repeating this process as needed, the scFv with the desired binding activity can be concentrated.
  • the cDNA is digested by restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA.
  • the restriction enzyme recognizes and cleaves nucleotide sequences that occur with low frequency in the nucleotide sequence of the antibody gene. And it is preferred to introduce restriction sites conferring sticky ends into the vector to insert a single copy of the digested fragment in the correct orientation.
  • the cDNA encoding the V region of the anti-BAFFR antibody was digested as described above and inserted into a suitable expression vector to construct an antibody expression vector. In this case, if the gene encoding the constant region (C region) of the antibody and the gene encoding the V region described above are fused in-frame, a chimeric antibody is obtained.
  • chimeric antibody means that the origin of the constant region is different from the origin of the variable region. Therefore, human-human allochimeric antibodies are also included in the chimeric antibodies of the present invention in addition to mouse-human heterozygous chimeric antibodies.
  • a chimeric antibody expression vector can be constructed by inserting the above V region gene into an expression vector already having a constant region. Specifically, for example, a recognition sequence of a restriction enzyme that excises the above-mentioned V region gene can be appropriately placed on the 5' side of an expression vector carrying DNA encoding a desired antibody constant region (C region). Chimeric antibody expression vectors were constructed by in-frame fusion of two genes digested with the same combination of restriction enzymes.
  • an anti-BAFFR monoclonal antibody the antibody gene is inserted into an expression vector such that the gene is expressed under the control of an expression control region.
  • Expression control regions used to express antibodies include, for example, enhancers and promoters.
  • an appropriate signal sequence can be appended to the amino terminus to allow secretion of the expressed antibody outside the cell.
  • other suitable signal sequences are attached.
  • the expressed polypeptide is cleaved at the carboxy-terminal portion of the above sequence, and the resulting polypeptide can be secreted out of the cell as a mature polypeptide.
  • recombinant cells expressing DNA encoding anti-BAFFR antibody can be obtained.
  • DNAs encoding the heavy chain (H chain) and light chain (L chain) of the antibody are inserted into different expression vectors, respectively.
  • Antibody molecules having H and L chains can be expressed by co-transfecting the same host cell with a vector into which H and L chains are inserted.
  • host cells can be transformed with a single expression vector into which DNAs encoding H chain and L chain are inserted (refer to International Publication WO 94/11523).
  • antibody gene expression systems using prokaryotic cells are also known.
  • bacterial cells such as Escherichia coli (E.coli) and Bacillus subtilis can be appropriately used.
  • An expression vector containing the antibody gene of interest is introduced into these cells by transformation.
  • the desired antibody can be obtained from the culture of transformed cells by culturing the transformed cells in vitro.
  • transgenic animals can also be used to produce recombinant antibodies. That is, the antibody can be obtained from an animal into which a gene encoding the desired antibody has been introduced.
  • an antibody gene can be constructed by in-frame insertion into a gene encoding a protein inherently produced in milk to construct a fusion gene.
  • a protein secreted into milk for example, goat B casein and the like can be used.
  • a DNA fragment containing the fusion gene into which the antibody gene is inserted is injected into a goat embryo, and the embryo is then introduced into a female goat.
  • the milk produced by the transgenic goat from the goat that received the embryo (or its progeny) can obtain the desired antibody as a fusion protein with the milk protein.
  • domains derived from genetic recombinant antibodies that have been artificially modified to reduce heterologous antigenicity to humans, etc. can be suitably used as containing antibody variable regions domain of an antigen-binding molecule.
  • Such genetically recombinant antibodies include, for example, humanized antibodies. These modified antibodies are suitably prepared by known methods.
  • Humanized antibodies specifically, humanized antibodies obtained by grafting CDRs of non-human animal antibodies, eg, mouse antibodies, onto human antibodies, and the like are known.
  • Conventional genetic recombination methods for obtaining humanized antibodies are also known.
  • a method of grafting mouse antibody CDRs to human FRs for example, overlap extension PCR (overlap extension PCR) is known.
  • overlap extension PCR overlap extension PCR
  • nucleotide sequences encoding the CDRs of the mouse antibody to be transplanted are appended to primers for synthesizing FRs of human antibodies. Primers were prepared separately for the 4 FRs.
  • mouse CDRs when mouse CDRs are transplanted into human FRs, it is beneficial to select human FRs with high identity to mouse FRs to maintain the function of CDRs. That is, it is generally preferable to use a human FR comprising an amino acid sequence having a high amino acid sequence identity with the FR adjacent to the mouse CDR to be transplanted.
  • the V regions of human antibodies are expressed as single-chain antibodies (scFv) on the surface of phage by phage display methods.
  • Phages can be selected that express scFvs that bind to the antigen.
  • the DNA sequence encoding the V region of a human antibody that binds an antigen can be determined by analyzing the genes of selected phages.
  • the DNA sequence of the scFv bound to the antigen was determined.
  • An expression vector can be produced by fusing the V region sequence with the desired human antibody C region sequence in frame and inserting it into an appropriate expression vector.
  • the expression vector is introduced into cells suitable for expression, such as those described above.
  • Human antibodies can be produced by expressing a gene encoding a human antibody in a cell.
  • the anti-BAFFR antibody provided by the invention can specifically bind to BAFFR, block the BAFFR-BAFF signaling pathway, remove excessively proliferated B lymphocytes, and inhibit the growth of B cells; drug problems.
  • Figure 1 ELISA method to detect the binding ability of anti-BAFFR murine antibody to human BAFFR protein.
  • Figure 2A FACS detection of the binding ability of anti-BAFFR murine antibody to human BAFFR-CHO-K1 cells.
  • Figure 2B FACS detection of the binding ability of anti-BAFFR chimeric antibodies to human BAFFR-CHO-K1 cells.
  • Figure 3 Ability of anti-BAFFR chimeric antibodies to block BAFF-BAFFR binding.
  • Figure 4 Binding activity of anti-BAFFR humanized antibody A, antibody B, antibody C, and antibody D to Human-BAFFR-His.
  • Figure 5 Binding activity of anti-BAFFR humanized antibody E, antibody F, and antibody G to Human-BAFFR-His.
  • Figure 6 Binding activity of anti-BAFFR humanized antibody H to Human-BAFFR-His.
  • mice immunization Take 3-5 6-week-old female Balb/c mice and use recombinant human BAFFR-Fc protein as immunogen for intraperitoneal or subcutaneous immunization. Negative serum was taken 3 days in advance, and for the first immunization, 50 ⁇ g of recombinant human BAFFR-Fc protein fully emulsified with Freund's complete adjuvant was injected intraperitoneally, and 25ug was fully emulsified with incomplete Freund's adjuvant by intraperitoneal injection on the 14th and 35th days. The recombinant human BAFFR-Fc protein was immunized for the second and third times.
  • the serum was diluted by titer, and the serum titer was detected by ELISA.
  • the mouse spleen and lymph nodes can be harvested for cell fusion and phage library construction.
  • Alpaca immunization Using recombinant human BAFFR-Fc protein as the immunogen, the antigen and adjuvant were emulsified 1:1 to form a homogeneous mixture. The total amount of antigen for each immunization was kept between 0.5-1 mg and the volume was below 1-2 mL . A small amount of the mixed antigen was injected into the left and right sides near the neck lymph nodes of the alpaca, and the immunization was performed once every 2 weeks, for a total of 7 immunizations. Blood was collected from the neck vein of the alpaca at an interval of 5-7 days after the 6th and 7th immunization. The upper serum was collected by centrifugation.
  • the lymphocytes can be separated with cell separation medium, and the number of cells can be calculated by using a hemocytometer. According to the number of cells, use RNAiso Plus to dissolve the isolated lymphocytes and prepare. Phage library construction was performed.
  • mice were sacrificed to remove the spleen and lymph nodes, and the cells were pressed through a mesh to obtain B lymphocytes and lymph node cells.
  • B lymphocytes/and lymph node cells were mixed with myeloma cells SP2/0 at a ratio of 2:1, the suspension was centrifuged at 1000 rpm for 8 minutes, the pellet was collected, and washed twice with electrofusion solution. Then take the precipitate and add electrofusion solution to 9 mL, and inject 9 mL of cell suspension into each electrode cell. It was fused with SP2/0 cells according to the conventional electroporation method.
  • the fusions were cultured in complete DMEM medium containing HAT in 8% CO 2 at 37°C.
  • the feeder cells used in the fusion selection culture were taken from macrophages in the peritoneal cavity of non-immunized animals to aid in the growth of new hybrid B lymphocyte hybridomas.
  • RNA of lymphocytes was extracted with RNAiso Plus reagent. Then reverse-transcribe cDNA, amplify specific antibody fragments from reverse-transcribed cDNA, use Taq DNA Polymerase Hot Start enzyme for PCR amplification, and run 1% agarose gel electrophoresis on the obtained PCR amplification product. The kb-sized band was cut into gel and recovered using a DNA purification and recovery kit according to the instructions. The specific antibody fragment was amplified again from the DNA fragment amplified and recovered by PCR in the previous step, and recovered by cutting the gel. The antibody gene sequence amplified in the previous step and the phage vector were digested with SfiI, and the antibody gene was ligated and cloned into the phage plasmid.
  • the plasmid was electrotransformed into SS320 competent cells.
  • 1 mL of 2YT medium was added to the electric shock cup for recovery.
  • the electric shock product was aspirated and the electric shock cup was washed with 2YT medium to obtain the recovery product, which was cultured at 37 ° C and 220 rpm until the OD600 reached 0.5.
  • the helper phage was added, the cells were incubated at 37°C for 30 minutes, kanamycin and 0.2 mM IPTG were added, and the cells were incubated at 30°C overnight.
  • the clones whose secreted antibodies can bind to human BAFFR protein were screened by ELISA method.
  • the binding activity of the secreted antibody to huBAFFR-CHO-K1 was detected by FACS method, and finally the species specificity of the hybridoma antibody binding to BAFFR was detected.
  • the culture supernatant of hybridoma cells was added to the ELISA plate, 50 ⁇ L/well, the supernatant of SP2/0 cells was used as a negative control, and the immune polyantibody serum was used as a positive control.
  • the lentiviral vector carrying the human BAFFR gene was transfected into CHO-K1 cells to obtain cells overexpressing human BAFFR protein, which were named as human BAFFR-CHO-K1 cells.
  • the human BAFFR-CHO-K1 cells in logarithmic growth phase were placed in a 1.5mL EP tube, washed with PBS, and centrifuged twice.
  • the positive cloned antibodies screened by ELISA were diluted to 40 ⁇ g/mL with PBS, and 8 cells were diluted 3 times.
  • the lentiviral vector carrying the mouse and cynomolgus monkey BAFFR gene was transfected into CHO-K1 cells to obtain cells overexpressing the mouse or cynomolgus monkey BAFFR protein, which were named as mouse/monkey BAFFR-CHO-K1 cells.
  • the binding activity of hybridoma antibody to mouse and cynomolgus monkey BAFFR protein was detected.
  • the eluted phage was amplified and then the screening process was repeated twice, and the amount of antibody coated on the immune tube was halved successively to obtain the eluted phage after 3 screenings.
  • the phage screened in the previous step was diluted 106 times, 100 ⁇ L was added to the SS320 bacterial solution with an OD600 of 0.5, incubated at 37 °C for 30 min, and then coated on a 2X YT plate containing tetracycline and ampicillin, and cultured at 37 °C overnight. Monoclonal colonies were obtained in two days.
  • 96 monoclonal colonies were selected and placed on a 96-well cell culture plate containing 2X YT medium containing tetracycline and ampicillin. After culturing at 37°C for 3-4 hours, kanamycin and 20:1 helper phage were added to the culture wells. Incubate overnight at 30°C. The cell fluid after overnight culture was centrifuged to obtain a supernatant. The phage supernatant obtained in the previous step was added to the 96-well ELISA plate coated with antigen overnight and blocked with 3% BSA, and incubated at room temperature for 1 h.
  • monoclonal antibodies are mainly obtained by in vitro culture method.
  • the cell line was expanded into a T75 culture flask, and cultured until the cell coverage was 80-90%, the cell supernatant was discarded, and 30 mL of hybridoma-SFM was added to culture at 37°C, 5% CO 2 . After 2-3 days of culture, add 30 mL of hybridoma-SFM, if the cell viability is less than 30%, fresh viable cells can be added. After culturing for 6-7 days, when the cell viability is lower than 20%, the culture supernatant is collected after low-speed centrifugation, stored at 4° C. for later use, and subsequent functional tests are performed to obtain candidate antibodies 1-8.
  • the DNA sequences of mouse antibody variable regions expressed by candidate hybridomas were determined using a degenerate primer PCR-based method.
  • the hybridoma cell lines were expanded and cultured respectively, the cells were collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol.
  • Trizol Trizol
  • the corresponding variable region DNA sequence was amplified by PCR using the first-strand cDNA as the subsequent template, and the PCR primers used were based on the Ig-primer set.
  • the purified PCR products are recovered and sequenced to obtain candidate hybridoma heavy chain variable region and light chain variable region sequences.
  • Example 8 Expression and purification of candidate sequence IgG chimeric antibody
  • the sequenced antibody fragments are synthesized into a human IgG framework, and then the antibody fragments are inserted into the vector using molecular cloning technology to construct mammalian
  • the cell expression plasmid was introduced into the host cell line CHO cells by liposome transfection, and the fermentation supernatant was obtained by cell fed-batch, and the fermentation supernatant was taken for a series of steps such as ProteinA affinity chromatography and ion exchange chromatography. purification, the purified IgG chimeric monoclonal antibody was finally obtained. Finally, the purity of the antibody was identified by SDS-PAGE, and the concentration of the antibody was determined by ultraviolet microspectrophotometer.
  • Example 9 FACS detection of anti-BAFFR antibody blocking the binding effect of BAFF and BAFFR
  • Anti-BAFFR antibody was mixed with biotin-labeled human BAFF (300 ng/mL) protein, and incubated at 4°C for 1 h. The cells were added to the flat bottom plate, centrifuged to remove the supernatant, 100 ⁇ L of antibody and human BAFF mixture dilution was added to the cells, incubated at 4°C for 1 h, and the cells were washed three times with FACS buffer. Add 5 ⁇ g/mL of NA-PE antibody and incubate at 4°C for 30 minutes. After washing the cells three times with FACS buffer, flow cytometry was used to verify that the antibody could block the binding of human BAFF to BAFFR on the surface of human BAFFR-CHO-K1 cells.
  • the AHC sensor was equilibrated with 0.02% PBST (0.02% Tween 20, pH 7.4, 1 ⁇ PBS) as buffer for 60 s, the BAFFR antibody in the sample plate was solidified for 300 s, and the secondary equilibration buffer was used for 180 s.
  • Human BAFFR-hi protein at 100 nm was bound to BAFFR antibody for 300 s and then dissociated for 600 s. After dissociation, 10 mM glycine (pH 2.0) was used as regeneration buffer for regeneration for 30 s.
  • the sensor was regenerated with 10 mM glycine (pH 2.0).
  • the affinity of antibodies 1-8 is about 10 nM, and the overall affinity is good.
  • Benchmark, anti-BAFFR antibody and isotype control purchased from Roche were taken and diluted to 10 ⁇ g/mL with PBS.
  • PBMC cells were taken, centrifuged (400G, 5 min), the supernatant was discarded, PBS was added to resuspend and wash, and PBS was resuspended after centrifugation again.
  • Set blank group PBMC cells
  • PBMC+secondary antibody APC anti-human IgG Fc, purchased from Biolegend
  • PBMC+CD20 group PBMC+CD20+secondary antibody group
  • PBMC+CD20+antibody+secondary antibody group The cell density is 1E6 cells/group, 100 ⁇ L/group
  • Anti-human CD20 (purchased from BD) was added to PBMC+CD20 group, PBMC+CD20+ secondary antibody group, and PBMC+CD20+ antibody+ secondary antibody group at 20 ⁇ L/well, and incubated at room temperature for 30 min in the dark.
  • the PBMC+CD20+antibody+secondary antibody group was added with 120 ⁇ L of antibody diluted to 10 ⁇ g/mL (final concentration 5 ⁇ g/mL), and incubated at room temperature for 1 h in the dark.
  • Blank group (PBMC), PBMC+secondary antibody group, PBMC+CD20 group, and PBMC+CD20+secondary antibody group were incubated with equal volume of PBS.
  • Antibody Mean-APC Benchmark 12642 isotype control 783 Antibody 1 13145 Antibody 2 12462 Antibody 3 9244 Antibody 4 9616 Antibody 5 12126 Antibody 6 10050 Antibody 7 12882 Antibody 8 14436
  • the sequence of antibody 5 was humanized to obtain humanized antibodies A, B, C, D, E, F, G and H.
  • the specific sequences of the antibodies are shown in Table 4.
  • Antibody A (reference antibody) SEQ ID NO: 150 SEQ ID NO: 158 Antibody B SEQ ID NO: 150 SEQ ID NO: 166 Antibody C SEQ ID NO: 163 SEQ ID NO: 167 Antibody D SEQ ID NO: 163 SEQ ID NO: 162 Antibody E SEQ ID NO: 163 SEQ ID NO: 165 Antibody F SEQ ID NO: 164 SEQ ID NO: 167 Antibody G SEQ ID NO: 164 SEQ ID NO: 162 Antibody H SEQ ID NO: 164 SEQ ID NO: 165
  • Blocking block with 3% nonfat milk powder, 300 ⁇ L/well, incubate at 37° C. for 1 hour, pour off the blocking solution, wash 4 times with a plate washer, and pat dry on flat paper.
  • Sample dilution Dilute the reference product and the test product to 10 ⁇ g/mL with 3% skimmed milk powder, and use this as the initial concentration for 3-fold dilution, a total of 11 gradients, and a blank well, only adding Diluent. 100 ⁇ L/well, incubate at 37°C for 1 hour. The liquid in the wells was discarded, washed 4 times in a plate washer, and patted dry on flat paper.
  • Enzyme-labeled secondary antibody Peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG was diluted 1:20000 with 3% nonfat milk powder, 100 ⁇ L/well, and incubated at 37°C for 1 hour. Wash in a plate washer 6 times and pat dry on flat paper.
  • Color development add TMB color development solution, 100 ⁇ L/well, wrap it with aluminum foil, and develop color at 37° C. for 8 minutes in the dark.
  • Stop color development Add stop solution 1M HCl to stop color reaction, 100 ⁇ L/well.
  • Humanized antibody C and antibody H were selected for affinity multi-concentration detection.
  • the AHC sensor was soaked with 0.02% PBST (0.02% Tween 20, pH 7.4, 1 ⁇ PBS) as a buffer for 600 s to remove the sucrose covered on the sensor surface.
  • PBST 0.02% Tween 20, pH 7.4, 1 ⁇ PBS
  • the experimental temperature is set to 30 °C
  • the shaking speed is set to 1000 rpm.
  • the AHC sensor was equilibrated with 0.02% PBST (0.02% Tween 20, pH 7.4, 1 ⁇ PBS) as buffer for 60 s, the BAFFR antibody in the sample plate was immobilized for 300 s, and the secondary equilibration buffer was 180 s.
  • 100 nM of human Human-BAFFR-His (KACTUS) protein binds to BAFFR antibody for 300 s and then dissociates for 600 s. After dissociation, 10 mM glycine (pH 2.0) was used as regeneration buffer for regeneration for 30 s.
  • the sensor was regenerated with 10 mM glycine (pH 2.0).

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Abstract

本发明公开了一种抗BAFFR抗体或其抗原结合片段及其用途以及药物组合物。本发明提供的抗BAFFR抗体能够特异性地与BAFFR结合，阻断BAFFR-BAFF信号通路，清除过度增殖的B淋巴细胞，抑制B细胞的生长；有效解决现有治疗B细胞淋巴瘤的药物的耐药性问题。

Description

一种抗BAFFR抗体及其应用 技术领域
本发明属于生物技术领域，具体涉及一种抗BAFFR抗体及其应用。
背景技术
BAFFR由TNFRSF13C基因编码，其它简称有：TNFRSF13C、BLyS-3、CD268等。BAFFR属于TNFR家族的跨膜蛋白，由184氨基酸组成，分为胞外部分、跨膜区和胞内部分，其胞外部分适合抗体药物的开发。
BAFFR是不同阶段B淋巴细胞特异性高表达标志物，在B细胞中表达图谱与CD20类似，表达于除浆细胞(plasma)、原B细胞(pro B)和前B(pre B)细胞外的其它B细胞的发育阶段，但是其表达丰度比CD20更高。BAFFR不表达于B细胞起源细胞骨髓干细胞，过度增殖的B细胞在被清除后，停药后B细胞仍会再次补充，并不影响人体的正常需求，安全性较好。
不同阶段B细胞增殖对应于不同的淋巴瘤适应症，BAFFR靶点阻断剂的适应症包含了除ALL(急性淋巴细胞白血病)和MM(多发性骨髓瘤)之外其它的淋巴瘤类型，如MCL(套细胞淋巴瘤)、DLBCL(弥漫大B淋巴瘤、慢淋、滤泡淋巴瘤)等。
BAFFR的唯一配体为BAFF，除作为B细胞特定发育阶段的标志物，BAFFR与BAFF结合后，共同作用激活B细胞中NF-kB信号通路，促进B细胞的增殖和活化。BAFF-BAFFR信号通路和BCR通路是Immature B细胞成熟和活化的关键通路，对双通路的阻断可以完全清除外周B淋巴细胞并抑制B淋巴细胞的发育和增殖。
目前用于治疗B细胞淋巴瘤的药物，大多为靶向CD19、CD20的药物，针对B细胞生长的BCR通路，但这类药物易产生耐药性。以CD19为例，根据现有报道，大约40％病人会由于癌细胞表面CD19抗原丢失而复发。BAFFR-BAFF信号通路位于BCR通路的下游，是细胞的必要生存通道，可以在下游进一步抑 制B细胞的生长。
发明内容
针对现有问题的不足，在本申请中，发明人开发了具有良好性能的抗BAFFR抗体，其能够特异性识别/结合BAFFR，阻断BAFFR-BAFF信号通路，清除过度增殖的B淋巴细胞，抑制B细胞的生长；有效解决现有治疗B细胞淋巴瘤的药物的耐药性问题。
本发明的第一个目的是提供一种抗BAFFR抗体或其抗原结合片段。
本发明的第二个目的是提供上述抗BAFFR抗体或其抗原结合片段的编码基因。
本发明的第三个目的是提供上述抗BAFFR抗体或其抗原结合片段的应用。
本发明的第四个目的是提供一种药物组合物。
本发明解决其技术问题采用的技术方案是：
一种抗BAFFR单克隆抗体或其抗原结合片段，包含重链和轻链，其特征在于，所述重链包含重链互补决定区CDR1、CDR2和CDR3，所述轻链可变区包含轻链互补决定区CDR1、CDR2和CDR3，其中，
(a)重链可变区的CDR1，选自SEQ ID NO：33-40的任一氨基酸序列，或与SEQ ID NO：33-40的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：33-40的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(b)重链可变区的CDR2，选自SEQ ID NO：41-48的任一氨基酸序列，或与SEQ ID NO：41-48的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：41-48的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(c)重链可变区的CDR3，选自SEQ ID NO：49-56的任一氨基酸序列，或与SEQ ID NO：49-56的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID  NO：49-56的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(d)轻链可变区的CDR1，选自SEQ ID NO：57-64的任一氨基酸序列，或与SEQ ID NO：57-64的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：57-64的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(e)轻链可变区的CDR2，选自SEQ ID NO：65-72的任一氨基酸序列，或与SEQ ID NO：65-72的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：65-72的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(f)轻链可变区的CDR3，选自SEQ ID NO：73-80的任一氨基酸序列；所述抗体结合siglec15，或与SEQ ID NO：73-80的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：73-80的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
优选地，所述重链和轻链包含重链可变区和轻链可变区，所述重链可变区包括框架区FR和所述重链可变区的CDR1、CDR2和CDR3；所述的框架区FR包括：
(a)选自SEQ ID NO：81-88任一氨基酸序列，
或与SEQ ID NO：81-88给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与SEQ ID NO：81-88的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR1，
(b)选自SEQ ID NO：89-96任一氨基酸序列，
或与SEQ ID NO：89-96给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与SEQ ID NO：89-96的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR2，
(c)选自SEQ ID NO：97-104任一氨基酸序列，
或与SEQ ID NO：97-104给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与SEQ ID NO：97-104的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR3，
和
(d)选自SEQ ID NO：105-112任一氨基酸序列，
或与SEQ ID NO：105-112给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与选自SEQ ID NO：105-112的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR4；
优选地，所述重链和轻链包含重链可变区和轻链可变区，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；所述的框架区FR包括：
(e)选自SEQ ID NO：113-120任一氨基酸序列，
或与SEQ ID NO：113-120给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与SEQ ID NO：113-120的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR1，
(f)选自SEQ ID NO：121-128任一氨基酸序列，
或与SEQ ID NO：121-128给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与SEQ ID NO：121-128的任一氨基酸序列相比具有一个或多个(优选1、 2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR2，
(g)选自SEQ ID NO：129-136任一氨基酸序列，
或与SEQ ID NO：129-136给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与SEQ ID NO：129-136的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR3，
和
(h)选自SEQ ID NO：137-144任一氨基酸序列，
或与SEQ ID NO：137-144给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与选自SEQ ID NO：137-144的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR4。
优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：33、SEQ ID NO：41和SEQ ID NO：49组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：57、SEQ ID NO：65和SEQ ID NO：73组成；
更优选地，所述重链可变区包括框架区FR和上述重链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：91所示的FR1、SEQ ID NO：101所示的FR2、SEQ ID NO：111所示的FR3和SEQ ID NO：121所示的FR4。
更优选地，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：131所示的FR1、SEQ ID NO：139所示的FR2、SEQ ID NO：147所示的FR3和SEQ ID NO：155所示的FR4。
优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：38、SEQ ID NO：48和SEQ ID NO：58组成，且所述轻链可变区的CDR1、 CDR2和CDR3分别由SEQ ID NO：68、SEQ ID NO：76和SEQ ID NO：84组成；
更优选地，所述重链可变区包括框架区FR和上述重链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：92所示的FR1、SEQ ID NO：102所示的FR2、SEQ ID NO：112所示的FR3和SEQ ID NO：122所示的FR4。
更优选地，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：132所示的FR1、SEQ ID NO：140所示的FR2、SEQ ID NO：148所示的FR3和SEQ ID NO：156所示的FR4。
优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：39、SEQ ID NO：49和SEQ ID NO：59组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：69、SEQ ID NO：77和SEQ ID NO：85组成；
更优选地，所述重链可变区包括框架区FR和上述重链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：93所示的FR1、SEQ ID NO：103所示的FR2、SEQ ID NO：113所示的FR3和SEQ ID NO：123所示的FR4。
更优选地，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：133所示的FR1、SEQ ID NO：141所示的FR2、SEQ ID NO：149所示的FR3和SEQ ID NO：157所示的FR4。
优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：40、SEQ ID NO：50和SEQ ID NO：60组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：70、SEQ ID NO：78和SEQ ID NO：86组成；
更优选地，所述重链可变区包括框架区FR和上述重链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：84所示的FR1、SEQ ID NO：94所示的FR2、SEQ ID NO：100所示的FR3和SEQ ID NO： 108所示的FR4。
更优选地，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：116所示的FR1、SEQ ID NO：124所示的FR2、SEQ ID NO：132所示的FR3和SEQ ID NO：140所示的FR4。
优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：37、SEQ ID NO：45和SEQ ID NO：53组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：61、SEQ ID NO：69和SEQ ID NO：77组成；
更优选地，所述重链可变区包括框架区FR和上述重链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：85所示的FR1、SEQ ID NO：93所示的FR2、SEQ ID NO：101所示的FR3和SEQ ID NO：109所示的FR4。
更优选地，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：117所示的FR1、SEQ ID NO：125所示的FR2、SEQ ID NO：133所示的FR3和SEQ ID NO：141所示的FR4。
优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：38、SEQ ID NO：46和SEQ ID NO：54组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：62、SEQ ID NO：70和SEQ ID NO：78组成；
更优选地，所述重链可变区包括框架区FR和上述重链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：86所示的FR1、SEQ ID NO：94所示的FR2、SEQ ID NO：102所示的FR3和SEQ ID NO：110所示的FR4。
更优选地，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：118所示的FR1、SEQ ID NO：126所示的FR2、SEQ ID NO：134所示的FR3和SEQ ID NO：142所示的FR4。
优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：39、SEQ ID NO：47和SEQ ID NO：55组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：63、SEQ ID NO：71和SEQ ID NO：79组成；
更优选地，所述重链可变区包括框架区FR和上述重链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：87所示的FR1、SEQ ID NO：95所示的FR2、SEQ ID NO：103所示的FR3和SEQ ID NO：111所示的FR4。
更优选地，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：119所示的FR1、SEQ ID NO：127所示的FR2、SEQ ID NO：135所示的FR3和SEQ ID NO：143所示的FR4。
优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：40、SEQ ID NO：48和SEQ ID NO：56组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：64、SEQ ID NO：72和SEQ ID NO：80组成。
更优选地，所述重链可变区包括框架区FR和上述重链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：88所示的FR1、SEQ ID NO：96所示的FR2、SEQ ID NO：104所示的FR3和SEQ ID NO：112所示的FR4。
更优选地，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；更加优选地，所述的框架区FR包括：SEQ ID NO：120所示的FR1、SEQ ID NO：128所示的FR2、SEQ ID NO：136所示的FR3和SEQ ID NO：144所示的FR4。
一种抗BAFFR单克隆抗体或其抗原结合片段，包括重链可变区和轻链可变区；其特征在于，其中：
(a)所述重链可变区具有SEQ ID NO：1-8给出的任一氨基酸序列，
或与SEQ ID NO：1-8给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与SEQ ID NO：1-8的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(b)所述轻链可变区具有SEQ ID NO：17-24给出的任一氨基酸序列；
或与SEQ ID NO：17-24给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
或与SEQ ID NO：17-24的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列。
优选地，所述重链可变区和轻链可变区选自如下(1)-(8)中的任一种氨基酸序列：
(1)SEQ ID NO：1和SEQ ID NO：17；
(2)SEQ ID NO：2和SEQ ID NO：18；
(3)SEQ ID NO：3和SEQ ID NO：19；
(4)SEQ ID NO：4和SEQ ID NO：20；
(5)SEQ ID NO：5和SEQ ID NO：21；
(6)SEQ ID NO：6和SEQ ID NO：22；
(7)SEQ ID NO：7和SEQ ID NO：23；
(8)SEQ ID NO：8和SEQ ID NO：24。
优选地，所述抗体为鼠源抗体、单域抗体、嵌合抗体、全人抗体或人源化抗体。
优选地，所述抗体或其抗原结合片段为单特异性抗体、双特异性抗体、多特异性抗体、抗体偶联物或细胞治疗的car分子。
优选地，所述抗体偶联物的细胞毒性药物为细胞毒性分子、放射性同位素、药物或细胞因子。
优选地，所述双特异性抗体包括上述任一所述的抗体或其抗原结合片段，以及针对其他抗原和/或其他抗原表位的抗体或抗原结合片段。
优选地，所述抗体为单克隆抗体。
优选地，所述抗体或其抗原结合片段结合至BAFFR蛋白。
优选地，所述BAFFR蛋白是人猴BAFFR蛋白。
优选地，所述BAFFR蛋白形成细胞的一部分。
优选地，所述BAFFR蛋白在所述细胞的表面上表达。
优选地，所述细胞是淋巴细胞。
优选地，所述细胞是B细胞。
优选地，所述细胞是癌细胞。
优选地，所述癌细胞是淋巴瘤细胞。
本发明还提供一种编码上述任一所述的抗BAFFR抗体或其抗原结合片段的基因。
优选地，所述基因选自如下(1)-(8)中的任一种：
(1)含有如SEQ ID NO：9所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：25所示的编码所述抗体轻链可变区的核苷酸序列；
(2)含有如SEQ ID NO：10所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：26所示的编码所述抗体轻链可变区的核苷酸序列；
(3)含有如SEQ ID NO：11所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：27所示的编码所述抗体轻链可变区的核苷酸序列；
(4)含有如SEQ ID NO：12所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：28所示的编码所述抗体轻链可变区的核苷酸序列；
(5)含有如SEQ ID NO：13所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：29所示的编码所述抗体轻链可变区的核苷酸序列；
(6)含有如SEQ ID NO：14所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：30所示的编码所述抗体轻链可变区的核苷酸序列；
(7)含有如SEQ ID NO：15所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：31所示的编码所述抗体轻链可变区的核苷酸序列；
(8)含有如SEQ ID NO：16所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：32所示的编码所述抗体轻链可变区的核苷酸序列。
本发明提供一种含有所述基因的重组载体、表达盒、转基因细胞系或重组菌。
本发明提供一种上述任一项所述的抗体或其抗原结合片段在如下(a)或(b)或(c)中的应用：
(a)制备抑制癌细胞迁移的药物中的应用；
(b)制备治疗癌症的药物中的应用；
(c)制备治疗自身免疫病的药物中的应用。
本发明提供一种多功能融合蛋白，其包含上述任一项所述的抗体或其抗原结合片段或抗体偶联药物。
优选地，其还包含一个或多个与其他抗原特异性结合的第二抗体或其抗原结合部分。
优选地，所述结合第二抗体或其抗原结合部分的抗原选自肿瘤相关抗原(TAA)或免疫检查点。
优选地，所述免疫检查点为CD3、4-1BB、CD40、OX40、ICOS。
优选地，所述多功能融合蛋白还包含细胞因子。
优选地，所述细胞因子选自IL8、IL10、IL15、IL18、TGF、VEGF、IFNγ、IFNα或GM-CSF。
本发明还提供上述任一项所述的抗体或其抗原结合片段、上述任一项所述的多功能融合蛋白，其在制备治疗癌症或自身免疫疾病的药物中的用途。
优选地，所述癌症是淋巴瘤、白血病或骨髓瘤。
优选地，所述淋巴瘤是套细胞淋巴瘤、滤泡淋巴瘤、弥漫性大B细胞淋巴瘤、边缘区淋巴瘤或伯基特氏淋巴瘤。
优选地，所述白血病是淋巴母细胞白血病、慢性淋巴细胞白血病或毛细胞白血病。
优选地，所述骨髓瘤是多发性骨髓瘤。
优选地，所述自身免疫疾病是系统性红斑狼疮、类风湿性关节炎、强直性脊柱炎、特发性血小板减少性紫癜、溃疡性结肠炎、干燥综合征。
优选地，所述药物与其他药物或疗法进行联合应用。
优选地，所述其他疗法选自：化疗、放疗、靶向治疗、基因治疗、细胞治疗、干细胞治疗。
本发明提供一种调节细胞功能的方法，包括将编码上述任一项的抗体或其抗原结合片段上述任一项所述的多功能融合蛋白的多核苷酸序列递送至所述细胞中。
优选地，所述方法用于基因治疗、CAR-T治疗或CRISPR治疗。
本发明提供一种一种药物组合物，其包含上述任一项所述的抗体或其抗原结合片段和可接受的载体、稀释剂或赋形剂。
本发明提供一种一种药物组合物，其包含方上述任一项所述的多功能融合蛋白和可接受的载体、稀释剂或赋形剂。
本发明提供一种抑制细胞增殖的方法，所述方法包括：
(1)将细胞与上述任一项所述的BAFFR抗体或上述任一项所述的多功能融合蛋白接触，从而形成接触细胞；以及
(2)使所述BAFFR抗体或多功能融合蛋白结合所述接触细胞上的BAFFR蛋白，从而抑制所述细胞增殖。
优选地，所述细胞是淋巴细胞。
优选地，所述细胞是B细胞。
优选地，所述细胞是癌细胞。
优选地，所述细胞是淋巴瘤细胞。
缩写及术语定义
在本文中使用以下缩写：
mAb：单克隆抗体；VH：抗体重链可变区；VL：抗体轻链可变区；CDR：免疫球蛋白可变区中的互补决定区；FR：抗体构架区，即抗体可变区中除CDR残基以外的氨基酸残基；IgG：免疫球蛋白G；ELISA：酶联免疫吸附测定；FACS：荧光激活细胞分选术；PCR：聚合酶链式反应。
本文中，术语“抗体”是指天然的免疫球蛋白或者通过部分或完全合成而制备的免疫球蛋白。抗体可从天然存在该抗体的血浆或血清等的天然资源、或者产生抗体的杂交瘤细胞的培养上清中、动物免疫血清中、噬菌体文库筛选进行重建得到分离。备选地，可通过使用基因重组等的技术部分或完全地合成。优选地抗体包括，例如，免疫球蛋白的同种型或这些同种型的亚类的抗体。已知人免疫球蛋白包括IgGl、IgG2、IgG3、IgG4、IgAl、IgA2、IgD、IgE、IgM这9种类别(同种型)。在这些同种型中，本发明的抗体可以包括IgGl、IgG2、IgG3、IgG4。
术语“抗原结合片段”是指抗体的多肽片段，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，亦称为“抗原结合部分”。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’)2、 Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体、线性抗体(linear antibody)、单域抗体、结构域抗体、和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
术语“载体”指能够增殖与其连接的另一种核酸的核酸分子。该术语包括作为自身复制型核酸结构的载体及并入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达，本文称为“表达载体”。
术语“药学上可接受的载体”包括任何标准药物载体，诸如磷酸盐缓冲盐水溶液、水和乳液，以及各种类型的润湿剂。
“百分比(％)氨基酸序列同一性”定义为比对序列并在必要时引入缺口以获取最大百分比序列同一性后，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式进行，例如使用公众可得到的计算机软件，诸如BLAST软件或FASTA程序包。
“特异性”表示参与特异性结合的分子之一不显示任何与不同于结合伙伴分子中的一个或数个的分子的显著结合。此外，在含抗体可变区的结构域对抗原中的多个表位中的特定表位具有特异性时，也使用该术语。当含抗体可变区的结构域所结合的表位被包含在数个不同抗原中时，包含含抗体可变区的结构域的抗原结合分子可以结合具有所述表位的各种抗原。
“细胞毒性”是由细胞或化学物质引起的单纯细胞杀伤事件，不依赖于凋亡或坏死的细胞死亡机理。有时需要进行特定物质细胞毒性的检测，比如药物筛选。细胞毒性检测主要是根据细胞膜通透性发生改变来进行的检测，常用以下几种方法：MTT、XTT法；LDH的方法；其它酶方法：如检测上清中碱性磷酸酶、酸性磷酸酶的活性等。
“表位”意指抗原中的抗原决定簇，并且是指在本说明书中公开的包含抗体可变区的抗原结合分子的结构域所结合的抗原位点。因此，可以根据其结构来定义表位。另外，也可以根据识别该表位的抗原结合分子中的抗原结合活性来定义该表位。当抗原是肽或多肽时，表位可以由形成表位的氨基酸残基指定；当表位是糖链时，表位可以通过其特定的糖链结构来确定。
“BMK”是Benchmark的缩写，指阳性对照抗体，能够结合靶点蛋白或是 表达靶点蛋白的天然或工程化细胞。
以下可以确认包含含有具有BAFFR结合活性的抗体可变区的结构域的测试抗原结合分子对表位的三维结构的识别。制备表达BAFFR的细胞以用于上述目的。例如，当包含含有具有BAFFR结合活性的抗体可变区的结构域的测试抗原结合分子接触表达BAFFR的细胞时，其与所述细胞紧密结合，但是另一方面，存在这样的情况，其中抗原结合分子基本上不结合固定的包含构成BAFFR的胞外结构域的氨基酸序列的线性肽。在这些情况中，“基本上不结合”是指，相对于对表达人BAFFR的细胞的结合活性，结合活性为80％以下，通常为50％以下，优选为30％以下，并且特别优选为15％以下。
作为测定含有BAFFR抗原结合结构域的测试抗原结合分子与表达BAFFR的细胞的结合活性的方法，例如可以举出：Antibodies:A Laboratory Manual记载的方法(Ed Harlow,David Lane,Cold Spring Harbor Laboratory(1988)359-420)。BR，可以基于使用表达BAFFR的细胞为抗原的ELISA或荧光激活细胞分选术(FACS)的原理进行评价。
本发明的抗原结合分子中的含抗体可变区的结构域可以结合相同的表位。本文中，相同的表位可以存在于包含SEQ ID NO：145氨基酸序列的蛋白质中。备选地，本发明的抗原结合分子中的含抗体可变区的结构域可以分别结合不同的表位。本文中，不同的表位可以存在于包含SEQ ID NO：145的氨基酸序列的蛋白质中。
更加优选地，本申请一种抗BAFFR抗体或其抗原结合片段，所述抗体结合表位，所述表位全部或部分包含在SEQ ID NO：145所述的氨基酸序列内。
抗BAFFR抗体可以使用公知的方法以多克隆或单克隆抗体的形式获得。作为抗BAFFR抗体，可以优选制备来源于哺乳动物的单克隆抗体。这些来源于哺乳动物的单克隆抗体包含由杂交瘤产生的抗体、噬菌体表面展示技术筛选得到产生或由宿主细胞所产生的抗体，所述宿主细胞通过基因工程技术用携带抗体基因的表达载体转化。
单克隆抗体的制作例如以如下方式来进行。首先，从常规生化试剂商店购买得到BAFFR蛋白质。将BAFFR蛋白用作致敏性抗原以用于哺乳动物中的免疫。BAFFR的部分肽也可被用作致敏性抗原。在此情况中，所述部分肽也可以通过 化学合成自人BAFFR氨基酸序列获得。此外，其也可以通过将BAFFR基因的一部分整合到表达载体中并将其表达而获得。此外，其也可以通过使用蛋白酶将BAFFR蛋白降解获得，但是用作部分肽的BAFFR肽的区域和尺寸不特别地受限于具体实施方案。组成被用作致敏性抗原的肽的氨基酸的数目为至少五个以上，或优选地例如，六个以上，或七个以上。更具体地，由8至50个残基或优选地10至30个残基组成的肽可被用作致敏性抗原。
备选地，可以将BAFFR蛋白质的所需的部分多肽或肽与不同的多肽融合而成的融合蛋白质作为致敏性抗原来利用。为了制备用作致敏性抗原的融合蛋白质，例如可以优选利用抗体的Fc片段和肽标签等。表达融合蛋白质的载体可以通过使编码所需的两种或其以上的多肽片段的基因在符合读框内进行融合，并将该融合基因如上述插入到表达载体中来制作。
作为用该致敏性抗原免疫的哺乳动物，不限于特定的动物。但优选考虑与用于细胞融合的亲本细胞的适合性来进行选择。一般来说优选使用啮齿类的动物如小鼠、大鼠和仓鼠、羊驼、兔和猴。
为了通过DNA免疫获得本发明的单克隆抗体，首先，对免疫动物施用表达BAFFR蛋白质的DNA。编码BAFFR的DNA可以通过PCR等公知的方法来合成。所得BAFFR被插入到适当的表达载体中，并对免疫动物进行施用。作为表达载体，可以适合利用例如PCDNA3.1等市售的表达载体。作为将载体施用到活体内的方法，可以利用通常使用的方法。例如，通过将吸附有表达载体的金颗粒用基因枪导入到免疫动物个体的细胞内来进行DNA免疫。
在如上所述免疫哺乳动物后，在血清中证实了BAFFR结合抗体滴度的增加。然后，从哺乳动物收集免疫细胞，然后进行细胞融合。作为优选地免疫细胞，特别是可以使用脾细胞。
作为与上述免疫细胞相融合的细胞，可以使用哺乳动物的骨髓瘤细胞。骨髓瘤细胞优选具备用于筛选的适当的选择标记。选择标记是指在特定的培养条件下能够(或者不能够)存活的特性。公知的选择标记有：缺乏次黄嘌呤-鸟嘌呤-磷酸核糖转移酶(以下省略为缺乏HGPRT)、或者缺乏胸苷激酶(以下省略为缺乏TK)等。具有缺乏HGPRT或TK的细胞具有次黄嘌呤-氨基蝶呤-胸苷敏感性(以下省略为HAT敏感性)。但如果与正常的细胞相融合，则利用正常细胞的补救途 径可以持续DNA的合成，因此在HAT选择培养中也可以增殖。
缺乏HGPRT或缺乏TK的细胞分别可以在含有6-硫鸟嘌呤、8-氮鸟嘌呤(以下省略为8AG)、或者5'-溴脱氧尿苷的培养基中选择。将这些嘧啶类似物摄入到DNA中的正常细胞将死亡。同时，不摄入这些嘧啶类似物的、缺乏这些酶的细胞可以在选择培养中存活。此外被称为G418抗性的选择标记通过新霉素抗性基因赋予对2-脱氧链霉胺类抗生素(庆大霉素类似物)的抗性。适合于细胞融合的各种骨髓瘤细胞是公知的。例如，可以优选使用包括以下细胞的骨髓瘤细胞：P3(P3x63Ag8.653)(J.Immunol.(1979)123(4),1548-1550)；NS-1(C.Eur.J.Immunol.(1976)6(7),511-519)；SP2/0(Nature(1978)276(5685),269-270)；R210(Nature(1979)277(5692),131-133)等。基本上是按照公知的方法、例如Kohler和Milstein等人的方法(Methods Enzymol.(1981)73,3-46)等，来进行上述免疫细胞与骨髓瘤细胞的细胞融合。
更具体而言，例如可以在细胞融合促进剂的存在下，在常规的营养培养液中实施上述细胞融合。融合促进剂包括例如聚乙二醇(PEG)和仙台病毒(HVJ)。根据需要可以添加例如二甲基亚砜等的辅助剂以进一步提高融合效率。
免疫细胞与骨髓瘤细胞的使用比例可以任意设定。例如，相对于骨髓瘤细胞优选使用1～10倍的免疫细胞。作为用于上述细胞融合的培养液，可以使用例如适合于上述骨髓瘤细胞株增殖的RPMI1640培养液、MEM培养液、以及该种的细胞培养中使用的常规的培养液。并且可以适合添加胎牛血清(FCS)等的血清补液。
就细胞融合而言，可以将上述免疫细胞与骨髓瘤细胞的规定量在上述培养液中充分混合。再以通常30％～60％(w/v)的浓度添加预先加热到37℃左右的PEG溶液。通过对混合液进行缓慢混合可以形成所需的融合细胞(杂交瘤)。接着，逐渐添加上述例举的适当培养液到细胞中，并且将其重复离心以除去上清。可以除去不利于杂交瘤生长的细胞融合剂等。
可以通过基于抗原抗体反应的公知筛选方法来实施所需抗体的筛选和单一克隆。例如，结合于BAFFR的单克隆抗体，可以结合于在细胞表面表达的BAFFR。这样的单克隆抗体，例如可以通过荧光激活细胞分选术(FACS)进行筛选。FACS是指通过用激光分析与荧光抗体接触的细胞，测定各细胞所发出的荧光进而使结 合于细胞表面的抗体的测定成为可能的系统。
为了通过FACS对产生本发明的单克隆抗体的杂交瘤进行筛选，首先制备表达BAFFR的细胞。用于筛选的优选细胞为强制表达BAFFR的哺乳动物细胞。用作对照，可以使用未转化的哺乳动物细胞作为宿主细胞来选择性地检测对细胞表面的BAFFR的抗体的结合活性。即，通过选择产生不结合于宿主细胞、结合于强制表达BAFFR的细胞的抗体的杂交瘤，可以获得产生BAFFR单克隆抗体的杂交瘤。
备选地，抗体对于固定的表达BAFFR的细胞的结合活性可以基于ELISA的原理进行评价。例如，使表达BAFFR的细胞固定在ELISA板的孔内。通过使杂交瘤的培养上清与孔内的固定的细胞相接触，来检测结合于固定细胞上的抗体。当单克隆抗体为小鼠由来的情况下，结合于细胞上的抗体可以通过抗小鼠免疫球蛋白抗体来检测。通过上述筛选而选择的、具有抗原结合能的、产生所需抗体的杂交瘤，可以通过有限稀释法等进行克隆。
将该杂交瘤按照常规的方法进行培养，可以从其培养上清中获得所需的单克隆抗体。或者将杂交瘤施用于与其具有适应性的哺乳动物并进行增殖，可以从其腹水中获得单克隆抗体。前者的方法适合于获得高纯度的抗体。
由从该杂交瘤等的抗体产生细胞克隆的抗体基因所编码的抗体也可以被适合地利用。通过将克隆的抗体基因插入到适当的载体并导入到宿主中，由该基因所编码的抗体得以表达。用于抗体基因的分离、向载体插入基因、以及宿主细胞的转化的方法，重组抗体的制备方法也是公知的。
例如，可以从表达抗BAFFR抗体的杂交瘤细胞中制备编码抗BAFFR抗体的可变区(V区)的cDNA。为此，通常先从杂交瘤中提取总RNA。作为用于从细胞提取mRNA的方法，可以利用胍超速离心法和AGPC法。
所提取的mRNA可以使用mRNA提纯试剂盒(GE Healthcare Bioscience)等来进行纯化。或者，如QuickPrep mRNA提纯试剂盒(GE Healthcare Bioscience)等这样的用于从细胞直接提取总mRNA的试剂盒也得以市售。可以使用这样的试剂盒从杂交瘤中获得mRNA。可以使用逆转录酶由所制备的mRNA合成编码抗体V区的cDNA。cDNA可以使用AMV Reverse Transcriptase First-strand cDNA Synthesis Kit(生化学工业社)等来合成。另外，为了合成和扩增cDNA，可以适 当利用SMART RACE cDNA扩增试剂盒和基于PCR的5'-RACE法。并且，在这样的cDNA的合成的过程中，可以在cDNA的两末端导入后述的适当的限制酶位点。
将所得PCR产物纯化作为目标的cDNA片段，接着使其与载体DNA相连接。如此，重组载体得以制作，将其导入到大肠杆菌等中。选择菌落后，可以由形成该菌落的大肠杆菌制备所需的重组载体。而且，关于该重组载体是否具有作为目标的cDNA核苷酸序列，可以利用公知的方法、例如双脱氧核苷酸链终止法等来确认。
为了分离编码可变区的基因，利用使用了可变区基因扩增用引物的5'-RACE法是简便的。首先以由杂交瘤细胞提取的RNA为模板合成cDNA，获得5'-RACE cDNA文库。可以适当使用SMART RACE cDNA扩增试剂盒等市售的试剂盒来合成5'-RACE cDNA文库。
以所得5'-RACE cDNA文库为模板，通过PCR法扩增抗体基因。基于公知的抗体基因序列可以设计小鼠抗体基因扩增用引物。这些引物的核苷酸序列依赖于免疫球蛋白的亚类而不同。因此，优选地是预先使用Iso Strip小鼠单克隆抗体同种型试剂盒(Roche Diagnostics)等的市售试剂盒来决定亚类。
具体而言，可以利用能够扩增编码作为重链的Y1、Y2a、Y2b、Y3、作为轻链的k链和&链的基因的引物以分离编码小鼠IgG的基因。为了扩增IgG的可变区基因，通常可以利用在与可变区相近的恒定区位点退火的引物作为3'侧引物。另一方面，连接到5'-RACE cDNA文库构建试剂盒的引物用作5'侧引物。
利用如此扩增的PCR产物，可以重构由重链和轻链的组合而组成的免疫球蛋白。以重构的免疫球蛋白的BAFFR结合活性为指标，可以筛选所需的抗体。例如以分离对抗BAFFR的抗体为目的时，进一步优选地是抗体与BAFFR的结合为特异性的。与BAFFR结合的抗体例如可以通过以下步骤进行筛选：
(1)将含有由杂交瘤分离的cDNA所编码的V区的抗体与BAFFR的细胞相接触；
(2)检测表达BAFFR的细胞与抗体结合；
(3)选择与表达BAFFR的细胞结合的抗体。
检测抗体与表达BAFFR的细胞结合的方法是公知的。具体而言，能够通过 前面所述的FACS等技术检测到抗体与表达BAFFR的细胞的结合。为了评价抗体的结合活性可以适当利用表达BAFFR的细胞的固定样品。
作为以结合活性为指标的抗体的筛选方法还包括使用噬菌体载体的淘选方法。在抗体基因是从表达多克隆抗体的细胞群的重链和轻链亚类文库分离的情况下，利用噬菌体载体的筛选方法是有利的。编码重链和轻链的可变区的基因可以通过用适当的接头序列进行连接来形成单链Fv(scFv)。通过将编码scFv的基因插入到噬菌体载体可以获得scFv呈现于表面的噬菌体。该噬菌体与目的抗原接触。通过收集与抗原结合的噬菌体,可以分离编码具有目标结合活性的scFv的DNA。根据需要重复该过程，可以浓缩具有所需结合活性的scFv。
分离编码目标抗BAFFR抗体的V区的cDNA后，通过识别插入到该cDNA两末端的限制酶位点的限制酶来消化该cDNA。优选地限制酶识别并切割出现在抗体基因的核苷酸序列中的频率低的核苷酸序列。并且优选将赋予粘性末端的限制酶切位点导入到载体中以将单拷贝的消化片段以正确的方向插入。如上所述消化编码抗BAFFR抗体的V区的cDNA，并将其插入合适的表达载体中以构建抗体表达载体。在这种情况下，如果编码抗体恒定区(C区)的基因和编码上述V区的基因符合读框地融合，则获得嵌合抗体。
本文中，“嵌合抗体”是指恒定区的起点不同于可变区的起点。因此，除了小鼠-人等的异种嵌合抗体之外，人-人同种嵌合抗体也包含在本发明的嵌合抗体中。通过向已经具有恒定区的表达载体中插入上述V区基因，可以构建嵌合抗体表达载体。具体地，例如，切除上述V区基因的限制酶的识别序列可以适当地置于携带编码所需抗体恒定区(C区)的DNA的表达载体的5'侧。通过使利用相同组合的限制酶消化的两基因符合读框的融合来构建嵌合抗体表达载体。
为了制备抗BAFFR单克隆抗体，抗体基因被插入到表达载体中使所述基因在表达控制区的控制下表达。用于表达抗体的表达控制区包含例如增强子和启动子。另外，可以在氨基末端附加适当的信号序列使得表达的抗体分泌到细胞外。此外还附接其他适合的信号序列。所表达的多肽在上述序列的羧基末端部分被切割，并且产生的多肽可以作为成熟多肽分泌到细胞外。接着，通过用该表达载体来转化适当的宿主细胞，可以获得表达编码抗BAFFR抗体的DNA的重组细胞。
为了表达抗体基因，编码抗体重链(H链)和轻链(L链)的DNA分别被 插入到不同的表达载体中。通过用插入有H链和L链的载体共转染相同的宿主细胞，可以表达具备H链和L链的抗体分子。或者能够利用其中插入编码H链和L链的DNA的单一表达载体转化宿主细胞(参照国际公开WO 94/11523)。
通过将所分离的抗体基因导入到适当的宿主用来制备抗体的宿主细胞/表达载体的各自组合是公知的。这些表达系统均可以应用于包含本发明的抗体可变区的结构域的分离。
另外，利用原核细胞的抗体基因表达系统也是公知的。例如，使用细菌细胞的情况下，可以适当利用大肠杆菌(E.coli)、枯草杆菌等的细菌细胞。通过转化向这些细胞中导入包含目标抗体基因的表达载体。通过将所转化的细胞在体外进行培养，可以从该转化细胞的培养物中获得所需的抗体。
除了上述宿主细胞外，转基因动物也可用于产生重组抗体。即，从导入了编码所需抗体的基因的动物可以获得该抗体。例如，抗体基因可以通过符合读框的插入到编码乳汁中固有产生的蛋白质的基因的内部来构建融合基因。作为分泌到乳汁中的蛋白质，可以利用例如山羊B酪蛋白等。含有插入了抗体基因的融合基因的DNA片段被注入到山羊的胚胎中，并且然后该胚胎被导入到雌性山羊体内。由接受了胚胎的山羊生出转基因山羊，从该转基因山羊(或其后代)产生的乳汁中可以作为与乳汁蛋白质的融合蛋白质获得所需抗体。
当将本文中所述的抗原结合分子施用于人时，来源于已被人工修饰而减小针对人等的异源抗原性的遗传重组抗体的结构域可被合适地用作包含抗体可变区的抗原结合分子的结构域。此种遗传重组的抗体包括，例如，人源化抗体。这些经修饰的抗体通过已知方法被合适地制备。
人源化抗体，具体而言，将非人动物抗体、例如小鼠抗体的CDR移植到人抗体的人源化抗体等是公知的。也已知为了获得人源化抗体的常规的基因重组方法。具体而言，作为将小鼠的抗体的CDR移植到人的FR的方法，例如重叠序列延伸PCR(overlap extension PCR)是公知的。在重叠序列延伸PCR中，在用于合成人抗体的FR的引物上附加编码想要移植的小鼠抗体CDR的核苷酸序列。对于4个FR分别准备引物。通常认为当将小鼠CDR移植到人FR中时,选择与小鼠的FR同一性高的人FR对于维持CDR的功能是有利的。即，通常优选利用包含与想要移植的小鼠CDR相邻的FR的氨基酸序列同一性高的氨基酸序列的 人FR。
此外，还已知使用人抗体文库通过淘选制备人抗体的技术。例如，通过噬菌体展示方法将人抗体的V区表达为噬菌体表面上的单链抗体(scFv)。可以选择表达与抗原结合的scFv的噬菌体。编码与抗原结合的人抗体V区的DNA序列可以通过分析所选噬菌体的基因来确定。测定与抗原结合的scFv的DNA序列。通过使该V区序列与所需人抗体C区的序列符合读框的融合并且插入到适当的表达载体中，能够制作表达载体。将表达载体导入适于表达的细胞，例如上述那些。可以通过在细胞中表达人编码抗体的基因来产生人抗体。
本发明提供的抗BAFFR抗体能够特异性地与BAFFR结合，阻断BAFFR-BAFF信号通路，清除过度增殖的B淋巴细胞，抑制B细胞的生长；有效解决现有治疗B细胞淋巴瘤的药物的耐药性问题。
附图说明
图1：ELISA法检测抗BAFFR鼠源抗体与人BAFFR蛋白的结合能力。
图2A：FACS检测抗BAFFR鼠源抗体与人BAFFR-CHO-K1细胞的结合能力。
图2B：FACS检测抗BAFFR嵌合抗体与人BAFFR-CHO-K1细胞的结合能力。
图3：抗BAFFR嵌合抗体阻断BAFF-BAFFR结合的能力。
图4：抗BAFFR人源化抗体A、抗体B、抗体C、抗体D与Human-BAFFR-His的结合活性。
图5：抗BAFFR人源化抗体E、抗体F、抗体G与Human-BAFFR-His的结合活性。
图6：抗BAFFR人源化抗体H与Human-BAFFR-His的结合活性。
具体实施方式
以下结合附图与具体实施例对本发明做进一步的描述，本发明的保护内容不局限于以下实施例。还应该理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。在不背离发明构思的精神和 范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求及其任何等同物为本发明的保护范围。在本发明的说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。实施本发明的过程、条件、试剂、实验方法等，除以下专门提及的内容之外，均为本领域技术人员的普遍知识和公知常识，本发明没有特别限制内容。
实施例1：动物免疫
小鼠免疫：取3～5只6周龄雌性Balb/c小鼠，以重组人BAFFR-Fc蛋白作为免疫原进行腹腔或是皮下免疫。提前3天采取阴性血清，首次免疫，以腹腔免疫注射50μg经弗氏完全佐剂充分乳化的重组人BAFFR-Fc蛋白，第14天、第35天腹腔注射25ug经弗氏不完全佐剂充分乳化的重组人BAFFR-Fc蛋白进行第二次、三次免疫。6天后，尾部采血，滴度稀释血清，用ELISA法检测血清效价。以重组人BAFFR-his蛋白包被ELISA板过夜，弃包被液，PBST洗涤，加入血清稀释液，PBST洗涤，加入辣根过氧化物酶标记的羊抗鼠二抗，PBST洗板。加入TMB显色液显色，于450nm下测光密度。当效价结果满足要求时，可收获小鼠脾脏和淋巴结，进行细胞融合与噬菌体文库构建。
羊驼免疫：以重组人BAFFR-Fc蛋白作为免疫原，将抗原和佐剂1:1乳化使其形成均匀混合物，每次免疫总的抗原量保持在0.5-1mg之间，体积在1-2mL以下。在羊驼颈部淋巴结附近分左右两侧多点少量注射混合好的抗原，每2周免疫一次，一共进行7次免疫。在第6、7次免疫后间隔5-7天从羊驼颈部静脉采血。离心分离取上层血清。滴度稀释血清，用ELISA法检测血清效价，当效价结果满足要求，可用细胞分离液分离淋巴细胞，使用血球计数板计算细胞数目，根据细胞数目使用RNAiso Plus溶解分离得到的淋巴细胞，准备进行噬菌体文库构建。
实施例2：细胞融合
将小鼠处死取出脾脏和淋巴结，将细胞压磨过网，得到B淋巴细胞和淋巴结细胞。将B淋巴细胞/和淋巴结细胞与骨髓瘤细胞SP2/0按2:1混合，将其悬浮液以1000转/分离心8分钟，取沉淀，用电融合液洗两次。再取沉淀加电融合液至9mL，向电极小池各注入9mL细胞悬浮液。按常规电转方法将其与SP2/0细胞融合。将融合体在含有HAT的DMEM完全培养基中置于8％CO 2，37℃条件 下培养。
融合体选择培养中所用的饲养细胞取自未经免疫的动物腹腔中的巨噬细胞，以辅助新的杂种B淋巴细胞杂交瘤生长。
实施例3：噬菌体文库的构建
首先用RNAiso Plus试剂提取淋巴细胞总RNA。而后反转录cDNA，从反转录的cDNA中扩增特定的抗体片段，使用Taq DNA Polymerase Hot Start酶进行PCR扩增，将得到的PCR扩增产物跑1％琼脂糖凝胶电泳，对0.7kb大小的条带切胶使用DNA纯化回收试剂盒按说明书进行回收。从上一步PCR扩增并回收后DNA片段中再次扩增特定的抗体片段，切胶回收。将上一步扩增得到的抗体基因序列和噬菌体载体使用SfiI酶切，将抗体基因连接克隆至噬菌体质粒。
接着将质粒电转化至SS320感受态细胞，电击之后立即向电击杯中加入1mL2YT培养基复苏，吸出电击产物并用2YT培养基洗净电击杯，获得复苏产物，37℃，220rpm培养直至OD600达到0.5。加入辅助噬菌体后继续37℃培养30分钟，加入卡那霉素和0.2mM的IPTG，30℃过夜培养。将过夜培养的细胞离心，将上清转移到新的离心管后加入1/4体积预冷的5X PEG8000/NaCl，在冰上孵育30分钟。离心去除上清后加入1mL PBS缓冲液溶解沉淀。再次加入250μL 5X PEG8000/NaCl后冰上孵育10分钟，离心去除上清并将沉淀溶解在1mL PBS中得到噬菌体库。
实施例4：筛选杂交瘤阳性克隆
采用ELISA方法筛选出所分泌的抗体可结合人BAFFR蛋白的克隆。再用FACS方法检测所分泌的抗体与huBAFFR-CHO-K1的结合活性，最后检测杂交瘤抗体结合BAFFR的种属特异性。
(1)ELISA检测杂交瘤抗BAFFR抗体与人BAFFR的结合
将杂交瘤细胞培养上清加入ELISA板中，50μL/孔，以SP2/0细胞上清作为阴性对照，免疫多抗血清作为阳性对照，37℃水浴2h；PBST洗涤3次；加入工作浓度的HRP标记的羊抗鼠IgG和IgM抗体，50μL/孔，37℃水浴1.5h；洗涤后，TMB显色10min，显示终止后酶标仪测定OD450读数。被测孔OD450读数 大于阴性对照两倍以上判定为阳性。
(2)FACS检测杂交瘤抗BAFFR抗体与人BAFFR-CHO-K1细胞的结合活性
用携带人BAFFR基因的慢病毒载体转导入CHO-K1细胞，得到过表达人BAFFR蛋白的细胞，命名为人BAFFR-CHO-K1细胞。取对数生长期的人BAFFR-CHO-K1细胞于1.5mL EP管内，用PBS洗涤、离心两次，将ELISA检测筛选出的阳性克隆抗体用PBS稀释至40μg/mL，并3倍稀释8个梯度，每管100μL，同时用PBS做空白对照，4℃孵育1小时；再用PBS洗涤、离心两次，然后加入PBS稀释的荧光抗体APC anti-human IgG Fc Antibody，每管100μL，4℃孵育45min后，用PBS洗涤、离心两次；最后用200μL PBS重悬细胞，流式上机检测分析。
(3)FACS检测杂交瘤抗BAFFR抗体结合BAFFR的种属特异性
用携带鼠、食蟹猴BAFFR基因的慢病毒载体转导入CHO-K1细胞，得到过表达鼠或是食蟹猴BAFFR蛋白的细胞，命名为鼠/猴BAFFR-CHO-K1细胞。检测杂交瘤抗体结合鼠、食蟹猴BAFFR蛋白的结合活性。
实施例5：噬菌体文库筛选抗BAFFR阳性抗体
将噬菌体500μL加入到1mL 3％BSA中，室温旋转孵育2h。同时往包被好人BAFFR-his的免疫管中加入2-3mL 3％BSA，室温旋转孵育2h。将封闭后的免疫管用含有0.01％吐温的PBS洗3次。将封闭后的噬菌体文库加入到封闭后的免疫管中，添加PBS直至2-3mL，室温旋转孵育1h。将抗原和噬菌体孵育后的免疫管用含有0.01％吐温的PBS洗20次。往免疫管中加入1mL 100mM三甲酰亚胺，室温孵育10分钟，加入1M Tris-HCl中和三甲酰亚胺，将最后1.5mL的洗脱噬菌体转移到新的离心管中。
将洗脱的噬菌体扩增后再重复筛选过程2次，逐次减半包被免疫管的抗体量，得到3次筛选后的洗脱噬菌体。将上一步筛选得到的噬菌体稀释106倍后，取100μL加入到OD600为0.5的SS320菌液中，37℃培养30min后涂布含有四环素和氨苄霉素的2X YT培养板上，37℃过夜培养第二天得到单克隆菌落。
挑选96个单克隆菌落到含有四环素和氨苄霉素的2X YT培养液的96孔细胞培养板上，37℃培养3-4h后往培养孔中加入卡那霉素和20:1的辅助噬菌体， 30℃过夜培养。将过夜培养后的细胞液离心，获得上清液。将过夜包被抗原和用3％BSA封闭过后的96孔ELISA板中加入上一步获得的噬菌体上清液，室温孵育1h。用含有0.05％吐温的PBS清洗3次后，用人BAFFR-his作为一抗，用相应的二抗anti-VHH-HRP或anti-mouse Fab-HRP，TMB显色后在波长450读取每个孔的吸光值。选取吸光值读数最高的SS320菌落送去测序，得到抗体的基因序列。
实施例6：杂交瘤细胞抗BAFFR单克隆抗体生产
获得稳定的杂交瘤细胞系后，主要采用体外培养法获取单克隆抗体。将细胞株扩增至在T75培养瓶内，培养至细胞覆盖率为80-90％，将细胞上清弃去，加入30mL hybridoma-SFM，37℃，5％CO 2培养。培养2-3天后添加30mL hybridoma-SFM，细胞活率低于30％则可添加新鲜的活细胞。培养6-7天，待细胞存活率低于20％，低速离心后收集培养上清，4℃储存备用，进行后续的功能试验，得到候选抗体1-8。
表1抗体序列表
候选抗体 重链可变区序列 轻链可变区序列 重链全长序列 轻链全长序列
抗体1 SEQ ID NO：1 SEQ ID NO：17 SEQ ID NO：146 SEQ ID NO：154
抗体2 SEQ ID NO：2 SEQ ID NO：18 SEQ ID NO：147 SEQ ID NO：155
抗体3 SEQ ID NO：3 SEQ ID NO：19 SEQ ID NO：148 SEQ ID NO：156
抗体4 SEQ ID NO：4 SEQ ID NO：20 SEQ ID NO：149 SEQ ID NO：157
抗体5 SEQ ID NO：5 SEQ ID NO：21 SEQ ID NO：150 SEQ ID NO：158
抗体6 SEQ ID NO：6 SEQ ID NO：22 SEQ ID NO：151 SEQ ID NO：159
抗体7 SEQ ID NO：7 SEQ ID NO：23 SEQ ID NO：152 SEQ ID NO：160
抗体8 SEQ ID NO：8 SEQ ID NO：24 SEQ ID NO：153 SEQ ID NO：161
实施例7：杂交瘤候选抗体可变区序列的获得
用基于简并引物PCR的方法，测定由候选杂交瘤表达的小鼠抗体可变区的DNA序列。将杂交瘤细胞株分别扩大培养，1000rpm离心收集细胞，并以Trizol提取总RNA。以此为模板，合成第一链cDNA后，以第一链cDNA为后续模板PCR扩增对应的可变区DNA序列，所用PCR引物基于Ig-引物组。回收纯化PCR产物，将扩增产物测序后，得到候选杂交瘤重链可变区和轻链可变区序列。
实施例8：候选序列IgG嵌合抗体表达与纯化
杂交瘤与噬菌体文库筛选得到的候选序列进行抗体基因测序后，将测序得到的抗体片段进行基因合成，构建到人IgG框架中，而后利用分子克隆技术，将抗体片段插入载体中，构建成哺乳动物细胞表达质粒，利用脂质体转染方式，导入宿主细胞株CHO细胞，利用细胞fed-batch获得发酵上清液，取发酵液上清进行ProteinA亲和层析、离子交换层析等一系列步骤的纯化，最终得到纯化后的IgG嵌合单克隆抗体。最后采用SDS-PAGE法鉴定抗体纯度，紫外微量分光光度计法测定抗体浓度。
实施例9：FACS检测抗BAFFR抗体阻断BAFF与BAFFR的结合作用
将抗BAFFR的抗体与生物素标记的人BAFF(300ng/mL)蛋白混合，4℃孵育1h。将细胞加入平底板中，离心去上清，加入100μL抗体与人BAFF混合物稀释液到细胞中，4℃孵育1h，FACS缓冲液清洗细胞3次。加入5μg/mL的NA-PE抗体并4℃孵育30分钟。FACS缓冲液洗涤细胞3次后，通过流式细胞仪检测验证抗体能够阻断人BAFF与人BAFFR-CHO-K1细胞表面的BAFFR的结合作用。
实施例10：抗BAFFR抗体与BAFFR亲和力验证
(1)固化及捕获：
AHC传感器以0.02％PBST(0.02％吐温20，pH7.4，1×PBS)作为缓冲液平衡60s，固化样品板中的BAFFR抗体300s，二次平衡缓冲液180s。100nm的人BAFFR-hi蛋白与BAFFR抗体结合300s，然后解离600s。解离后以10mM甘氨酸(pH2.0)作为再生缓冲液，再生30s。
(2)再生：
用10mM甘氨酸(pH2.0)再生传感器。
(3)数据分析：
从测试结果图中减去参考通道H1的结果图。实验数据符合1:1结合模型。计算抗体亲和力。
表2抗BAFFR嵌合抗体与BAFFR亲和力检测结果
固化物 KD值
Benchmark 3.15×10 -8
抗体1 2.92×10 -8
抗体2 3.03×10 -8
抗体3 3.50×10 -8
抗体4 5.30×10 -8
抗体5 3.45×10 -8
抗体6 3.57×10 -8
抗体7 4.45×10 -8
抗体8 1.23×10 -8
抗体1-8的亲和力在10nM左右，整体亲和力较好。
实施例11：抗BAFFR抗体与PBMC中的B细胞结合验证
(1)抗体稀释
取Benchmark、抗BAFFR抗体和同型对照(购自罗氏)，用PBS稀释至10μg/mL。
(2)细胞分组
取PBMC细胞，离心(400G，5min)后弃上清，加入PBS重悬清洗，再次离心后PBS重悬。设空白组(PBMC细胞)，PBMC+二抗(APC anti-human IgG Fc，购自Biolegend)组，PBMC+CD20组，PBMC+CD20+二抗组，PBMC+CD20+抗体+二抗组。细胞密度为1E6个/组，100μL/组
(3)CD20标记PBMC
Anti-human CD20(购自BD)以20μL/孔加入PBMC+CD20组，PBMC+CD20+二抗组，PBMC+CD20+抗体+二抗组，室温避光孵育30min。
(4)抗体孵育
PBMC+CD20+抗体+二抗组分别加入120μL稀释至10μg/mL的抗体(终浓度5μg/mL)，室温避光孵育1h。空白组(PBMC)，PBMC+二抗组，PBMC+CD20组，PBMC+CD20+二抗组加入等体积的PBS进行孵育。
(5)二抗孵育
PBS清洗2次后，加入100μL PBS重悬，空白组(PBMC)、PBMC+CD20组加入5μL PBS，其余各组加入5μL二抗，室温避光孵育30min。
(6)上机检测
PBS清洗2次后，加入100μL PBS重悬，将各样品组的细胞依次进行流式 检测。
(7)数据分析
抗BAFFR抗体与PBMC中的B细胞结合检测结果见表3。结果表明，抗体1-8与B细胞结合活性较好。
表3抗BAFFR抗体与PBMC中的B细胞结合检测结果
抗体 Mean-APC
Benchmark 12642
同型对照 783
抗体1 13145
抗体2 12462
抗体3 9244
抗体4 9616
抗体5 12126
抗体6 10050
抗体7 12882
抗体8 14436
实施例12：序列人源化
对抗体5的序列进行人源化，得到人源化抗体A、B、C、D、E、F、G和H，抗体具体序列见表4。
表4人源化抗体序列表
候选抗体 重链全长序列 轻链全长序列
抗体A(参照抗体) SEQ ID NO：150 SEQ ID NO：158
抗体B SEQ ID NO：150 SEQ ID NO：166
抗体C SEQ ID NO：163 SEQ ID NO：167
抗体D SEQ ID NO：163 SEQ ID NO：162
抗体E SEQ ID NO：163 SEQ ID NO：165
抗体F SEQ ID NO：164 SEQ ID NO：167
抗体G SEQ ID NO：164 SEQ ID NO：162
抗体H SEQ ID NO：164 SEQ ID NO：165
实施例13：人源化抗体与Human-BAFFR-His的结合活性测定
(1)包被：用包被液(1×PBS，pH7.4)将Human-BAFFR-His稀释为1μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤4次，并在平板纸上拍干。
(2)封闭：用3％脱脂奶粉封闭，300μL/孔，37℃孵育1小时，倒掉封闭液，洗板机洗涤4次，平板纸上拍干。
(3)样品稀释：用3％脱脂奶粉将参比品和供试品稀释为10μg/mL，以此为初始浓度进行3倍稀释，共稀释11个梯度，另设1个空白孔，只加稀释液。100μL/孔，37℃孵育1小时。弃去孔中液体，洗板机洗涤4次，平板纸上拍干。
(4)加酶标二抗：用3％脱脂奶粉将Peroxidase-conjugated AffiniPure F(ab’)2Fragment Goat Anti-Human IgG按1:20000稀释，100μL/孔，37℃孵育1小时。洗板机洗涤6次，平板纸上拍干。
(5)显色：加入TMB显色液，100μL/孔，并用铝箔纸包好，37℃避光显色8分钟。
(6)终止显色：加入终止液1M HCl终止显色反应，100μL/孔。
(7)在酶标仪上450nm处读数。
(8)数据分析。
人源化抗体与Human-BAFFR-His的结合活性检测结果如图4-6所示。结果表明，人源化抗体B、C、D、E、F、G和H与Human-BAFFR-His的结合活性均优于参照抗体A。
实施例14：人源化抗体的亲和力测定
选择人源化抗体C和抗体H进行亲和力多浓度检测。
设备：OCTET Red96e(Fortebio)。
传感器：AHC。
(1)实验设置：
传感器准备：在使用前以0.02％PBST(0.02％吐温20，pH7.4，1×PBS)作为缓冲液浸润AHC传感器600s，除去传感器表面覆盖的蔗糖。
根据样品的实际加样位置设置样品板及传感器位置。
设置需要进行的步骤，并设置时间和转速，实验温度设置为30℃，震荡速度为1000rpm。
(2)固化及捕获：
AHC传感器以0.02％PBST(0.02％吐温20，pH7.4，1×PBS)作为缓冲液平 衡60s，固化样品板中的BAFFR抗体300s，二次平衡缓冲液180s。100nM的人Human-BAFFR-His(KACTUS)蛋白与BAFFR抗体结合300s，然后解离600s。解离后以10mM甘氨酸(pH2.0)作为再生缓冲液，再生30s。
(3)再生：
用10mM甘氨酸(pH2.0)再生传感器。
(4)数据分析：
人源化抗体的亲和力多浓度检测结果如表5所示。结果表明，人源化抗体H的亲和力高于参照抗体A。
表5抗体C和抗体H的亲和力多浓度检测结果
Figure PCTCN2022080548-appb-000001
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

Claims (43)

  1. 一种抗BAFFR单克隆抗体或其抗原结合片段，包含重链和轻链，其特征在于，所述重链包含重链互补决定区CDR1、CDR2和CDR3，所述轻链可变区包含轻链互补决定区CDR1、CDR2和CDR3，其中，
    (a)重链可变区的CDR1，选自SEQ ID NO：33-40的任一氨基酸序列，或与SEQ ID NO：33-40的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：33-40的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (b)重链可变区的CDR2，选自SEQ ID NO：41-48的任一氨基酸序列，或与SEQ ID NO：41-48的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：41-48的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (c)重链可变区的CDR3，选自SEQ ID NO：49-56的任一氨基酸序列，或与SEQ ID NO：49-56的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：49-56的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (d)轻链可变区的CDR1，选自SEQ ID NO：57-64的任一氨基酸序列，或与SEQ ID NO：57-64的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：57-64的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (e)轻链可变区的CDR2，选自SEQ ID NO：65-72的任一氨基酸序列，或与SEQ ID NO：65-72的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：65-72的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (f)轻链可变区的CDR3，选自SEQ ID NO：73-80的任一氨基酸序列；所述抗体结合siglec15，或与SEQ ID NO：73-80的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：73-80的任一氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：33、SEQ ID NO：41和SEQ ID NO：49组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：57、SEQ ID NO：65和SEQ ID NO：73组成；
    优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：34、SEQ ID NO：42和SEQ ID NO：50组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：58、SEQ ID NO：66和SEQ ID NO：74组成；
    优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：35、SEQ ID NO：43和SEQ ID NO：51组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：59、SEQ ID NO：67和SEQ ID NO：75组成；
    优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：36、SEQ ID NO：44和SEQ ID NO：52组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：60、SEQ ID NO：68和SEQ ID NO：76组成；
    优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：37、SEQ ID NO：45和SEQ ID NO：53组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：61、SEQ ID NO：69和SEQ ID NO：77组成；
    优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：38、SEQ ID NO：46和SEQ ID NO：54组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：62、SEQ ID NO：70和SEQ ID NO：78组成；
    优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：39、SEQ ID NO：47和SEQ ID NO：55组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：63、SEQ ID NO：71和SEQ ID NO：79组成；
    优选地，所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：40、SEQ ID NO：48和SEQ ID NO：56组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：64、SEQ ID NO：72和SEQ ID NO：80组成。
  2. 根据权利要求1所述的一种抗BAFFR单克隆抗体或其抗原结合片段，其特征在于，所述重链和轻链包含重链可变区和轻链可变区，所述重链可变区包括框架区FR和所述重链可变区的CDR1、CDR2和CDR3；所述的框架区FR包括：
    (a)选自SEQ ID NO：81-88任一氨基酸序列，
    或与SEQ ID NO：81-88给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
    或与SEQ ID NO：81-88的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR1，
    (b)选自SEQ ID NO：89-96任一氨基酸序列，
    或与SEQ ID NO：89-96给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
    或与SEQ ID NO：89-96的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR2，
    (c)选自SEQ ID NO：97-104任一氨基酸序列，
    或与SEQ ID NO：97-104给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
    或与SEQ ID NO：97-104的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR3，
    和
    (d)选自SEQ ID NO：105-112任一氨基酸序列，
    或与SEQ ID NO：105-112给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
    或与选自SEQ ID NO：105-112的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR4；
    优选地，所述重链和轻链包含重链可变区和轻链可变区，所述轻链可变区包括框架区FR和上述轻链可变区的CDR1、CDR2和CDR3；所述的框架区FR包括：
    (e)选自SEQ ID NO：113-120任一氨基酸序列，
    或与SEQ ID NO：113-120给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
    或与SEQ ID NO：113-120的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR1，
    (f)选自SEQ ID NO：121-128任一氨基酸序列，
    或与SEQ ID NO：121-128给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
    或与SEQ ID NO：121-128的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR2，
    (g)选自SEQ ID NO：129-136任一氨基酸序列，
    或与SEQ ID NO：129-136给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
    或与SEQ ID NO：129-136的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR3，
    和
    (h)选自SEQ ID NO：137-144任一氨基酸序列，
    或与SEQ ID NO：137-144给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
    或与选自SEQ ID NO：137-144的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列所示的FR4。
  3. 一种抗BAFFR单克隆抗体或其抗原结合片段，包括重链可变区和轻链可变区；其特征在于，其中：
    (a)所述重链可变区具有SEQ ID NO：1-8给出的任一氨基酸序列，
    或与SEQ ID NO：1-8给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，
    或与SEQ ID NO：1-8的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (b)所述轻链可变区具有SEQ ID NO：17-24给出的任一氨基酸序列；
    或与SEQ ID NO：17-24给出的任一氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：17-24的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列。
  4. 根据权利要求3所述的一种抗BAFFR单克隆抗体或其抗原结合片段，其特征在于，所述重链可变区和轻链可变区选自如下(1)-(8)中的任一种氨基酸序列：
    (1)SEQ ID NO：1和SEQ ID NO：17；
    (2)SEQ ID NO：2和SEQ ID NO：18；
    (3)SEQ ID NO：3和SEQ ID NO：19；
    (4)SEQ ID NO：4和SEQ ID NO：20；
    (5)SEQ ID NO：5和SEQ ID NO：21；
    (6)SEQ ID NO：6和SEQ ID NO：22；
    (7)SEQ ID NO：7和SEQ ID NO：23；
    (8)SEQ ID NO：8和SEQ ID NO：24。
  5. 根据权利要求1-4任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体为鼠源抗体、单域抗体、嵌合抗体、全人抗体或人源化抗体。
  6. 根据权利要求1-4任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段为单特异性抗体、双特异性抗体、多特异性抗体、抗体偶联物或细胞治疗的car分子；优选地，所述抗体偶联物的细胞毒性药物为细胞毒性分子、放射性同位素、药物或细胞因子。
  7. 根据权利要求6所述的抗体或其抗原结合片段，其特征在于，所述双特异性抗体包括权利要求1-4任一项所述的抗体或其抗原结合片段。
  8. 根据权利要求1-4任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体为单克隆抗体。
  9. 根据权利要求1-8任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段结合至BAFFR蛋白。
  10. 根据权利要求9所述的抗体或其抗原结合片段，其中所述BAFFR蛋白是人猴BAFFR蛋白。
  11. 根据权利要求9或10所述的抗体或其抗原结合片段，其中所述BAFFR蛋白形成细胞的一部分。
  12. 根据权利要求11所述的抗体或其抗原结合片段，其中所述BAFFR蛋白在所述细胞的表面上表达。
  13. 根据权利要求11或12所述的抗体或其抗原结合片段，其中所述细胞是淋巴细胞。
  14. 根据权利要求11-13任一项所述的抗体或其抗原结合片段，其中所述细胞是B细胞。
  15. 根据权利要求11-14任一项所述的抗体或其抗原结合片段，其中所述细胞是癌细胞。
  16. 根据权利要求15所述的抗体或其抗原结合片段，其中所述癌细胞是淋巴瘤细胞。
  17. 编码权利要求1-16任一所述的抗BAFFR抗体或其抗原结合片段的基因。
  18. 根据权利要求17所述的基因，其特征在于，所述基因选自如下(1)-(8)中 的任一种：
    (1)含有如SEQ ID NO：9所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：25所示的编码所述抗体轻链可变区的核苷酸序列；
    (2)含有如SEQ ID NO：10所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：26所示的编码所述抗体轻链可变区的核苷酸序列；
    (3)含有如SEQ ID NO：11所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：27所示的编码所述抗体轻链可变区的核苷酸序列；
    (4)含有如SEQ ID NO：12所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：28所示的编码所述抗体轻链可变区的核苷酸序列；
    (5)含有如SEQ ID NO：13所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：29所示的编码所述抗体轻链可变区的核苷酸序列；
    (6)含有如SEQ ID NO：14所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：30所示的编码所述抗体轻链可变区的核苷酸序列；
    (7)含有如SEQ ID NO：15所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：31所示的编码所述抗体轻链可变区的核苷酸序列；
    (8)含有如SEQ ID NO：16所述的编码抗体重链可变区的核苷酸序列，以及如SEQ ID NO：32所示的编码所述抗体轻链可变区的核苷酸序列。
  19. 含有权利要求18所述基因的重组载体、表达盒、转基因细胞系或重组菌。
  20. 权利要求1-16任一项所述的抗体或其抗原结合片段在如下(a)或(b)或(c)中的应用：
    (a)制备抑制癌细胞迁移的药物中的应用；
    (b)制备治疗癌症的药物中的应用；
    (c)制备治疗自身免疫病的药物中的应用。
  21. 一种多功能融合蛋白，其包含权利要求1-16任一项所述的抗体或其抗原结合片段或抗体偶联药物。
  22. 根据权利要求21所述的多功能融合蛋白，其还包含一个或多个与其他抗原特异性结合的第二抗体或其抗原结合部分。
  23. 根据权利要求22所述的多功能融合蛋白，其中所述结合第二抗体或其抗原结合部分的抗原选自肿瘤相关抗原(TAA)或免疫检查点。
  24. 根据权利要求23所述的多功能融合蛋白，所述免疫检查点为CD3、4-1BB、 CD40、OX40、ICOS。
  25. 根据权利要求22-24任一项所述的多功能融合蛋白，其还包含细胞因子。
  26. 根据权利要求25所述的多功能融合蛋白，所述细胞因子选自IL8、IL10、IL15、IL18、TGF、VEGF、IFNγ、IFNα或GM-CSF。
  27. 权利要求1-16任一项所述的抗体或其抗原结合片段、权利要求22-26任一项所述的多功能融合蛋白，其在制备治疗癌症或自身免疫疾病的药物中的用途。
  28. 根据权利要求27所述的用途，其中所述癌症是淋巴瘤、白血病或骨髓瘤。
  29. 根据权利要求28所述的用途，其中所述淋巴瘤是套细胞淋巴瘤、滤泡淋巴瘤、弥漫性大B细胞淋巴瘤、边缘区淋巴瘤或伯基特氏淋巴瘤。
  30. 根据权利要求28所述的用途，其中所述白血病是淋巴母细胞白血病、慢性淋巴细胞白血病或毛细胞白血病。
  31. 根据权利要求28所述的用途，其中所述骨髓瘤是多发性骨髓瘤。
  32. 根据权利要求27所述的用途，其中所述自身免疫疾病是系统性红斑狼疮、类风湿性关节炎、强直性脊柱炎、特发性血小板减少性紫癜、溃疡性结肠炎、干燥综合征。
  33. 根据权利要求27-32任一项所述的用途，其中所述药物与其他药物或疗法进行联合应用。
  34. 根据权利要求33所述的用途，其中所述其他疗法选自：化疗、放疗、靶向治疗、基因治疗、细胞治疗、干细胞治疗。
  35. 调节细胞功能的方法，包括将编码权利要求1-16中任一项的抗体或其抗原结合片段权利要求21-26任一项所述的多功能融合蛋白的多核苷酸序列递送至所述细胞中。
  36. 根据权利要求35的方法，其中所述方法用于基因治疗、CAR-T治疗或CRISPR治疗。
  37. 一种药物组合物，其包含权利要求1-16任一项所述的抗体或其抗原结合片段和可接受的载体、稀释剂或赋形剂。
  38. 一种药物组合物，其包含权利要求21-26任一项所述的多功能融合蛋白和可接受的载体、稀释剂或赋形剂。
  39. 一种抑制细胞增殖的方法，所述方法包括：
    (1)将细胞与权利要求1-16中任一项所述的BAFFR抗体或权利要求21- 26任一项所述的多功能融合蛋白接触，从而形成接触细胞；以及
    (2)使所述BAFFR抗体或多功能融合蛋白结合所述接触细胞上的BAFFR蛋白，从而抑制所述细胞增殖。
  40. 根据权利要求39所述的方法，其中所述细胞是淋巴细胞。
  41. 根据权利要求39或40所述的方法，其中所述细胞是B细胞。
  42. 根据权利要求39-41中任一项所述的方法，其中所述细胞是癌细胞。
  43. 根据权利要求39-42中任一项所述的方法，其中所述细胞是淋巴瘤细胞。
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