WO2021209066A1 - Specific antigen binding molecule, and preparation method and pharmaceutical use therefor - Google Patents

Specific antigen binding molecule, and preparation method and pharmaceutical use therefor Download PDF

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Publication number
WO2021209066A1
WO2021209066A1 PCT/CN2021/088092 CN2021088092W WO2021209066A1 WO 2021209066 A1 WO2021209066 A1 WO 2021209066A1 CN 2021088092 W CN2021088092 W CN 2021088092W WO 2021209066 A1 WO2021209066 A1 WO 2021209066A1
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seq
heavy chain
antigen
variable region
chain variable
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PCT/CN2021/088092
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French (fr)
Chinese (zh)
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花海清
包如迪
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上海翰森生物医药科技有限公司
江苏豪森药业集团有限公司
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Priority to CN202180021516.2A priority Critical patent/CN115335402A/en
Publication of WO2021209066A1 publication Critical patent/WO2021209066A1/en

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    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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

Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention relates to an anti-BCMA antibody, its antigen-binding fragment, a bispecific antigen-binding molecule that binds BCMA and CD3, its preparation method, and its medical use.
  • B cells are lymphocytes, which play an important role in humoral immunity and the production of antibodies that specifically recognize antigens.
  • the three subtypes of B cells are naive B cells, memory B cells, and plasma cells.
  • variable domains encoded by B cells of different lineages are further changed, resulting in up to 109 unique B cell lineages, resulting in specific Antibody.
  • B cells Malignant transformation of B cells leads to cancer, including lymphomas (such as multiple myeloma and Hodgkin's lymphoma). Autoimmune diseases also involve B cells, including systemic lupus erythematosus (SLE) and IgA nephropathy. Cancers and autoimmune diseases involving B cells are considered to be abnormal in B cell function, so a possible strategy to control such diseases is to use antibodies that target pathological B cells.
  • lymphomas such as multiple myeloma and Hodgkin's lymphoma
  • SLE systemic lupus erythematosus
  • IgA nephropathy IgA nephropathy
  • BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily, which is a non-glycosylated inner membrane receptor for the ligands BAFF (B cell activating factor) and APRIL (proliferation inducing ligand). BCMA and its corresponding ligands can regulate humoral immunity, B cell development and homeostasis. BCMA was detected in the spleen, lymph nodes, thymus, adrenal glands and liver, and tonsil memory B cells and germinal center B cells also expressed BCMA. Analysis of various B cell lines showed that the expression level of BCMA increased after maturation. BCMA is highly expressed in B-cell lymphoma and multiple myeloma.
  • the current therapies for BCMA are mainly divided into three categories: chimeric antigen receptor T cell therapy (CAR-T), bispecific antibodies (BsAb) and antibody-conjugated drugs (ADC).
  • CAR-T chimeric antigen receptor T cell therapy
  • BsAb bispecific antibodies
  • ADC antibody-conjugated drugs
  • GSK's ADC drug Blenrep was approved by the U.S. FDA in August 2020, becoming the first immune-related drug approved for anti-BCMA, but its treatment has disadvantages such as visual impairment and late failure, making it bispecific Sexual antibody strategy has become another popular research and development direction for this target.
  • Amgen's double antibody AMG-420 has priority to enter the late clinical stage.
  • the antibody is designed with BiTE, which has good penetration, but has a small molecular weight and short half-life, which reduces patient compliance.
  • an anti-BCMA antibody or antigen-binding fragment thereof which comprises:
  • an antibody heavy chain variable region comprising at least one HCDR selected from the following sequences:
  • SEQ ID NO: 3 SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 9;
  • An antibody light chain variable region comprising at least one LCDR selected from the following sequence: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO : 10.
  • the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof includes: HCDR1 shown in SEQ ID NO: 3, and shown in SEQ ID NO: 4 or SEQ ID NO: 9.
  • the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof includes: LCDR1 shown in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 10. LCDR2 shown and LCDR3 shown in SEQ ID NO: 8.
  • an anti-BCMA antibody or antigen-binding fragment thereof which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and
  • the light chain variable region includes: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8,
  • the heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9 and HCDR3 shown in SEQ ID NO: 5; and
  • the light chain variable region includes: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8,
  • the heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9 and HCDR3 shown in SEQ ID NO: 5;
  • the light chain variable region includes LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8,
  • the heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and
  • the light chain variable region includes: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8.
  • the anti-BCMA antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG4 or a variant thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises the heavy chain constant region of human IgG1.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region variant of human IgG1.
  • the human IgG1 heavy chain constant region variant has reduced ADCC toxicity compared with wild IgG1 heavy chain constant region.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 31, or a heavy chain constant region variant as shown in SEQ ID NO: 42, Or a heavy chain constant region variant as shown in SEQ ID NO: 33, or a heavy chain constant region variant as shown in SEQ ID NO: 34.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 31 or a heavy chain constant region variant as shown in SEQ ID NO: 42.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region variant as shown in SEQ ID NO:42.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody kappa chain, lambda chain or a variant thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody kappa chain.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO: 32.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequences, or at least 70%, 75%, 80% compared with the following sequences , 85%, 90%, 95% or 99% identical heavy chain variable regions: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and/or,
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the following sequences, or has at least 70%, 75%, 80%, 85%, 90%, 95% compared with the following sequences Or a light chain variable region with 99% identity: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.
  • the anti-BCMA antibody or antigen-binding fragment thereof contains a heavy chain as shown in the following sequence, or at least 80%, 85%, 90%, 95% or 99% compared with the following sequence % Identity heavy chain: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 43; and/or,
  • the anti-BCMA antibody or antigen-binding fragment thereof contains a light chain as shown in the following sequence, or a light chain with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises:
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises:
  • an anti-CD3 antibody or an antigen-binding fragment thereof which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37;
  • the light chain variable region includes LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.
  • an anti-CD3 antibody or antigen-binding fragment thereof which comprises a heavy chain variable region shown in SEQ ID NO: 24 and a light chain variable region shown in SEQ ID NO: 25 .
  • an antigen-binding fragment of an anti-CD3 antibody which is a scFv.
  • the scFv is shown in SEQ ID NO: 41.
  • the first binding region that specifically binds to BCMA is the first binding region that specifically binds to BCMA.
  • the second binding region that specifically binds to CD3.
  • the first binding region is selected from the aforementioned anti-BCMA antibodies or antigen-binding fragments thereof.
  • the second binding region is selected from anti-CD3 antibodies or antigen-binding fragments thereof.
  • a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, said anti-BCMA antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising SEQ ID NO: HCDR1, HCDR1, SEQ ID NO: 4, and HCDR3, SEQ ID NO: 5;
  • the light chain variable region includes LCDR1, SEQ ID NO: 6 and LCDR1, SEQ ID NO : LCDR2 shown in 10 and LCDR3 shown in SEQ ID NO: 8.
  • a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 said anti-BCMA antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO: 11 and SEQ ID NO: 14 The variable region of the light chain.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1.
  • a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 said anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region variant of human IgG1, said human IgG1 heavy chain constant region variant It has reduced ADCC toxicity compared with human IgG1 heavy chain constant region.
  • a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 said anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO: 31.
  • the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention comprises a heavy chain constant as shown in SEQ ID NO: 42 Area variants.
  • a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 said anti-BCMA antibody or antigen-binding fragment thereof further comprises a human antibody kappa chain, a light chain constant region of a lambda chain or a variant thereof, preferably a human antibody
  • the light chain constant region of the kappa chain is most preferably the light chain constant region shown in SEQ ID NO: 32.
  • a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 said anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 17 or SEQ ID NO: 43 and Such as the light chain shown in SEQ ID NO: 20.
  • a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 said anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 43 and a heavy chain as shown in SEQ ID NO: 20 The light chain shown.
  • bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37;
  • the light chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof includes LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.
  • a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 contains the heavy chain variable region shown in SEQ ID NO: 24 and SEQ ID NO: 25 The variable region of the light chain.
  • the heavy chain variable region and the light chain variable region contained in the second binding region are connected by a peptide linker.
  • the peptide linker connecting the heavy chain variable region and the light chain variable region of the second binding region is selected from (GGGGS) n , n is selected from an integer of 1-5, preferably, n is 3.
  • the second binding region comprises the sequence shown in SEQ ID NO: 41.
  • the N-terminus of the heavy chain variable region of the second binding region is connected to the C-terminus of the light chain constant region of the first binding region.
  • the N-terminus of the heavy chain variable region of the second binding region is connected to the C-terminus of the light chain constant region of the first binding region by a peptide linker selected from (GGGGS) n , n is selected from an integer of 1-5, preferably n is 3.
  • the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 comprises a first binding region that specifically binds BCMA and a second binding region that specifically binds CD3, wherein:
  • the first binding region includes a heavy chain variable region and a light chain variable region
  • the heavy chain variable region of the first binding region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5;
  • the light chain variable region of the first binding region includes LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8, and
  • the second binding region includes a heavy chain variable region and a light chain variable region
  • the heavy chain variable region of the second binding region includes HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37;
  • the light chain variable region of the second binding region includes LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.
  • bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein:
  • the first binding region includes the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14, and
  • the second binding region includes the heavy chain variable region shown in SEQ ID NO: 24 and the light chain variable region shown in SEQ ID NO: 25.
  • bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein:
  • the first binding region includes a heavy chain as shown in SEQ ID NO: 17 or SEQ ID NO: 43 and a light chain as shown in SEQ ID NO: 20, and
  • the second binding region includes the heavy chain variable region shown in SEQ ID NO: 24 and the light chain variable region shown in SEQ ID NO: 25.
  • the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention wherein the first binding region comprises a heavy chain as shown in SEQ ID NO: 43 and a heavy chain as shown in SEQ ID NO
  • the light chain shown in: 20 and the second binding region include the heavy chain variable region shown in SEQ ID NO: 24 and the light chain variable region shown in SEQ ID NO: 25.
  • the bispecific antibody or antigen-binding fragment thereof that binds to BCMA and CD3, wherein the heavy chain variable region and the light chain variable region contained in the second binding region are connected by a peptide linker, and the second binding region
  • the peptide linker of the heavy chain variable region and the light chain variable region is selected from (GGGGS) n , n is selected from an integer of 1-5 (1, 2, 3, 4, 5), preferably, n is 3.
  • the N-terminus of the heavy chain variable region of the second binding region is connected to the C-terminus of the light chain constant region of the first binding region; preferably, the The N terminal of the heavy chain variable region of the second binding region is connected to the C terminal of the light chain constant region of the first binding region through a peptide linker.
  • the first binding region comprises at least one Fab fragment of an anti-BCMA antibody
  • the second binding region comprises at least one scFv fragment of an anti-CD3 antibody .
  • the first binding region comprises two Fab fragments of anti-BCMA antibodies
  • the second binding region comprises two scFv fragments of anti-CD3 antibodies .
  • the first binding region is selected from the above-mentioned anti-BCMA antibodies comprising light and heavy chains, and the second binding region comprises two of the above-mentioned anti-BCMA antibodies.
  • the N-terminus of the scFv fragment is connected to the Fab fragment or the C-terminus of the light chain constant region of the anti-BCMA antibody; preferably, the scFv The N-terminus of the fragment is connected to the Fab fragment through a peptide linker or to the C-terminus of the light chain constant region of the anti-BCMA antibody.
  • the first binding region is selected from an anti-BCMA antibody comprising a light chain and a heavy chain
  • the second binding region comprises two anti-CD3 antibodies The scFv fragment.
  • the N-terminus of the scFv fragment is connected to the C-terminus of the light chain constant region of the Fab fragment or the anti-BCMA antibody through a peptide linker, and the peptide linker is selected from (GGGGS) n , n is selected from an integer of 1-5, preferably n is 3.
  • the bispecific antibody or antigen-binding fragment thereof of the present invention has a four-chain structure, comprising two identical first chains and two identical second chains, wherein:
  • the first chain includes the heavy chain variable region of the first binding region and the heavy chain constant region of the first binding region from the N-terminus to the C-terminus;
  • the second chain from the N-terminus to the C-terminus, contains the light chain variable region of the first binding region, the light chain constant region of the first binding region and the first peptide linker, and the heavy chain variable region and the first peptide linker of the second binding region.
  • the light chain variable region of the dipeptide linker and the second binding region contains the light chain variable region of the first binding region, the light chain constant region of the first binding region and the first peptide linker, and the heavy chain variable region and the first peptide linker of the second binding region.
  • the heavy chain variable region of the second binding region in the second chain and the light chain variable region of the second binding region are selected from the anti-CD3 antibody of the present invention or its
  • the variable region of the heavy chain and the variable region of the light chain of the antigen-binding fragment, the second peptide linker is selected from (GGGGS) n , n is selected from an integer of 1-5, preferably, n is 3.
  • the first binding region includes an anti-BCMA antibody Fab fragment
  • the second binding region includes an anti-CD3 antibody scFv fragment.
  • the bispecific antibody or antigen-binding fragment thereof of the present invention comprises:
  • the first polypeptide comprises the heavy chain variable region of the first binding region and the heavy chain constant region of the first binding region from the N-terminus to the C-terminus;
  • the second polypeptide includes the light chain variable region of the first binding region and the light chain constant region of the first binding region from the N-terminus to the C-terminus;
  • the third polypeptide from the N-terminus to the C-terminus, includes the heavy chain variable region of the second binding region, the first peptide linker, the light chain variable region of the second binding region, the second peptide linker, and the heavy chain of the first binding region. Chain constant region.
  • the second peptide linker in the third polypeptide is selected from (GGGGS)n, n is selected from an integer of 1-5, preferably, n is 2.
  • n 3 when the peptide linker (ie, the first peptide linker) connects the heavy chain variable region of the second binding region and the light chain variable region of the second binding region, n is 3.
  • n is 2 when the peptide linker (ie, the second peptide linker) connects the light chain variable region and the heavy chain constant region of the second binding region.
  • the heavy chain variable region of the first binding region of the first polypeptide is selected from the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof of the present invention.
  • the light chain variable region of the first binding region of the second polypeptide is selected from the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof of the present invention.
  • the heavy chain variable region of the second binding region and the light chain variable region of the second binding region of the third polypeptide are respectively selected from the anti-CD3 antibody of the present invention Or the variable region of the heavy chain and the variable region of the light chain of the antigen binding fragment thereof; the first peptide linker is selected from (GGGGS)n, and n is selected from an integer of 1-5.
  • the heavy chain constant region of the first polypeptide comprises a heavy chain constant region derived from human IgG
  • the heavy chain constant region of the third polypeptide comprises a heavy chain constant region derived from human IgG
  • the heavy chain constant region domain of the IgG preferably, the heavy chain constant region domain of the IgG comprises (from N to C-terminus) hinge region, CH2, and CH3.
  • human IgG is selected from IgG1, IgG2, IgG3, IgG4 or variants thereof, and IgG1 is preferred.
  • the heavy chain constant regions of the first polypeptide and the third polypeptide include one or more cysteine residues in the hinge region to Forms disulfide bonds.
  • the heavy chain constant regions of the first polypeptide and the third polypeptide further comprise one or more cysteine residues outside the hinge region , To form disulfide bonds.
  • the cysteine residue is located at a position or combination selected from: 349, 354 or equivalent positions.
  • the number sequence can be compared to determine equivalent residue positions.
  • the Kabat numbering rule was developed based on the location of the same hypervariable area.
  • the Chothia numbering scheme (Al-Lazikani, 1997) is the same as Kabat's scheme, but the first VH CDR is corrected.
  • IMGT Lefranc, 2003
  • AHo Hegger and Plückthun, 2001
  • TCR T cell receptors
  • the Eu numbering rule was established based on the purified human IgG1 (named Eu) by Gerald M. Edelman et al. Gerald M. Edelman et al. determined the amino acid sequence of IgG1 and numbered it. In the prior art, many tools can be used to determine or compare the amino acid numbers of antibodies, such as but not limited to ANARCI and abYsis.
  • the amino acid positions in the constant region of the heavy chain follow the EU numbering rules, and the positions 349 or 354 refer to the amino acid positions under the EU numbering rules.
  • the skilled person can clearly determine that the cysteine residue at the equivalent position is also expected to perform the same function (ie, promote the formation of a disulfide bond between the first polypeptide and the third polypeptide).
  • the numbering rules for determining the amino acid positions of antibodies also include: Kabat, Chothia, IMGT, and the technician can determine the corresponding equivalent positions in different numbering rules.
  • a technician can determine such an equivalent site by comparing and analyzing the amino acid sequence.
  • the heavy chain constant regions of the first polypeptide and the third polypeptide comprise 349C and 354C, respectively.
  • the heavy chain constant region of the first polypeptide comprises 354C
  • the heavy chain constant region of the third polypeptide comprises 349C
  • the heavy chain constant regions of the first polypeptide and the third polypeptide comprise knob and socket domains, respectively, and The knob domain interaction promotes the heterodimerization of the first polypeptide and the third polypeptide.
  • the heavy chain constant region comprises a knob domain, preferably a tryptophan residue at 366 or equivalent.
  • the heavy chain constant region comprises a hole domain, preferably an amino acid residue selected from the group consisting of: 366 or an equivalent position of S, 368 or an equivalent position of A, 407 or an equivalent position of V .
  • a hole domain preferably an amino acid residue selected from the group consisting of: 366 or an equivalent position of S, 368 or an equivalent position of A, 407 or an equivalent position of V .
  • the first polypeptide heavy chain constant region comprises a knob domain
  • the third polypeptide heavy chain constant region comprises Mortar domain
  • amino acid residue positions of the mutations in the constant region of the heavy chain are numbered according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) .
  • the first polypeptide heavy chain constant region comprises the sequence shown in SEQ ID NO: 33
  • the third polypeptide heavy chain constant region comprises SEQ ID NO: the sequence shown in 34.
  • the bispecific antibody or antigen-binding fragment thereof of the present invention contains the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27.
  • the bispecific antibody or antigen-binding fragment thereof of the present invention has a four-chain structure, comprising two (preferably identical) first chains and two (preferably identical) second chains.
  • the first chain includes the sequence shown in SEQ ID NO: 26; the second chain includes the sequence shown in SEQ ID NO: 27.
  • the bispecific antibody or antigen-binding fragment thereof of the present invention contains the sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 20.
  • the bispecific antibody or antigen-binding fragment thereof of the present invention is a three-chain antibody
  • the first polypeptide contains the sequence shown in SEQ ID NO: 28
  • the second polypeptide contains The sequence shown in SEQ ID NO: 20
  • the third polypeptide contains the sequence shown in SEQ ID NO: 29
  • the present invention also provides a polynucleotide, which encodes the anti-BCMA antibody or antigen-binding fragment thereof of the present invention, the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3.
  • the present invention also provides an expression vector, which contains the polynucleotide of the present invention.
  • the present invention also provides a host cell which is introduced into or contains the expression vector of the present invention.
  • the host cell is bacteria, preferably E. coli.
  • the host cell is yeast, preferably P. pastoris.
  • the host cell is a mammalian cell, preferably a CHO cell or HEK293 cell.
  • the present invention provides a method for producing an anti-BCMA antibody or an antigen-binding fragment thereof, a bispecific antibody that binds BCMA and CD3, or an antigen-binding fragment thereof, including the steps:
  • the antibody is purified.
  • the present invention also provides a pharmaceutical composition, which contains:
  • anti-BCMA antibody or its antigen-binding fragment of the present invention or the bispecific antibody or its antigen-binding fragment that binds BCMA and CD3;
  • the present invention provides a use, comprising the anti-BCMA antibody or antigen-binding fragment thereof, bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, or the pharmaceutical composition as described above For the treatment or prevention of BCMA-mediated diseases or conditions.
  • the disease or condition is cancer; preferably BCMA-expressing cancer; more preferably lymphoma and myeloma.
  • the disease or condition is an autoimmune disease; preferably lupus erythematosus, IgA nephropathy and rheumatoid arthritis.
  • the anti-BCMA antibody or its antigen-binding fragment provided by the present invention can specifically bind to the BCMA antigen and cells expressing BCMA, and has strong targeting ability.
  • the bispecific antibody provided by the present invention can maintain the affinity to each antigen (or epitope), and has obvious tumor inhibitory effects in vitro and in vivo. Compared with AMG-420, the bispecific antibody has better affinity activity and prolongs the drug effect in vivo. Better sex.
  • the chains of the bispecific antibody of the present invention can be correctly paired, are easy to express, have simple preparation process, stable properties, good druggability, and broad clinical application prospects.
  • Figure 1 Schematic model of the bispecific antibody, A: first chain; B: second chain.
  • FIG. 1 Schematic model of the bispecific antibody, C: the first polypeptide; D: the second polypeptide; E: the third polypeptide.
  • antibody in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain of the present invention may further include a light chain constant region, and the light chain constant region includes human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues of the VL region and VH region of the antibody or antigen-binding fragment of the present invention comply with the known Kabat numbering rules and Kabat or ABM definition rules.
  • BCMA includes any variant or isoform of BCMA expressed by a cell.
  • the antibodies of the present invention or fragments thereof can cross-react with BCMA derived from non-human species.
  • the antibody may also be specific for human BCMA, and may not cross-react with BCMA of other species.
  • BCMA or any variants or isoforms thereof can be isolated from the cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques commonly used in the art and those described herein.
  • anti-BCMA antibodies or fragments thereof target human BCMA with glycosylation modification.
  • Specifically binds to BCMA means that the antibody or fragment thereof of the present invention recognizes and binds to BCMA or its epitope.
  • CD3 includes any variant or isoform of CD3 expressed by a cell.
  • the antibodies or fragments thereof of the present invention can cross-react with CD3 derived from non-human species.
  • the antibody may also be specific for human CD3, and may not cross-react with CD3 of other species.
  • CD3 or any variants or isoforms thereof can be isolated from cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques commonly used in the art and those described herein.
  • Specifically binds to CD3 means that the antibody or fragment thereof of the present invention recognizes and binds to CD3 or its epitope.
  • recombinant human antibody includes human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:
  • Antibodies isolated from host cells transformed to express antibodies such as transfectomas;
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
  • murine antibody in the present invention refers to a monoclonal antibody to human BCMA or its epitope prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with BCMA antigen or its fragments, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
  • the murine BCMA antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, and/or further comprise murine IgG1 , IgG2, IgG3 or IgG4 or variants of the heavy chain constant region.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies”) .
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by grafting CDR sequences of non-human species into the framework of a human antibody variable region.
  • Humanized antibodies can overcome the shortcomings of immune responses induced by chimeric antibodies that carry a large amount of heterologous protein components.
  • the variable region of the human antibody can be reversely mutated to maintain activity.
  • chimeric antibody refers to an antibody formed by fusing the variable region of an antibody of the first species with the constant region of an antibody of the second species, which can reduce the immune response induced by the heterologous antibody.
  • a chimeric antibody it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed.
  • the mouse variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations to enhance ADCC (antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1 heavy chain constant region.
  • ADCC antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • antigen-binding fragment refers to antigen-binding fragments and antibody analogs of antibodies, which usually include at least part of the antigen-binding region or variable region (for example, one or more CDRs) of a parental antibody.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a mole basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
  • antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.
  • Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.
  • the "Fab fragment” consists of the CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc” region is two heavy chain fragments containing CH2 and CH3 domains.
  • the two heavy chain fragments are held together by one or more disulfide bonds.
  • Fab'fragment contains a light chain and a part of a heavy chain (which includes the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); an interchain two can be formed between the two Fab' fragments Sulfur bonds to form F(ab') 2 molecules.
  • the "Fv region” contains the variable regions of the heavy and light chains, but lacks the constant region.
  • single-chain antibody is a single-chain recombinant protein formed by connecting the VH and VL of the heavy chain variable region of the antibody through a connecting peptide. The smallest antibody fragment at the antigen binding site.
  • scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • a suitable prior art linker consists of a repeated GGGGS (SEQ ID NO: 23) amino acid sequence or variants thereof, for example, using 1-4 (including 1, 2, 3, or 4) repeated variants ( Holliger et al. (1993), Proc Natl Acad Sci USA.
  • domain antibody fragment is an immunoglobulin fragment with immunological functions that only contains a heavy chain variable region or a light chain variable region chain.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment.
  • the two VH regions of the bivalent domain antibody fragment can target the same or different antigens.
  • binding with BCMA in the present invention refers to the ability to interact with human BCMA.
  • antigen-binding site refers to a three-dimensional site recognized by the antibody or antigen-binding fragment of the present invention.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent.
  • Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • specific binding and “selective binding” as used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody is approximately 10 -7 M or even lower than the equilibrium dissociation smaller dissociation constant ( K D ) binds to a predetermined antigen, and its binding affinity to the predetermined antigen is at least twice its binding affinity to non-specific antigens (such as BSA, etc.) other than the predetermined antigen or closely related antigens.
  • K D equilibrium dissociation smaller dissociation constant
  • the term "antibody that recognizes an antigen” can be used interchangeably with the term “antibody that specifically binds” herein.
  • Bispecific antibodies or bifunctional antibodies are hybrid antibodies that have two different epitope binding sites.
  • the two epitopes can be from the same antigen or from different antigens.
  • the two epitopes are from BCMA and CD3, respectively.
  • Bispecific antibodies can be produced by well-known methods (including but not limited to hybridoma fusion or linking Fab' fragments). See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).
  • cross-reactivity refers to the ability of the antibodies of the present invention to bind to BCMA from different species.
  • the antibody of the present invention that binds to human BCMA can also bind to BCMA of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express BCMA.
  • binding assays such as SPR and ELISA
  • Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • Inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with anti-BCMA antibodies compared to ligands not contacted with anti-BCMA antibodies.
  • inhibition of growth is intended to include any measurable decrease in cell growth.
  • the term "50 EC” or "half maximal effective concentration” means the concentration after exposure for a certain time, able to induce 50% of maximal response of the molecule (e.g., an antibody of the present application or antigen binding fragment).
  • a method for determining the EC 50 are known in the art, for example, the concentration plotted using software GraphPad Prism - effect curve fit, thus calculated EC 50.
  • ADCC namely antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • Fc receptors are directly killed by recognizing the Fc segment of antibodies and are coated with antibodies.
  • the target cell The ADCC effect function of the antibody can be enhanced or reduced or eliminated by modifying the Fc segment of IgG.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody.
  • mice can be immunized with human BCMA or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • the human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) database, or from the Journal of Immunoglobulin, 2001 ISBN012441351.
  • the engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods.
  • the cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the FC region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • the antibody of the present invention refers to a monoclonal antibody.
  • the monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage cloned cell line.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as containing any one of the antibodies of the present invention, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in the treated patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present invention may be ineffective in alleviating the symptoms of the target disease that each patient has, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • an "effective amount” includes an amount sufficient to improve or prevent the symptoms of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Lupus erythematosus refers to a chronic autoimmune disease in which the immune system becomes overactive and attacks normal tissues. This attack triggers inflammation. Lupus erythematosus is a "non-organ specific" type of autoimmune disease.
  • IgA nephropathy used herein is characterized by the deposition of IgA on mesangial cells. IgA nephropathy belongs to proliferative nephritis caused by an immune response to glomerular mesangial cells.
  • rheumatoid arthritis refers to an autoimmune disease that attacks one's own joints or body parts due to abnormalities in the immune system and causes inflammation. It shows pain and pain in various joints such as hands, feet, wrists, and knees. The symptoms of swelling may also cause abnormal systemic chronic inflammatory diseases to the muscles, skin, lungs, eyes and other organs.
  • lymphoma refers to a malignant tumor that originates from the lymphoid hematopoietic system.
  • the classification of lymphoma is carried out in accordance with the 2016 WHO Lymphoma Classification standard.
  • myeloma (also known as plasmacytoma) as used herein is a malignant tumor that originates from plasma cells in the bone marrow.
  • the classification of myeloma is carried out in accordance with the standards of WHO (2013) Classification of Bone Tumors.
  • Exogenous refers to a substance that is produced outside an organism, cell, or human body according to the background.
  • Endogenous refers to a substance produced in an organism, cell, or human body according to the background.
  • Identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% identical. Generally speaking, the comparison is made when two sequences are aligned to obtain the maximum percent identity.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably, and all such names include their progeny. Therefore, the words “transformant” and “transformed cell” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where the intent is different, it is clearly visible from the context.
  • “Pharmaceutical composition” means containing one or more antibodies or antigen-binding fragments thereof described herein, and other components such as physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • the following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention.
  • the experimental methods that do not specify specific conditions in the examples of the present invention usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the human BCMA (BCMA-His) protein encoding His tag was synthesized by SinoBiologics (Cat No.: 10620-H08H).
  • Example 2 Obtaining mouse hybridoma and antibody sequence
  • the immunized mouse serum was evaluated by the indirect ELISA method as described in Example 3 to evaluate the serum titer and the ability to bind to cell surface antigens, and the cell fusion was performed in accordance with the titer detection situation (more than 100,000-fold dilution).
  • the immunized mice with strong serum titer, affinity and FACS binding were selected for a final immunization and then the mice were sacrificed. After fusion of spleen cells and SP2/0 myeloma cells, hybridomas are obtained; the target hybridomas are screened by indirect ELISA, and the cell line is established as a monoclonal cell line by the limiting dilution method.
  • the obtained positive antibody strains are further screened using indirect ELISA to select hybridomas that bind the recombinant protein.
  • the logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara #2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced, and finally the sequence of the mouse antibody M1 was obtained.
  • the heavy chain and light chain variable region sequences of murine monoclonal antibody M1 are as follows:
  • BCMA His protein (Sino Biological Inc., cat#10428-H08H) with pH7.4 PBS to a concentration of 1 ⁇ g/ml, add 100 ⁇ l/well to a 96-well high-affinity ELISA plate, and incubate in a refrigerator at 4°C Overnight (16-20 hours). After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), add 150 ⁇ l/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate for 1 hour at room temperature for blocking. After the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer.
  • BSA bovine serum albumin
  • Collect cells with high BCMA expression (HEK-293T cells overexpressing BCMA and tumor cells expressing BCMA, NCI-H929 myeloma cell line); after adjusting the cell density, spread the cells on a 96-well U bottom plate, 1 ⁇ per well 10 5 to 2 ⁇ 10 5 cells. Centrifuge at 1200g for 5min and remove the supernatant; add 100 ⁇ l of diluted antibody solution or mouse immune serum and incubate at 4°C for 60min; centrifuge at 1200g for 5min to remove the supernatant; after washing the cells twice with PBS, add a fluorescently labeled secondary antibody (PE -GAM or PE-GAH) 100 ⁇ l per well, incubate at 4°C for 60min.
  • PE -GAM or PE-GAH fluorescently labeled secondary antibody
  • Humanization of murine anti-human BCMA monoclonal antibodies is carried out as disclosed in many documents in the field.
  • a human constant domain is used instead of the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the murine antibody and the human antibody.
  • the present invention humanizes the murine antibody M1.
  • the heavy and light chain variable region sequences are compared with the human antibody germline database to obtain a human germline template with high homology.
  • the CDR regions of the murine antibody M1 were grafted onto the selected humanized template. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH are back-mutated, and the CDR region Optimization of chemically unstable amino acid residues.
  • the sequence is as follows:
  • sequence of the humanized heavy chain variable region HCVR was selected.
  • the sequence is as follows:
  • the sequence of the humanized light chain variable region LCVR was selected, and the sequence is as follows:
  • the designed heavy chain and light chain variable region sequences are respectively connected with the heavy chain constant region and light chain constant region sequences of the human antibody.
  • it is connected to the human IgG1 heavy chain constant region (SEQ ID NO: 31), the human IgG1 heavy chain constant region variant (SEQ ID NO: 42) and the human antibody kappa chain constant region sequence (SEQ ID NO: 32) .
  • Exemplary obtained heavy chain and light chain sequences are as follows:
  • CDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of the above humanized antibodies, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20).
  • the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days.
  • Collect the cell culture fluid after centrifugal filtration, load the sample to the antibody purification affinity column, wash the column with phosphate buffer, elution with glycine hydrochloric acid buffer (pH 2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH 9.0, And phosphate buffer dialysis to obtain the humanized antibody of the present invention.
  • glycine hydrochloric acid buffer pH 2.7 0.1M Gly-HCl
  • An antigen-binding fragment that specifically binds to CD3 The CD3HCVR and CD3LCVR shown in the following sequence are connected into a single-stranded fragment through a linking sequence; the linking sequence is a well-known linker in the art, and an exemplary linker can be (GGGGS) n , n Selected from 1, 2, 3, 4, or 5.
  • the CDR sequence of the anti-CD3 antibody or its antigen-binding fragment is as follows:
  • the antigen-binding fragment (first binding region) that specifically binds to BCMA and the antigen-binding fragment (second binding region) that specifically bind to CD3 are connected in different ways to obtain a bispecific antibody containing the following sequence:
  • Ab4 antibody is as follows (arrangement order from N-terminal to C-terminal) (the structure is shown in Figure 1):
  • the first chain, the heavy chain variable region and the heavy chain constant region of the first binding region are connected to
  • Ab5 antibody is as follows (arrangement order from N-terminal to C-terminal) (the structure is shown in Figure 2):
  • the first polypeptide includes the heavy chain variable region and the heavy chain constant region of the first binding region from the N-terminus to the C-terminus;
  • the second polypeptide includes the light chain variable region and the light chain constant region of the first binding region from the N-terminus to the C-terminus;
  • the third polypeptide includes the heavy chain variable region of the second binding region, the peptide linker, the light chain variable region of the second binding region, the peptide linker and the heavy chain constant region from the N-terminus to the C-terminus.
  • the peptide linker is a peptide linker used to connect the antigen binding domain
  • an exemplary linker sequence may be (GGGGS)n, where n is selected from 1, 2, 3, 4, or 5.
  • the heavy chain constant region can be selected from a human antibody heavy chain constant region (such as a human IgG1 heavy chain constant region) or a variant thereof (such as a variant that reduces ADCC activity).
  • the constant region of the heavy chain can be a constant region (or a constant region domain) of the same sequence, or a constant region (or a constant region domain) containing a Knob and a hole (Hole) spatial structure. Based on the sequence of the Hole constant region (or constant region domain), disulfide bonds are further introduced to promote the formation of dimers.
  • the sequence of the heavy chain constant region contained in the exemplary bispecific antibody of the present invention is as follows:
  • the sequence of the constructed bispecific antibody is as follows:
  • a cDNA fragment was synthesized based on the amino acid sequence of the above bispecific antibody and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20).
  • the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days.
  • CD3D/CD3E heterodimer protein (Acrobiosystems, cat#CDD-H82W0) to a concentration of 1 ⁇ g/ml with pH7.4 PBS, add 100 ⁇ l/well to a 96-well high-affinity ELISA plate at 4°C Incubate overnight in the refrigerator (16-20 hours). After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), add 150 ⁇ l/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate for 1 hour at room temperature for blocking. After the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer.
  • BSA bovine serum albumin
  • Example 8 Tumor cell killing effect mediated by bispecific antibody
  • the BCMA binding part of the bispecific antibody binds to tumor cells expressing BCMA, and the CD3 binding part binds to the TCR receptor on the surface of effector T cells, and forms an immune synapse under the guidance of the bispecific antibody.
  • T cells pass through granzyme and perforin. A series of cytokines will lyse and kill tumor cells.
  • the frozen PBMC was quickly thawed in a 37°C water bath, and the cell suspension was transferred to complete cell culture medium (90% RPMI-1640 containing 10% FBS, 100U/100ug/mL penicillin/streptomycin, 2mM glutamine), Suspend the cells, centrifuge at 2000rpm/min for 5min at room temperature, discard the supernatant; add fresh complete medium, resuspend the cell pellet; count, adjust the cell density to 1.5x10 5 /mL, use the EasySep Human T Cell Iso Kit (Stemcell, cat#: 17951) Isolate human total T cell population; add 33.3 ⁇ l T cell suspension 5 ⁇ 10 3 cells/well to a fully transparent 96-well U-shaped bottom plate (Corning, Cat#: 3799).
  • complete cell culture medium 90% RPMI-1640 containing 10% FBS, 100U/100ug/mL penicillin/streptomycin, 2mM glutamine
  • NCI-H929-LUC (Cobioer catalog number CBP30061L) in cell complete medium and count; adjust the cell density to 1.5x10 6 /mL.
  • Cell killing efficiency% (1-signal value of experimental group/signal value of control group) ⁇ 100%.
  • NCG mice are immunodeficient mice lacking T, B, and NK immune cells. Select 8-9 weeks old NCG female mice weighing 18-22 g.
  • 4 ⁇ 10 6 human PBMC cells were injected into each mouse.
  • 1 ⁇ 10 7 NCI-H929 cells were injected into the back of the mouse subcutaneously.
  • the mouse IgG1 control antibody or bispecific antibody was administered by intraperitoneal injection at a dose of 20 ⁇ g/kg body weight once every 3 days. After 14 days, the tumor volume of each mouse was measured and the tumor inhibition rate TGI was calculated as shown in the table below.
  • Tumor inhibition rate TGI 100%-(tumor volume of the administration group on day 14-tumor volume of the administration group on day 0)/(tumor volume of the control group on day 14-tumor volume of the control group on day 0) ⁇ 100%.
  • mice Through intraperitoneal injection, 5 ⁇ 10 6 human PBMC cells were injected into each mouse, and 1 ⁇ 10 7 RPMI-8226 cells were injected into the subcutaneous back of the mouse. After the tumor volume grows to about 95mm3, the mice are divided into 3 groups with 8 mice in each group.
  • the mouse IgG1 control antibody or bispecific antibody was administered by intraperitoneal injection at a dose of 20 ⁇ g/kg body weight once every 3 days. After 21 days, the tumor volume of each mouse was measured and the tumor inhibition rate TGI was calculated as shown in the table below.
  • Tumor inhibition rate TGI 100%-(tumor volume of the administration group on day 21-tumor volume of the administration group on day 0)/(tumor volume of the control group on day 21-tumor volume of the control group on day 0) ⁇ 100%.
  • Example 10 Affinity test of BCMA/CD3 bispecific antibody at the cellular level
  • the purpose of this example is to evaluate the difference in the affinity of the BCMA binding part of the candidate BCMA/CD3 bispecific antibody to the BCMA antigen on the surface of tumor cells.
  • NCI-H929 ATCC, CRL-9068 TM
  • the cell suspension was centrifuged at 300 g for 5 min; 5 ml of 2% FBS buffer was added to resuspend the cells, and the cell density was adjusted to 1 ⁇ 10 6 cells/ml.
  • Example 11 In vitro tumor cell killing effect mediated by BCMA/CD3 bispecific antibody
  • Preparation of human total T cell population of effector cells quickly thaw the frozen PBMC in a 37°C water bath, and transfer the cell suspension to complete cell culture medium (90% RPMI-1640 contains 10% FBS, 100U/100 ⁇ g/mL penicillin/chain Tetracycline, 2mM L-glutamine), resuspend the cells and centrifuge at 400g/min for 5min at room temperature; discard the supernatant and add fresh complete medium; resuspend the cell pellet, count, and adjust the cell density to 1x10 7 /mL; use EasySep Human T Cell Iso Kit (Stemcell, cat#: 17951): Follow the instructions to separate the total human T cell population, count the cells, and adjust the cell density to 1.2x10 6 /mL.
  • complete cell culture medium 90% RPMI-1640 contains 10% FBS, 100U/100 ⁇ g/mL penicillin/chain Tetracycline, 2mM L-glutamine
  • BCMA/CD3 bispecific antibody mediates the killing of RPMI-8226 cells by T cells

Abstract

Provided are a BCMA antibody, a bispecific antigen binding molecule that binds to BCMA and CD3 and a pharmaceutical composition thereof, and a use thereof in preparing an anti-cancer drug or a drug for treating auto-immune diseases.

Description

特异性抗原结合分子，其制备方法及医药用途Specific antigen binding molecule, its preparation method and medical use
本申请要求2020年04月17日提交的专利申请202010307360.1和2021年01月28日提交的专利申请202110119870.0的优先权。This application claims the priority of the patent application 202010307360.1 filed on April 17, 2020 and the patent application 202110119870.0 filed on January 28, 2021.
技术领域Technical field
本发明涉及生物医药领域，具体地，本发明涉及抗BCMA抗体、其抗原结合片段、结合BCMA和CD3的双特异抗原结合分子，其制备方法，及其医药用途。The present invention relates to the field of biomedicine. Specifically, the present invention relates to an anti-BCMA antibody, its antigen-binding fragment, a bispecific antigen-binding molecule that binds BCMA and CD3, its preparation method, and its medical use.
背景技术Background technique
B细胞是淋巴细胞，其在体液免疫和特异性识别抗原的抗体的产生中发挥重要的作用。B细胞的三种亚类是幼稚B细胞、记忆B细胞和浆细胞。B cells are lymphocytes, which play an important role in humoral immunity and the production of antibodies that specifically recognize antigens. The three subtypes of B cells are naive B cells, memory B cells, and plasma cells.
VDJ重组的过程，DNA发生重组以产生抗体可变结构域的组合阵列，通过不同谱系的B细胞编码的可变结构域进一步改变，导致多达10 9种独特B细胞谱系，产生具有特异性的抗体。 In the process of VDJ recombination, DNA is recombined to produce a combinatorial array of antibody variable domains. The variable domains encoded by B cells of different lineages are further changed, resulting in up to 109 unique B cell lineages, resulting in specific Antibody.
多种疾病涉及B细胞。B细胞的恶性转化导致癌症，包括淋巴瘤(如多发性骨髓瘤和霍奇金氏淋巴瘤)。自身免疫性疾病也会涉及到B细胞，包括系统性红斑狼疮(SLE)和IgA肾病。涉及B细胞的癌症和自家免疫性疾病被认为B细胞功能异常，因此控制这类疾病的可能策略是使用靶向病理性B细胞的抗体。Many diseases involve B cells. Malignant transformation of B cells leads to cancer, including lymphomas (such as multiple myeloma and Hodgkin's lymphoma). Autoimmune diseases also involve B cells, including systemic lupus erythematosus (SLE) and IgA nephropathy. Cancers and autoimmune diseases involving B cells are considered to be abnormal in B cell function, so a possible strategy to control such diseases is to use antibodies that target pathological B cells.
BCMA(CD269或TNFRSF17)是TNF受体超家族的成员，其是对配体BAFF(B细胞激活因子)和APRIL(增殖诱导配体)的非糖基化的内在膜受体。BCMA和它的相应配体可以调节体液免疫、B细胞发育和稳态。在脾脏、淋巴结、胸腺、肾上腺和肝脏检测到BCMA，扁桃体记忆B细胞和生发中心B细胞也表达BCMA。多种B细胞系的分析表明，成熟后BCMA的表达水平增加。BCMA在B细胞淋巴瘤和多发性骨髓瘤中高度表达。BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily, which is a non-glycosylated inner membrane receptor for the ligands BAFF (B cell activating factor) and APRIL (proliferation inducing ligand). BCMA and its corresponding ligands can regulate humoral immunity, B cell development and homeostasis. BCMA was detected in the spleen, lymph nodes, thymus, adrenal glands and liver, and tonsil memory B cells and germinal center B cells also expressed BCMA. Analysis of various B cell lines showed that the expression level of BCMA increased after maturation. BCMA is highly expressed in B-cell lymphoma and multiple myeloma.
当前针对BCMA的疗法主要分为三大类：嵌合抗原受体T细胞疗法(CAR-T)、双特异性抗体(BsAb)以及抗体偶联药物(ADC)。其中GSK的ADC药物Blenrep已于2020年8月获美国FDA批准，成为首个获批用于抗BCMA的免疫相关药，但其治疗存在视力损伤的不良反应，以及后期失效等缺点，使得双特异性抗体策略成为该靶点的另一个热门研发方向。其中，安进的双抗AMG-420优先进入临床后期。该抗体采用BiTE设计，穿透性好，但分子量小且半衰期短，降低了患者的依从性。The current therapies for BCMA are mainly divided into three categories: chimeric antigen receptor T cell therapy (CAR-T), bispecific antibodies (BsAb) and antibody-conjugated drugs (ADC). Among them, GSK's ADC drug Blenrep was approved by the U.S. FDA in August 2020, becoming the first immune-related drug approved for anti-BCMA, but its treatment has disadvantages such as visual impairment and late failure, making it bispecific Sexual antibody strategy has become another popular research and development direction for this target. Among them, Amgen's double antibody AMG-420 has priority to enter the late clinical stage. The antibody is designed with BiTE, which has good penetration, but has a small molecular weight and short half-life, which reduces patient compliance.
发明内容Summary of the invention
根据本发明的一些实施方案，提供了一种抗BCMA抗体或其抗原结合片段，其包含：According to some embodiments of the present invention, there is provided an anti-BCMA antibody or antigen-binding fragment thereof, which comprises:
抗体重链可变区，所述的抗体重链可变区包含至少1个选自以下序列所示的HCDR：An antibody heavy chain variable region, said antibody heavy chain variable region comprising at least one HCDR selected from the following sequences:
SEQ ID NO：3，SEQ ID NO：4，SEQ ID NO：5或SEQ ID NO：9；和SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 9; and
抗体轻链可变区，所述的抗体轻链可变区包含至少1个选自以下序列所述的LCDR： SEQ ID NO：6，SEQ ID NO：7，SEQ ID NO：8或SEQ ID NO：10。An antibody light chain variable region, said antibody light chain variable region comprising at least one LCDR selected from the following sequence: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO : 10.
在本发明一个具体实施方案中，所述的抗BCMA抗体或其抗原结合片段的重链可变区包含：SEQ ID NO：3所示的HCDR1、SEQ ID NO：4或SEQ ID NO：9所示的HCDR2和SEQ ID NO：5所示的HCDR3。In a specific embodiment of the present invention, the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof includes: HCDR1 shown in SEQ ID NO: 3, and shown in SEQ ID NO: 4 or SEQ ID NO: 9. HCDR2 shown in SEQ ID NO: 5 and HCDR3 shown in SEQ ID NO: 5.
在本发明一个具体实施方案中，所述的抗BCMA抗体或其抗原结合片段的轻链可变区包含：SEQ ID NO：6所示的LCDR1、SEQ ID NO：7或SEQ ID NO：10所示的LCDR2和SEQ ID NO：8所示的LCDR3。In a specific embodiment of the present invention, the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof includes: LCDR1 shown in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 10. LCDR2 shown and LCDR3 shown in SEQ ID NO: 8.
在本发明一个具体实施方案中，提供了一种抗BCMA抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：In a specific embodiment of the present invention, there is provided an anti-BCMA antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein:
i)i)
所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：4所示的HCDR2和SEQ ID NO：5所示的HCDR3；以及The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and
所述轻链可变区包含：SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2和SEQ ID NO：8所示的LCDR3，The light chain variable region includes: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8,
或ii)Or ii)
所述重链可变区包含：SEQ ID NO：3所示的HCDR1、SEQ ID NO：9所示的HCDR2和SEQ ID NO：5所示的HCDR3；以及The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9 and HCDR3 shown in SEQ ID NO: 5; and
所述的轻链可变区包含：SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2和SEQ ID NO：8所示的LCDR3，The light chain variable region includes: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8,
或iii)Or iii)
所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：9所示的HCDR2和SEQ ID NO：5所示的HCDR3；The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9 and HCDR3 shown in SEQ ID NO: 5;
所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：10所示的LCDR2和SEQ ID NO：8所示的LCDR3，The light chain variable region includes LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8,
或iv)Or iv)
所述重链可变区包含：SEQ ID NO：3所示的HCDR1、SEQ ID NO：4所示的HCDR2和SEQ ID NO：5所示的HCDR3；以及The heavy chain variable region includes: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and
所述的轻链可变区包含：SEQ ID NO：6所示的LCDR1、SEQ ID NO：10所示的LCDR2和SEQ ID NO：8所示的LCDR3。The light chain variable region includes: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8.
在本发明一个具体实施方案中，所述的抗BCMA抗体或其抗原结合片段为鼠源抗体或其抗原结合片段。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof.
在本发明一个具体实施方案中，所述的抗BCMA抗体或其抗原结合片段为嵌合抗体或其抗原结合片段。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof.
在本发明一个具体实施方案中，所述的抗BCMA抗体或其抗原结合片段为人抗体或其抗原结合片段。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.
在本发明一个具体实施方案中，所述的抗BCMA抗体或其抗原结合片段为人源化抗 体或其抗原结合片段。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.
在本发明一个具体实施方案中，所述的抗BCMA抗体或其抗原结合片段进一步包含人IgG1、IgG2、IgG3、IgG4的重链恒定区或其变体。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.
在本发明一个具体实施方案中，所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1、IgG2、IgG4的重链恒定区或其变体。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG4 or a variant thereof.
在本发明一个具体实施方案中，所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1的重链恒定区。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises the heavy chain constant region of human IgG1.
在本发明一个具体实施方案中，所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1的重链恒定区变体。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region variant of human IgG1.
在本发明一个具体实施方案中，其中，所述人IgG1重链恒定区变体具有与野生IgG1重链恒定区相比降低的ADCC毒性。In a specific embodiment of the present invention, wherein the human IgG1 heavy chain constant region variant has reduced ADCC toxicity compared with wild IgG1 heavy chain constant region.
在本发明一个具体实施方案中，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO：31的重链恒定区，或如SEQ ID NO：42所示的重链恒定区变体，或如SEQ ID NO：33所示的重链恒定区变体，或如SEQ ID NO：34所示的重链恒定区变体。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 31, or a heavy chain constant region variant as shown in SEQ ID NO: 42, Or a heavy chain constant region variant as shown in SEQ ID NO: 33, or a heavy chain constant region variant as shown in SEQ ID NO: 34.
在本发明一个具体实施方案中，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO：31的重链恒定区或如SEQ ID NO：42所示的重链恒定区变体。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 31 or a heavy chain constant region variant as shown in SEQ ID NO: 42.
在本发明一个具体实施方案中，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO：42所示的重链恒定区变体。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region variant as shown in SEQ ID NO:42.
在本发明一些实施方案中，所述抗BCMA抗体或其抗原结合片段进一步包含源自人抗体κ链、λ链的轻链恒定区或其变体。In some embodiments of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody kappa chain, lambda chain or a variant thereof.
在本发明一个具体实施方案中，所述抗BCMA抗体或其抗原结合片段进一步包含源自人抗体κ链的轻链恒定区。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody kappa chain.
在本发明一个具体实施方案中，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO：32所示的轻链恒定区。In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO: 32.
在本发明一个具体实施方案中，所述的抗BCMA抗体或其抗原结合片段包含选自以下序列所示的重链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区：SEQ ID NO：11、SEQ ID NO：12或SEQ ID NO：13；和/或，In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequences, or at least 70%, 75%, 80% compared with the following sequences , 85%, 90%, 95% or 99% identical heavy chain variable regions: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and/or,
所述的抗BCMA抗体或其抗原结合片段包含选自以下序列所示的轻链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区：SEQ ID NO：14、SEQ ID NO：15或SEQ ID NO：16。The anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the following sequences, or has at least 70%, 75%, 80%, 85%, 90%, 95% compared with the following sequences Or a light chain variable region with 99% identity: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.
在本发明一个具体实施方案中，所述的抗BCMA抗体或其抗原结合片段含有如下序列所示的重链，或与以下序列相比具有至少80％，85％，90％，95％或99％同一性的重链：SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19或SEQ ID NO：43；和/或，In a specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof contains a heavy chain as shown in the following sequence, or at least 80%, 85%, 90%, 95% or 99% compared with the following sequence % Identity heavy chain: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 43; and/or,
所述的抗BCMA抗体或其抗原结合片段含有如下序列所示的轻链，或与以下序列相比具有至少80％，85％，90％，95％或99％同一性的轻链：SEQ ID NO：20、SEQ ID NO： 21或SEQ ID NO：22。The anti-BCMA antibody or antigen-binding fragment thereof contains a light chain as shown in the following sequence, or a light chain with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.
在本发明一个更具体实施方案中，所述的抗BCMA抗体或其抗原结合片段，其包含：In a more specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises:
SEQ ID NO：11所示的重链可变区和SEQ ID NO：14所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14; or,
SEQ ID NO：12所示的重链可变区和SEQ ID NO：15所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 12 and the light chain variable region shown in SEQ ID NO: 15; or,
SEQ ID NO：11所示的重链可变区和SEQ ID NO：16所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 16; or,
SEQ ID NO：13所示的重链可变区和SEQ ID NO：16所示的轻链可变区。The heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 16.
在本发明一个更具体实施方案中，所述的抗BCMA抗体或其抗原结合片段，其包含：In a more specific embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises:
SEQ ID NO：17所示的重链和SEQ ID NO：20所示的轻链；或，The heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20; or,
SEQ ID NO：18所示的重链和SEQ ID NO：21所示的轻链；或，The heavy chain shown in SEQ ID NO: 18 and the light chain shown in SEQ ID NO: 21; or,
SEQ ID NO：19所示的重链和SEQ ID NO：22所示的轻链；或，The heavy chain shown in SEQ ID NO: 19 and the light chain shown in SEQ ID NO: 22; or,
SEQ ID NO：43所示的重链和SEQ ID NO：22所示的轻链；或，The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 22; or,
SEQ ID NO：43所示的重链和SEQ ID NO：20所示的轻链。The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 20.
根据本发明的一些实施方案，提供了一种抗CD3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：According to some embodiments of the present invention, there is provided an anti-CD3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein:
所述重链可变区包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2和SEQ ID NO：37所示的HCDR3；The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37;
所述轻链可变区包含SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2和SEQ ID NO：40所示的LCDR3。The light chain variable region includes LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.
根据本发明的一些实施方案，提供了一种抗CD3抗体或其抗原结合片段，其包含SEQ ID NO：24所示的重链可变区和SEQ ID NO：25所示的轻链可变区。According to some embodiments of the present invention, there is provided an anti-CD3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region shown in SEQ ID NO: 24 and a light chain variable region shown in SEQ ID NO: 25 .
根据本发明的一些实施方案，提供了一种抗CD3抗体的抗原结合片段，其是scFv。在具体的实施方案中，scFv如SEQ ID NO：41所示。According to some embodiments of the present invention, there is provided an antigen-binding fragment of an anti-CD3 antibody, which is a scFv. In a specific embodiment, the scFv is shown in SEQ ID NO: 41.
根据本发明的一些实施方案，提供了一种结合BCMA和CD3的双特异性抗体或其抗原结合片段，其包含：According to some embodiments of the present invention, there is provided a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, which comprises:
特异性结合BCMA的第一结合区；和The first binding region that specifically binds to BCMA; and
特异性结合CD3的第二结合区。The second binding region that specifically binds to CD3.
在一些具体实施方案中，所述第一结合区选自如前所述的抗BCMA抗体或其抗原结合片段。In some specific embodiments, the first binding region is selected from the aforementioned anti-BCMA antibodies or antigen-binding fragments thereof.
在一些具体实施方案中，所述第二结合区选自抗CD3抗体或其抗原结合片段。In some specific embodiments, the second binding region is selected from anti-CD3 antibodies or antigen-binding fragments thereof.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：4所示的HCDR2和SEQ ID NO：5所示的HCDR3；所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：10所示的LCDR2和SEQ ID NO：8所示的LCDR3。According to the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, said anti-BCMA antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising SEQ ID NO: HCDR1, HCDR1, SEQ ID NO: 4, and HCDR3, SEQ ID NO: 5; the light chain variable region includes LCDR1, SEQ ID NO: 6 and LCDR1, SEQ ID NO : LCDR2 shown in 10 and LCDR3 shown in SEQ ID NO: 8.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段包含SEQ ID NO：11所示的重链可变区和SEQ ID NO：14所示的 轻链可变区。According to the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, said anti-BCMA antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO: 11 and SEQ ID NO: 14 The variable region of the light chain.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1、IgG2、IgG3、IgG4的重链恒定区或其变体。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1的重链恒定区。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1的重链恒定区变体，所述人IgG1重链恒定区变体具有与人IgG1重链恒定区相比降低的ADCC毒性。According to the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, said anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region variant of human IgG1, said human IgG1 heavy chain constant region variant It has reduced ADCC toxicity compared with human IgG1 heavy chain constant region.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段包含如SEQ ID NO：31所示的重链恒定区。According to the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, said anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO: 31.
在本发明优选的实施方案中，根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段包含如SEQ ID NO：42所示的重链恒定区变体。In a preferred embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, said anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain constant as shown in SEQ ID NO: 42 Area variants.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段进一步包含人抗体κ链、λ链的轻链恒定区或其变体，优选人抗体κ链的轻链恒定区，最优选如SEQ ID NO：32所示的轻链恒定区。According to the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, said anti-BCMA antibody or antigen-binding fragment thereof further comprises a human antibody kappa chain, a light chain constant region of a lambda chain or a variant thereof, preferably a human antibody The light chain constant region of the kappa chain is most preferably the light chain constant region shown in SEQ ID NO: 32.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述所述抗BCMA抗体或其抗原结合片段包含如SEQ ID NO：17或如SEQ ID NO：43所示的重链和如SEQ ID NO：20所示的轻链。According to the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, said anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 17 or SEQ ID NO: 43 and Such as the light chain shown in SEQ ID NO: 20.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述所述抗BCMA抗体或其抗原结合片段包含如SEQ ID NO：43所示的重链和如SEQ ID NO：20所示的轻链。According to the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, said anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 43 and a heavy chain as shown in SEQ ID NO: 20 The light chain shown.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，其中，抗CD3抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：The bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
所述抗CD3抗体或其抗原结合片段的重链可变区包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2和SEQ ID NO：37所示的HCDR3；The heavy chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37;
所述抗CD3抗体或其抗原结合片段的轻链可变区包含SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2和SEQ ID NO：40所示的LCDR3。The light chain variable region of the anti-CD3 antibody or antigen-binding fragment thereof includes LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述抗CD3抗体或其抗原结合片段含有SEQ ID NO：24所示的重链可变区和SEQ ID NO：25所示的轻链可变区。According to the present invention, a bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, said anti-CD3 antibody or antigen-binding fragment thereof contains the heavy chain variable region shown in SEQ ID NO: 24 and SEQ ID NO: 25 The variable region of the light chain.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第二结合区包含的重链可变区和轻链可变区通过肽接头连接。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the heavy chain variable region and the light chain variable region contained in the second binding region are connected by a peptide linker.
所述连接第二结合区的重链可变区和轻链可变区的肽接头选自(GGGGS) n，n选自1-5的整数，优选地，n为3。 The peptide linker connecting the heavy chain variable region and the light chain variable region of the second binding region is selected from (GGGGS) n , n is selected from an integer of 1-5, preferably, n is 3.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第二结合区包含如SEQ ID NO：41所示的序列。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the second binding region comprises the sequence shown in SEQ ID NO: 41.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第二结合区的重链可变区的N端连接于第一结合区的轻链恒定区的C端。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 of the present invention, the N-terminus of the heavy chain variable region of the second binding region is connected to the C-terminus of the light chain constant region of the first binding region.
在一些实施方案中，所述第二结合区的重链可变区的N端通过肽接头连接于第一结合区的轻链恒定区的C端，所述肽接头选自(GGGGS) n，n选自1-5的整数，优选地，n为3。 In some embodiments, the N-terminus of the heavy chain variable region of the second binding region is connected to the C-terminus of the light chain constant region of the first binding region by a peptide linker selected from (GGGGS) n , n is selected from an integer of 1-5, preferably n is 3.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，其包含特异性结合BCMA的第一结合区和特异性结合CD3的第二结合区，其中：The bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention comprises a first binding region that specifically binds BCMA and a second binding region that specifically binds CD3, wherein:
所述第一结合区包含重链可变区和轻链可变区，The first binding region includes a heavy chain variable region and a light chain variable region,
第一结合区的重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：4所示的HCDR2和SEQ ID NO：5所示的HCDR3；The heavy chain variable region of the first binding region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5;
所述第一结合区的轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：10所示的LCDR2和SEQ ID NO：8所示的LCDR3，以及The light chain variable region of the first binding region includes LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8, and
所述第二结合区包含重链可变区和轻链可变区，The second binding region includes a heavy chain variable region and a light chain variable region,
第二结合区的重链可变区包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2和SEQ ID NO：37所示的HCDR3；The heavy chain variable region of the second binding region includes HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37;
第二结合区的轻链可变区包含SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2和SEQ ID NO：40所示的LCDR3。The light chain variable region of the second binding region includes LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，其中：The bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein:
第一结合区包含SEQ ID NO：11所示的重链可变区和SEQ ID NO：14所示的轻链可变区，以及The first binding region includes the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14, and
第二结合区包含SEQ ID NO：24所示的重链可变区和SEQ ID NO：25所示的轻链可变区。The second binding region includes the heavy chain variable region shown in SEQ ID NO: 24 and the light chain variable region shown in SEQ ID NO: 25.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，其中：The bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein:
第一结合区包含如SEQ ID NO：17或如SEQ ID NO：43所示的重链和如SEQ ID NO：20所示的轻链，以及The first binding region includes a heavy chain as shown in SEQ ID NO: 17 or SEQ ID NO: 43 and a light chain as shown in SEQ ID NO: 20, and
第二结合区包含SEQ ID NO：24所示的重链可变区和SEQ ID NO：25所示的轻链可变区。The second binding region includes the heavy chain variable region shown in SEQ ID NO: 24 and the light chain variable region shown in SEQ ID NO: 25.
在本发明优选的实施方案中，根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，其中，第一结合区包含如SEQ ID NO：43所示的重链和如SEQ ID NO：20所示的轻链，以及第二结合区包含SEQ ID NO：24所示的重链可变区和SEQ ID NO：25所示的轻链可变区。In a preferred embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, wherein the first binding region comprises a heavy chain as shown in SEQ ID NO: 43 and a heavy chain as shown in SEQ ID NO The light chain shown in: 20 and the second binding region include the heavy chain variable region shown in SEQ ID NO: 24 and the light chain variable region shown in SEQ ID NO: 25.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，其中，第二结合区包含的重链可变区和轻链可变区通过肽接头连接，所述连接第二结合区的重链可变区和轻链可变区的肽接头选自(GGGGS) n，n选自1-5的整数(1、2、3、4、5)，优选 地，n为3。 According to the present invention, the bispecific antibody or antigen-binding fragment thereof that binds to BCMA and CD3, wherein the heavy chain variable region and the light chain variable region contained in the second binding region are connected by a peptide linker, and the second binding region The peptide linker of the heavy chain variable region and the light chain variable region is selected from (GGGGS) n , n is selected from an integer of 1-5 (1, 2, 3, 4, 5), preferably, n is 3.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，第二结合区的重链可变区的N端连接于第一结合区的轻链恒定区的C端；优选地，所述第二结合区的重链可变区的N端通过肽接头连接于第一结合区的轻链恒定区的C端。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 of the present invention, the N-terminus of the heavy chain variable region of the second binding region is connected to the C-terminus of the light chain constant region of the first binding region; preferably, the The N terminal of the heavy chain variable region of the second binding region is connected to the C terminal of the light chain constant region of the first binding region through a peptide linker.
根据本发明的结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一结合区包含至少一个抗BCMA抗体的Fab片段，所述第二结合区包含至少一个抗CD3抗体的scFv片段。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the first binding region comprises at least one Fab fragment of an anti-BCMA antibody, and the second binding region comprises at least one scFv fragment of an anti-CD3 antibody .
根据本发明的结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一结合区包含两个抗BCMA抗体的Fab片段，所述第二结合区包含两个抗CD3抗体的scFv片段。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the first binding region comprises two Fab fragments of anti-BCMA antibodies, and the second binding region comprises two scFv fragments of anti-CD3 antibodies .
根据本发明的结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一结合区选自上述包含轻链和重链的抗BCMA抗体，所述第二结合区包含两个上述抗CD3抗体或其抗原结合片段的scFv片段。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the first binding region is selected from the above-mentioned anti-BCMA antibodies comprising light and heavy chains, and the second binding region comprises two of the above-mentioned anti-BCMA antibodies. A scFv fragment of a CD3 antibody or antigen-binding fragment thereof.
根据本发明的结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述scFv片段的N端连接于Fab片段或连接于抗BCMA抗体的轻链恒定区C端；优选地，所述scFv片段的N端通过肽接头连接于Fab片段或连接于抗BCMA抗体的轻链恒定区C端。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the N-terminus of the scFv fragment is connected to the Fab fragment or the C-terminus of the light chain constant region of the anti-BCMA antibody; preferably, the scFv The N-terminus of the fragment is connected to the Fab fragment through a peptide linker or to the C-terminus of the light chain constant region of the anti-BCMA antibody.
根据本发明的结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一结合区选自包含轻链和重链的抗BCMA抗体，所述第二结合区包含两个抗CD3抗体的scFv片段。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the first binding region is selected from an anti-BCMA antibody comprising a light chain and a heavy chain, and the second binding region comprises two anti-CD3 antibodies The scFv fragment.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述scFv片段的N端通过肽接头连接于Fab片段或抗BCMA抗体的轻链恒定区C端，所述肽接头选自(GGGGS) n，n选自1-5的整数，优选地，n为3。 According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the N-terminus of the scFv fragment is connected to the C-terminus of the light chain constant region of the Fab fragment or the anti-BCMA antibody through a peptide linker, and the peptide linker is selected from (GGGGS) n , n is selected from an integer of 1-5, preferably n is 3.
根据本发明的一个具体实施方案，本发明的双特异性抗体或其抗原结合片段为四链结构，包含2条相同的第一链和2条相同的第二链，其中：According to a specific embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention has a four-chain structure, comprising two identical first chains and two identical second chains, wherein:
第一链，从N端到C端包含第一结合区的重链可变区和第一结合区的重链恒定区；The first chain includes the heavy chain variable region of the first binding region and the heavy chain constant region of the first binding region from the N-terminus to the C-terminus;
第二链，从N端到C端包含第一结合区的轻链可变区、第一结合区的轻链恒定区和第一肽接头，以及第二结合区的重链可变区、第二肽接头和第二结合区的轻链可变区。The second chain, from the N-terminus to the C-terminus, contains the light chain variable region of the first binding region, the light chain constant region of the first binding region and the first peptide linker, and the heavy chain variable region and the first peptide linker of the second binding region. The light chain variable region of the dipeptide linker and the second binding region.
根据本发明的双特异性抗体或其抗原结合片段，所述第二链中第二结合区的重链可变区和第二结合区的轻链可变区选自本发明抗CD3抗体或其抗原结合片段的重链可变区和轻链可变区，第二肽接头选自(GGGGS) n，n选自1-5的整数，优选地，n为3。 According to the bispecific antibody or antigen-binding fragment thereof of the present invention, the heavy chain variable region of the second binding region in the second chain and the light chain variable region of the second binding region are selected from the anti-CD3 antibody of the present invention or its The variable region of the heavy chain and the variable region of the light chain of the antigen-binding fragment, the second peptide linker is selected from (GGGGS) n , n is selected from an integer of 1-5, preferably, n is 3.
根据本发明的结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一结合区包含一个抗BCMA抗体的Fab片段，所述第二结合区包含一个抗CD3抗体的scFv片段。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the first binding region includes an anti-BCMA antibody Fab fragment, and the second binding region includes an anti-CD3 antibody scFv fragment.
根据本发明的一个具体实施方案，本发明的双特异性抗体或其抗原结合片段包含：According to a specific embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention comprises:
第一多肽，从N端到C端包含第一结合区的重链可变区和第一结合区的重链恒定区；The first polypeptide comprises the heavy chain variable region of the first binding region and the heavy chain constant region of the first binding region from the N-terminus to the C-terminus;
第二多肽，从N端到C端包含第一结合区的轻链可变区和第一结合区的轻链恒定区；The second polypeptide includes the light chain variable region of the first binding region and the light chain constant region of the first binding region from the N-terminus to the C-terminus;
第三多肽，从N端到C端包含第二结合区的重链可变区、第一肽接头、第二结合区的轻链可变区、第二肽接头和第一结合区的重链恒定区。The third polypeptide, from the N-terminus to the C-terminus, includes the heavy chain variable region of the second binding region, the first peptide linker, the light chain variable region of the second binding region, the second peptide linker, and the heavy chain of the first binding region. Chain constant region.
根据本发明的结合BCMA和CD3的双特异性抗体或其抗原结合片段，第三多肽中的第二肽接头选自(GGGGS)n，n选自1-5的整数，优选地，n为2。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 of the present invention, the second peptide linker in the third polypeptide is selected from (GGGGS)n, n is selected from an integer of 1-5, preferably, n is 2.
在一些实施方案中，当肽接头(即第一肽接头)连接第二结合区的重链可变区和第二结合区的轻链可变区时，n为3。In some embodiments, when the peptide linker (ie, the first peptide linker) connects the heavy chain variable region of the second binding region and the light chain variable region of the second binding region, n is 3.
在一些实施方案中，当肽接头(即第二肽接头)连接第二结合区的轻链可变区和重链恒定区时，n为2。In some embodiments, n is 2 when the peptide linker (ie, the second peptide linker) connects the light chain variable region and the heavy chain constant region of the second binding region.
根据本发明的双特异性抗体或其抗原结合片段，所述第一多肽的第一结合区的重链可变区选自本发明抗BCMA抗体或其抗原结合片段的重链可变区。According to the bispecific antibody or antigen-binding fragment thereof of the present invention, the heavy chain variable region of the first binding region of the first polypeptide is selected from the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof of the present invention.
根据本发明的双特异性抗体或其抗原结合片段，所述第二多肽的第一结合区的轻链可变区选自本发明抗BCMA抗体或其抗原结合片段的轻链可变区。According to the bispecific antibody or antigen-binding fragment thereof of the present invention, the light chain variable region of the first binding region of the second polypeptide is selected from the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof of the present invention.
根据本发明的双特异性抗体或其抗原结合片段，所述第三多肽的第二结合区的重链可变区和第二结合区的轻链可变区分别选自本发明抗CD3抗体或其抗原结合片段的重链可变区和轻链可变区；第一肽接头选自(GGGGS)n，n选自1-5的整数。According to the bispecific antibody or antigen-binding fragment thereof of the present invention, the heavy chain variable region of the second binding region and the light chain variable region of the second binding region of the third polypeptide are respectively selected from the anti-CD3 antibody of the present invention Or the variable region of the heavy chain and the variable region of the light chain of the antigen binding fragment thereof; the first peptide linker is selected from (GGGGS)n, and n is selected from an integer of 1-5.
根据本发明的双特异性抗体或其抗原结合片段，所述第一多肽中重链恒定区包含源自人IgG的重链恒定区，第三多肽中重链恒定区包含源自人IgG的重链恒定区结构域，优选地，所述IgG的重链恒定区结构域包含(从N至C端)铰链区、CH2、和CH3。其中，人IgG选自IgG1、IgG2、IgG3或IgG4或其变体，优选IgG1。According to the bispecific antibody or antigen-binding fragment thereof of the present invention, the heavy chain constant region of the first polypeptide comprises a heavy chain constant region derived from human IgG, and the heavy chain constant region of the third polypeptide comprises a heavy chain constant region derived from human IgG The heavy chain constant region domain of the IgG, preferably, the heavy chain constant region domain of the IgG comprises (from N to C-terminus) hinge region, CH2, and CH3. Among them, human IgG is selected from IgG1, IgG2, IgG3, IgG4 or variants thereof, and IgG1 is preferred.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一多肽和第三多肽的重链恒定区在铰链区包含一个或多个半胱氨酸残基，以形成二硫键。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 of the present invention, the heavy chain constant regions of the first polypeptide and the third polypeptide include one or more cysteine residues in the hinge region to Forms disulfide bonds.
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一多肽和第三多肽的重链恒定区在铰链区外进一步包含一个或多个半胱氨酸残基，以形成二硫键。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the heavy chain constant regions of the first polypeptide and the third polypeptide further comprise one or more cysteine residues outside the hinge region , To form disulfide bonds.
在一些实施方案中，半胱氨酸残基位于选自以下的位点或组合：349、354或等同位点。In some embodiments, the cysteine residue is located at a position or combination selected from: 349, 354 or equivalent positions.
针对抗体氨基酸的编号，存在多种不同的编号规则。可以进行编号规则的比对，以便确定等同的残基位置。Kabat编号规则是基于相同高变区的位置而开发的。Chothia编号规则(Al-Lazikani，1997)与Kabat的方案相同，但对第一个VH CDR进行了校正。IMGT(Lefranc，2003)和AHo(Honegger和Plückthun，2001)定义了抗体和T细胞受体(TCR)的可变结构域，因此可以比较二者的等同残基位置。There are many different numbering rules for the numbering of antibody amino acids. The number sequence can be compared to determine equivalent residue positions. The Kabat numbering rule was developed based on the location of the same hypervariable area. The Chothia numbering scheme (Al-Lazikani, 1997) is the same as Kabat's scheme, but the first VH CDR is corrected. IMGT (Lefranc, 2003) and AHo (Honegger and Plückthun, 2001) define the variable domains of antibodies and T cell receptors (TCR), so the equivalent residue positions of the two can be compared.
Eu编号规则的建立基于Gerald M.Edelman等人所纯化得到人IgG1(命名为Eu)，Gerald M.Edelman等人测定了IgG1的氨基酸序列并对其进行遍号。现有技术中，众多工具可用于确定或比对抗体的氨基酸编号，例如但不限于ANARCI、abYsis。The Eu numbering rule was established based on the purified human IgG1 (named Eu) by Gerald M. Edelman et al. Gerald M. Edelman et al. determined the amino acid sequence of IgG1 and numbered it. In the prior art, many tools can be used to determine or compare the amino acid numbers of antibodies, such as but not limited to ANARCI and abYsis.
在本申请的上下文中，重链恒定区中氨基酸位点遵守EU编号规则，位点349或354是指在EU编号规则下的氨基酸位点。技术人员能够显而易见地确定，在等同位点上的 半胱氨酸残基也预期能够实现相同的功能(即，在第一多肽和第三多肽之间促进二硫键的形成)。例如，确定抗体氨基酸位点的编号规则还包括：Kabat、Chothia、IMGT，技术人员可以确定不同编号规则中对应的等同位点。再比如，技术人员通过氨基酸序列的比对分析，可以确定这样的等同位点。In the context of this application, the amino acid positions in the constant region of the heavy chain follow the EU numbering rules, and the positions 349 or 354 refer to the amino acid positions under the EU numbering rules. The skilled person can clearly determine that the cysteine residue at the equivalent position is also expected to perform the same function (ie, promote the formation of a disulfide bond between the first polypeptide and the third polypeptide). For example, the numbering rules for determining the amino acid positions of antibodies also include: Kabat, Chothia, IMGT, and the technician can determine the corresponding equivalent positions in different numbering rules. For another example, a technician can determine such an equivalent site by comparing and analyzing the amino acid sequence.
在一个具体的实施方案中，所述第一多肽和第三多肽的重链恒定区分别包含349C和354C。In a specific embodiment, the heavy chain constant regions of the first polypeptide and the third polypeptide comprise 349C and 354C, respectively.
在一个具体的实施方案中，根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一多肽的重链恒定区包含354C，第三多肽的重链恒定区包含349C。In a specific embodiment, according to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the heavy chain constant region of the first polypeptide comprises 354C, and the heavy chain constant region of the third polypeptide comprises 349C.
在另一些实施方案中，根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一多肽和第三多肽重链恒定区分别包含杵和臼结构域，臼和杵结构域相互作用促进第一多肽和第三多肽的异二聚化。In other embodiments, according to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the heavy chain constant regions of the first polypeptide and the third polypeptide comprise knob and socket domains, respectively, and The knob domain interaction promotes the heterodimerization of the first polypeptide and the third polypeptide.
在一个具体的实施方案中，重链恒定区包含杵结构域，优选包含366或等同位点的色氨酸残基。In a specific embodiment, the heavy chain constant region comprises a knob domain, preferably a tryptophan residue at 366 or equivalent.
在一个具体的实施方案中，重链恒定区包含臼结构域，优选包含选自以下的氨基酸残基：366或等同位点的S，368或等同位点的A，407或等同位点的V。同上所述，技术人员能够确定这样的等同位点。In a specific embodiment, the heavy chain constant region comprises a hole domain, preferably an amino acid residue selected from the group consisting of: 366 or an equivalent position of S, 368 or an equivalent position of A, 407 or an equivalent position of V . As mentioned above, the skilled person can determine such equivalent sites.
在一个具体的实施方案中，根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一多肽重链恒定区包含杵结构域，第三多肽重链恒定区包含臼结构域。In a specific embodiment, according to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 according to the present invention, the first polypeptide heavy chain constant region comprises a knob domain, and the third polypeptide heavy chain constant region comprises Mortar domain.
所述重链恒定区的突变，其氨基酸残基位点根据Kabat等,Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health,Bethesda,MD(1991)的EU索引进行编号。The amino acid residue positions of the mutations in the constant region of the heavy chain are numbered according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) .
根据本发明结合BCMA和CD3的双特异性抗体或其抗原结合片段，所述第一多肽重链恒定区包含如SEQ ID NO：33所示的序列，第三多肽重链恒定区包含如SEQ ID NO：34所示的序列。According to the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3 of the present invention, the first polypeptide heavy chain constant region comprises the sequence shown in SEQ ID NO: 33, and the third polypeptide heavy chain constant region comprises SEQ ID NO: the sequence shown in 34.
在本发明优选的实施方案中，本发明的双特异性抗体或其抗原结合片段含有SEQ ID NO：26和SEQ ID NO：27所示的序列。In a preferred embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention contains the sequences shown in SEQ ID NO: 26 and SEQ ID NO: 27.
根据本发明优选的实施方案，本发明的双特异性抗体或其抗原结合片段为四链结构，包含2条(优选相同的)第一链和2条(优选相同的)第二链，所述第一链包含如SEQ ID NO：26所示的序列；所述第二链包含如SEQ ID NO：27所示的序列。According to a preferred embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention has a four-chain structure, comprising two (preferably identical) first chains and two (preferably identical) second chains. The first chain includes the sequence shown in SEQ ID NO: 26; the second chain includes the sequence shown in SEQ ID NO: 27.
在本发明一个更具体实施方案中，本发明的双特异性抗体或其抗原结合片段含有SEQ ID NO：28，SEQ ID NO：29和SEQ ID NO：20所示的序列。In a more specific embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention contains the sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 20.
根据本发明的一个具体实施方案，本发明的双特异性抗体或其抗原结合片段为三链抗体，所述第一多肽含有如SEQ ID NO：28所示的序列，第二多肽含有如SEQ ID NO：20所示的序列，第三多肽含有如SEQ ID NO：29所示的序列According to a specific embodiment of the present invention, the bispecific antibody or antigen-binding fragment thereof of the present invention is a three-chain antibody, the first polypeptide contains the sequence shown in SEQ ID NO: 28, and the second polypeptide contains The sequence shown in SEQ ID NO: 20, and the third polypeptide contains the sequence shown in SEQ ID NO: 29
在本发明还提供了一种多核苷酸，其编码本发明的抗BCMA抗体或其抗原结合片段、 结合BCMA和CD3的双特异性抗体或其抗原结合片段。The present invention also provides a polynucleotide, which encodes the anti-BCMA antibody or antigen-binding fragment thereof of the present invention, the bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3.
本发明还提供了一种表达载体，其含有本发明所述的多核苷酸。The present invention also provides an expression vector, which contains the polynucleotide of the present invention.
本发明还提供了一种宿主细胞，其导入或含有本发明所述的表达载体。The present invention also provides a host cell which is introduced into or contains the expression vector of the present invention.
在本发明优选的方案中，所述的宿主细胞为细菌，优选为大肠杆菌(E.coli)。In a preferred embodiment of the present invention, the host cell is bacteria, preferably E. coli.
在本发明优选的方案中，所述的宿主细胞为酵母菌，优选为毕赤酵母(P.pastoris)。In a preferred embodiment of the present invention, the host cell is yeast, preferably P. pastoris.
在本发明优选的方案中，所述的宿主细胞为哺乳动物细胞，优选为CHO细胞或HEK293细胞。In a preferred embodiment of the present invention, the host cell is a mammalian cell, preferably a CHO cell or HEK293 cell.
另一方面，本发明提供了一种生产抗BCMA抗体或其抗原结合片段、结合BCMA和CD3的双特异性抗体或其抗原结合片段的方法，包括步骤：In another aspect, the present invention provides a method for producing an anti-BCMA antibody or an antigen-binding fragment thereof, a bispecific antibody that binds BCMA and CD3, or an antigen-binding fragment thereof, including the steps:
培养如上所述的宿主细胞；Culturing host cells as described above;
从培养物中分离抗体；以及Isolate the antibody from the culture; and
对所述抗体进行纯化。The antibody is purified.
本发明还提供了一种药物组合物，其含有：The present invention also provides a pharmaceutical composition, which contains:
本发明所述的抗BCMA抗体或其抗原结合片段或结合BCMA和CD3的双特异性抗体或其抗原结合片段；以及The anti-BCMA antibody or its antigen-binding fragment of the present invention or the bispecific antibody or its antigen-binding fragment that binds BCMA and CD3; and
可药用的赋形剂、稀释剂或载体。Pharmaceutically acceptable excipients, diluents or carriers.
另一方面，本发明提供了一种用途，包括本发明所述的抗BCMA抗体或其抗原结合片段、结合BCMA和CD3的双特异性抗体或其抗原结合片段，或如上所述的药物组合物用于治疗或预防BCMA介导的疾病或病症。In another aspect, the present invention provides a use, comprising the anti-BCMA antibody or antigen-binding fragment thereof, bispecific antibody or antigen-binding fragment thereof that binds BCMA and CD3, or the pharmaceutical composition as described above For the treatment or prevention of BCMA-mediated diseases or conditions.
在本发明优选的实施方案中，所述的疾病或病症为癌症；优选为表达BCMA的癌症；更优选淋巴瘤和骨髓瘤。In a preferred embodiment of the present invention, the disease or condition is cancer; preferably BCMA-expressing cancer; more preferably lymphoma and myeloma.
在本发明优选的实施方案中，所述的疾病或病症为自身免疫疾病；优选红斑狼疮，IgA肾病和风湿性关节炎。In a preferred embodiment of the present invention, the disease or condition is an autoimmune disease; preferably lupus erythematosus, IgA nephropathy and rheumatoid arthritis.
本发明提供的抗BCMA抗体或其抗原结合片段，能够特异性结合BCMA抗原和表达BCMA的细胞，靶向性强。The anti-BCMA antibody or its antigen-binding fragment provided by the present invention can specifically bind to the BCMA antigen and cells expressing BCMA, and has strong targeting ability.
本发明提供的双特异性抗体能够保持与各抗原(或表位)的亲和力，体外和体内肿瘤抑制作用明显，相较于AMG-420具有更好亲和活性的同时，体内药效延长，依从性更优。本发明的双特异性抗体的各链能够正确配对，易于表达，制备工艺简单，性质稳定，具有良好的成药性，临床应用前景广阔。The bispecific antibody provided by the present invention can maintain the affinity to each antigen (or epitope), and has obvious tumor inhibitory effects in vitro and in vivo. Compared with AMG-420, the bispecific antibody has better affinity activity and prolongs the drug effect in vivo. Better sex. The chains of the bispecific antibody of the present invention can be correctly paired, are easy to express, have simple preparation process, stable properties, good druggability, and broad clinical application prospects.
附图说明Description of the drawings
图1：双特异抗体的示意性模型，A：第一链；B：第二链。Figure 1: Schematic model of the bispecific antibody, A: first chain; B: second chain.
图2：双特异抗体的示意性模型，C：第一多肽；D：第二多肽；E：第三多肽。Figure 2: Schematic model of the bispecific antibody, C: the first polypeptide; D: the second polypeptide; E: the third polypeptide.
具体实施方式Detailed ways
术语the term
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.
本发明所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。The three-letter codes and one-letter codes of amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968).
本发明所述的术语“抗体”指免疫球蛋白，是由两条重链和两条轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。The term "antibody" in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, and γ chain. , Α chain and ε chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into a kappa chain or a lambda chain by the difference of the constant region. Each of the five types of Ig can have a kappa chain or a lambda chain.
在本发明中，本发明所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。In the present invention, the antibody light chain of the present invention may further include a light chain constant region, and the light chain constant region includes human or murine κ, λ chains or variants thereof.
在本发明中，本发明所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。In the present invention, the antibody heavy chain of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2，和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则和Kabat或ABM定义规则。The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions. The sequence from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues of the VL region and VH region of the antibody or antigen-binding fragment of the present invention comply with the known Kabat numbering rules and Kabat or ABM definition rules.
术语“BCMA”包括由细胞表达的BCMA的任何变体或同种型。本发明的抗体或其片段可与得自非人物种的BCMA交叉反应。作为另一种选择，该抗体也可以是人BCMA特异性的，可不与其他物种的BCMA交叉反应。BCMA或其任何变体或同种型可从天然表达它们的细胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。作为一个示例，抗BCMA抗体或其片段靶向具有糖基化修饰的人源BCMA。The term "BCMA" includes any variant or isoform of BCMA expressed by a cell. The antibodies of the present invention or fragments thereof can cross-react with BCMA derived from non-human species. As another option, the antibody may also be specific for human BCMA, and may not cross-react with BCMA of other species. BCMA or any variants or isoforms thereof can be isolated from the cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques commonly used in the art and those described herein. As an example, anti-BCMA antibodies or fragments thereof target human BCMA with glycosylation modification.
“特异性结合BCMA”是指本发明的抗体或其片段识别并结合BCMA或其表位。"Specifically binds to BCMA" means that the antibody or fragment thereof of the present invention recognizes and binds to BCMA or its epitope.
术语“CD3”包括由细胞表达的CD3的任何变体或同种型。本发明的抗体或其片段可与得自非人物种的CD3交叉反应。作为另一种选择，该抗体也可以是人CD3特异性的，可不与其他物种的CD3交叉反应。CD3或其任何变体或同种型可从天然表达它们的细胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。The term "CD3" includes any variant or isoform of CD3 expressed by a cell. The antibodies or fragments thereof of the present invention can cross-react with CD3 derived from non-human species. As another option, the antibody may also be specific for human CD3, and may not cross-react with CD3 of other species. CD3 or any variants or isoforms thereof can be isolated from cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques commonly used in the art and those described herein.
“特异性结合CD3”是指本发明的抗体或其片段识别并结合CD3或其表位。"Specifically binds to CD3" means that the antibody or fragment thereof of the present invention recognizes and binds to CD3 or its epitope.
术语“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及 的技术和方法在本领域中是熟知的，诸如：The term "recombinant human antibody" includes human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:
1.从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；1. Antibodies isolated from transgenes of human immunoglobulin genes, transchromosomal animals (such as mice) or hybridomas prepared therefrom;
2.从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；2. Antibodies isolated from host cells transformed to express antibodies, such as transfectomas;
3.从重组组合人抗体文库中分离的抗体；以及3. Antibodies isolated from the recombinant combinatorial human antibody library; and
4.通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。4. Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。Such recombinant human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人BCMA或其表位的单克隆抗体。制备时用BCMA抗原或其片段注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源BCMA抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，和/或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。The term "murine antibody" in the present invention refers to a monoclonal antibody to human BCMA or its epitope prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with BCMA antigen or its fragments, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated. In a preferred embodiment of the present invention, the murine BCMA antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, and/or further comprise murine IgG1 , IgG2, IgG3 or IgG4 or variants of the heavy chain constant region.
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies") .
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人物种的CDR序列移植到人的抗体可变区框架中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量异源蛋白成分，从而诱导的免疫应答反应的缺点。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行反向突变，以保持活性。The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by grafting CDR sequences of non-human species into the framework of a human antibody variable region. Humanized antibodies can overcome the shortcomings of immune responses induced by chimeric antibodies that carry a large amount of heterologous protein components. In order to avoid the decrease of immunogenicity and the decrease of activity at the same time, the variable region of the human antibody can be reversely mutated to maintain activity.
术语“嵌合抗体(chimeric antibody)”，是将第一物种抗体的可变区与第二物种抗体的恒定区融合而成的抗体，可以减轻异源抗体诱发的免疫应答反应。作为一个示例，建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变后增强ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1重链恒定区。The term "chimeric antibody" refers to an antibody formed by fusing the variable region of an antibody of the first species with the constant region of an antibody of the second species, which can reduce the immune response induced by the heterologous antibody. As an example, to establish a chimeric antibody, it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed. The mouse variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations to enhance ADCC (antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1 heavy chain constant region.
术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段 保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。抗原结合片段实例包括但不限于：Fab、Fab’、F(ab’)2、Fv片段、线性抗体(linear antibody)、单链抗体、纳米抗体、结构域抗体和多特异性抗体。工程改造的抗体变体综述于Holliger和Hudson，2005，Nat.Biotechnol.23：1126-1136中。The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which usually include at least part of the antigen-binding region or variable region (for example, one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a mole basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.
“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。The "Fab fragment" consists of the CH1 and variable regions of one light chain and one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.
“Fc”区是含有CH2和CH3结构域的两个重链片段。两个重链片段由一个或多个二硫键保持在一起。The "Fc" region is two heavy chain fragments containing CH2 and CH3 domains. The two heavy chain fragments are held together by one or more disulfide bonds.
“Fab’片段”含有一条轻链和一条重链的一部分(其包含VH结构域、CH1结构域、以及CH1和CH2结构域之间区域)；可在两个Fab’片段之间形成链间二硫键以形成F(ab’) 2分子。 "Fab'fragment" contains a light chain and a part of a heavy chain (which includes the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains); an interchain two can be formed between the two Fab' fragments Sulfur bonds to form F(ab') 2 molecules.
“Fv区”包含重链和轻链的可变区，但缺少恒定区。The "Fv region" contains the variable regions of the heavy and light chains, but lacks the constant region.
术语“单链抗体”、“单链Fv”或“scFv”是由抗体的重链可变区VH和轻链可变区VL通过一段连接肽连接而成的单链重组蛋白，它是具有完全抗原结合位点的最小抗体片段。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS(SEQ ID NO：23)氨基酸序列或其变体组成，例如使用1-4个(包括1个、2个、3个或4个)重复的变体(Holliger等人(1993)，Proc Natl Acad Sci USA.90：6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur J Immuno.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J Mol Biol.293：41-56和Roovers等人(2001)，Cancer Immunol Immunother.50：51-59.描述。The term "single-chain antibody", "single-chain Fv" or "scFv" is a single-chain recombinant protein formed by connecting the VH and VL of the heavy chain variable region of the antibody through a connecting peptide. The smallest antibody fragment at the antigen binding site. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. A suitable prior art linker consists of a repeated GGGGS (SEQ ID NO: 23) amino acid sequence or variants thereof, for example, using 1-4 (including 1, 2, 3, or 4) repeated variants ( Holliger et al. (1993), Proc Natl Acad Sci USA. 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur J Immuno. 31: 94-106, Hu et al. (1996), Cancer Res .56: 3055-3061, Kipriyanov et al. (1999), J Mol Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol Immunother. 50: 51-59. Description.
术语“结构域抗体片段”是仅含有重链可变区或轻链可变区链的具有免疫学功能的免疫球蛋白片段。在某些情况下，两个或多个VH区与肽接头共价连接以形成二价结构域抗体片段。二价结构域抗体片段的两个VH区可靶向相同或不同抗原。The term "domain antibody fragment" is an immunoglobulin fragment with immunological functions that only contains a heavy chain variable region or a light chain variable region chain. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same or different antigens.
本发明的术语“与BCMA结合”，指能与人BCMA相互作用。The term "bind with BCMA" in the present invention refers to the ability to interact with human BCMA.
本发明的术语“抗原结合位点”指本发明抗体或抗原结合片段识别的三维空间位点。The term "antigen-binding site" of the present invention refers to a three-dimensional site recognized by the antibody or antigen-binding fragment of the present invention.
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent. Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
本发明所用的术语“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用人BCMA作为分析物并使用抗体作为配体，在仪器中通过表面等 离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。 The terms "specific binding" and "selective binding" as used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when using human BCMA used as the analyte and the antibody as the ligand in the instrument by surface plasmon resonance (SPR) measurement technique, the antibody is approximately 10 -7 M or even lower than the equilibrium dissociation smaller dissociation constant ( K D ) binds to a predetermined antigen, and its binding affinity to the predetermined antigen is at least twice its binding affinity to non-specific antigens (such as BSA, etc.) other than the predetermined antigen or closely related antigens. The term "antibody that recognizes an antigen" can be used interchangeably with the term "antibody that specifically binds" herein.
“双特异性”抗体或“双功能”抗体是具有两个不同表位结合位点的杂交抗体。两个表位可以来自同一抗原，或来自不同抗原。作为一个示例，两个表位分别来自BCMA和CD3。双特异性抗体可通过公知的方法(包括但不限于杂交瘤融合或连接Fab'片段)而产生。例如参见Songsivilai等人，Clin.Exp.Immunol.,79:315-321(1990)；Kostelny等人，J.Immunol.,148:1547-1553(1992)中公开的方法。"Bispecific" antibodies or "bifunctional" antibodies are hybrid antibodies that have two different epitope binding sites. The two epitopes can be from the same antigen or from different antigens. As an example, the two epitopes are from BCMA and CD3, respectively. Bispecific antibodies can be produced by well-known methods (including but not limited to hybridoma fusion or linking Fab' fragments). See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).
术语“交叉反应”是指本发明的抗体与来自不同物种的BCMA结合的能力。例如，结合人BCMA的本发明的抗体也可以结合另一物种的BCMA。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达BCMA的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。The term "cross-reactivity" refers to the ability of the antibodies of the present invention to bind to BCMA from different species. For example, the antibody of the present invention that binds to human BCMA can also bind to BCMA of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express BCMA. Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。配体的抑制/阻断优选地降低或改变无抑制或阻断的情况下发生配体结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗BCMA抗体接触时，与未与抗BCMA抗体接触的配体相比，任何可测量的配体结合亲和力降低。The terms "inhibition" or "blocking" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with anti-BCMA antibodies compared to ligands not contacted with anti-BCMA antibodies.
术语“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。The term "inhibition of growth" (eg, in relation to cells) is intended to include any measurable decrease in cell growth.
如此处所用，术语“EC 50”或“半数最大有效浓度”是指在持续特定的暴露时间后，能够诱导50％最大反应的分子的浓度(例如，本申请的抗体或其抗原结合片段)。用于确定EC 50的方法是本领域公知的，例如可以用软件GraphPad Prism绘制浓度-效应的拟合曲线，从而计算得出EC 50。 As used herein, the term "50 EC" or "half maximal effective concentration" means the concentration after exposure for a certain time, able to induce 50% of maximal response of the molecule (e.g., an antibody of the present application or antigen binding fragment). A method for determining the EC 50 are known in the art, for example, the concentration plotted using software GraphPad Prism - effect curve fit, thus calculated EC 50.
本发明中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，增强或降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变。In the present invention, "ADCC", namely antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity, means that cells expressing Fc receptors are directly killed by recognizing the Fc segment of antibodies and are coated with antibodies. The target cell. The ADCC effect function of the antibody can be enhanced or reduced or eliminated by modifying the Fc segment of IgG. The modification refers to mutations in the constant region of the heavy chain of the antibody.
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，小鼠可以用人BCMA或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)数据库得到，或者从免疫球蛋白杂志，2001 ISBN012441351上获得。The methods of producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human BCMA or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions. The human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) database, or from the Journal of Immunoglobulin, 2001 ISBN012441351.
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。相应抗体的cDNA序列可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是 在FC区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。The engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies. The antibody-secreted culture medium can be purified and collected by conventional techniques. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
本发明的抗体指单克隆抗体。本发明所述的单克隆抗体(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术、合成技术(如CDR-grafting)、或其它现有技术进行重组得到。The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage cloned cell line. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。"Administration", "administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact. "Administration", "administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells. "Administration", "administration" and "treatment" also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的任一种抗体，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本发明的实施方案(例如治疗方法或制品)在缓解每个患都有的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。"Treatment" means administering an internal or external therapeutic agent, such as containing any one of the antibodies of the present invention, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in the treated patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Through any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated. As far as the embodiments of the present invention (such as treatment methods or products) may be ineffective in alleviating the symptoms of the target disease that each patient has, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
“有效量”包含足以改善或预防医学病症的症状的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。An "effective amount" includes an amount sufficient to improve or prevent the symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
本文使用的表述“红斑狼疮”是指其中免疫系统变得过度活跃并攻击正常组织的慢性自身免疫性疾病。这种攻击引发炎症。红斑狼疮是“非器官特异”类型的自身免疫性疾病。The expression "lupus erythematosus" as used herein refers to a chronic autoimmune disease in which the immune system becomes overactive and attacks normal tissues. This attack triggers inflammation. Lupus erythematosus is a "non-organ specific" type of autoimmune disease.
本文使用的表述“IgA肾病”以IgA沉积在肾小球膜细胞上为特征。IgA肾病属于由针对肾小球系膜细胞的免疫反应所引起的增生性肾炎。The expression "IgA nephropathy" used herein is characterized by the deposition of IgA on mesangial cells. IgA nephropathy belongs to proliferative nephritis caused by an immune response to glomerular mesangial cells.
本文使用的表述“类风湿性关节炎”是指，因免疫系统的异常而攻击自身的关节或身体部分并引起炎症的自体免疫性疾病，表现出手、脚、腕、膝等各关节的疼痛及肿胀的症状，还可能对肌肉、皮肤、肺、眼等器官造成异常的全身性的慢性炎症性疾病。The expression "rheumatoid arthritis" as used herein refers to an autoimmune disease that attacks one's own joints or body parts due to abnormalities in the immune system and causes inflammation. It shows pain and pain in various joints such as hands, feet, wrists, and knees. The symptoms of swelling may also cause abnormal systemic chronic inflammatory diseases to the muscles, skin, lungs, eyes and other organs.
本文使用的表述“淋巴瘤”是指起源于淋巴造血系统的恶性肿瘤。淋巴瘤的分类按照2016 WHO Lymphoma Classification的标准进行。The expression "lymphoma" as used herein refers to a malignant tumor that originates from the lymphoid hematopoietic system. The classification of lymphoma is carried out in accordance with the 2016 WHO Lymphoma Classification standard.
本文使用的表述“骨髓瘤”(又称浆细胞瘤)是起源于骨髓中浆细胞的恶性肿瘤。骨髓瘤的分类按照WHO(2013)Classification of Bone Tumors的标准进行。The expression "myeloma" (also known as plasmacytoma) as used herein is a malignant tumor that originates from plasma cells in the bone marrow. The classification of myeloma is carried out in accordance with the standards of WHO (2013) Classification of Bone Tumors.
“外源性”指根据背景在生物、细胞或人体外产生的物质。"Exogenous" refers to a substance that is produced outside an organism, cell, or human body according to the background.
“内源性”指根据背景在生物、细胞或人体内产生的物质。"Endogenous" refers to a substance produced in an organism, cell, or human body according to the background.
“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同一性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同一性。一般而言，当比对两个序列而得到最大的同一性百分率时进行比较。"Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position . The percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% identical. Generally speaking, the comparison is made when two sequences are aligned to obtain the maximum percent identity.
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意图指代不同的情况下，其由上下文清楚可见。As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Therefore, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where the intent is different, it is clearly visible from the context.
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。"Optional" or "optionally" means that the event or environment described later can but does not have to occur, and the description includes the occasion where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.
“药物组合物”表示含有一种或多种本文所述抗体或其抗原结合片段，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。"Pharmaceutical composition" means containing one or more antibodies or antigen-binding fragments thereof described herein, and other components such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
具体实施方式Detailed ways
以下结合实施例用于进一步描述本发明，但这些实施例并非限制着本发明的范围。本发明实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention. The experimental methods that do not specify specific conditions in the examples of the present invention usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer. The reagents without specific sources are the conventional reagents purchased on the market.
实施例1：抗原准备Example 1: Antigen preparation
编码带His标签的人BCMA(BCMA-His)蛋白由SinoBiologics公司合成(Cat No.：10620-H08H)。The human BCMA (BCMA-His) protein encoding His tag was synthesized by SinoBiologics (Cat No.: 10620-H08H).
BCMA-His序列：BCMA-His sequence:
Figure PCTCN2021088092-appb-000001
Figure PCTCN2021088092-appb-000001
实施例2：鼠杂交瘤及抗体序列的获得Example 2: Obtaining mouse hybridoma and antibody sequence
用人抗原BCMA-His进行动物免疫，共5只Balb/c和5只A/J小鼠，雌性，10周龄，使用Sigma完全弗氏佐剂(CFA)和Sigma不完全弗氏佐剂(IFA)，免疫原和免疫佐剂以1：1的比例充分混合乳化，制成稳定“油包水”液体；注射剂量25μg/200μL/小鼠。Animals were immunized with human antigen BCMA-His, a total of 5 Balb/c and 5 A/J mice, female, 10 weeks old, using Sigma Complete Freund’s Adjuvant (CFA) and Sigma Incomplete Freund’s Adjuvant (IFA) ), the immunogen and the immune adjuvant are fully mixed and emulsified at a ratio of 1:1 to make a stable "water-in-oil" liquid; the injection dose is 25μg/200μL/mouse.
表1.免疫方案Table 1. Immunization Scheme
第1天Day 1 第一次免疫，完全弗氏佐剂。The first immunization, complete Freund's adjuvant.
第21天Day 21 第二次免疫，不完全弗氏佐剂。The second immunization, incomplete Freund's adjuvant.
第35天Day 35 第三次免疫，不完全弗氏佐剂。The third immunization, incomplete Freund's adjuvant.
第42天Day 42 采血和血清效价检测(第三次免疫后收集的血液样本)Blood sampling and serum titer test (blood sample collected after the third immunization)
第49天Day 49 第四次免疫，不完全弗氏佐剂。The fourth immunization, incomplete Freund's adjuvant.
第56天Day 56 采血和血清效价检测(第四次免疫后收集的血液样本)Blood sampling and serum titer test (a blood sample collected after the fourth immunization)
对免疫小鼠血清使用如实施例3所述的间接ELISA法评估血清效价及结合细胞表面抗原的能力，对照效价检测情况(大于10万倍稀释度)进行细胞融合。选择血清效价、亲和力和FACS结合强的免疫小鼠进行一次终免疫后处死小鼠。取脾细胞和SP2/0骨髓瘤细胞融合后，获得杂交瘤；通过间接ELISA筛选到目标杂交瘤，并通过有限稀释法，将细胞株建立为单克隆细胞株。得到的阳性抗体株进一步使用间接ELISA进行筛选，从而选定结合重组蛋白的杂交瘤。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,15596-018)提取RNA并反转录(PrimeScript TM Reverse Transcriptase，Takara#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen，TB326 Rev.B 0503)进行PCR扩增后测序，最终得到鼠源抗体M1的序列。 The immunized mouse serum was evaluated by the indirect ELISA method as described in Example 3 to evaluate the serum titer and the ability to bind to cell surface antigens, and the cell fusion was performed in accordance with the titer detection situation (more than 100,000-fold dilution). The immunized mice with strong serum titer, affinity and FACS binding were selected for a final immunization and then the mice were sacrificed. After fusion of spleen cells and SP2/0 myeloma cells, hybridomas are obtained; the target hybridomas are screened by indirect ELISA, and the cell line is established as a monoclonal cell line by the limiting dilution method. The obtained positive antibody strains are further screened using indirect ELISA to select hybridomas that bind the recombinant protein. The logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara #2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced, and finally the sequence of the mouse antibody M1 was obtained.
鼠单抗M1的重链和轻链可变区序列如下：The heavy chain and light chain variable region sequences of murine monoclonal antibody M1 are as follows:
M1 HCVRM1 HCVR
Figure PCTCN2021088092-appb-000002
Figure PCTCN2021088092-appb-000002
M1 LCVRM1 LCVR
Figure PCTCN2021088092-appb-000003
Figure PCTCN2021088092-appb-000003
表2.鼠单抗M1的重链和轻链可变区CDR序列Table 2. Murine monoclonal antibody M1 heavy chain and light chain variable region CDR sequences
名称name 序列sequence 编号serial number
HCDR1HCDR1 GYTFTNYVMHGYTFTNYVMH SEQ ID NO：3SEQ ID NO: 3
HCDR2HCDR2 YIIPYNDGTKYNEKFKGKAYIIPYNDGTKYNEKFKGKA SEQ ID NO：4SEQ ID NO: 4
HCDR3HCDR3 TRLIFDGYYFDYTRLIFDGYYFDY SEQ ID NO：5SEQ ID NO: 5
LCDR1LCDR1 RASKSVSTSGFSYMHRASKSVSTSGFSYMH SEQ ID NO：6SEQ ID NO: 6
LCDR2LCDR2 SLASNLESSLASNLES SEQ ID NO：7SEQ ID NO: 7
LCDR3LCDR3 QHSRELPWTQHSRELPWT SEQ ID NO：8SEQ ID NO: 8
实施例3：抗体的体外结合活性检测方法Example 3: In vitro binding activity detection method of antibody
1.体外间接ELISA结合实验：1. In vitro indirect ELISA binding experiment:
用pH7.4的PBS将BCMA His蛋白(Sino Biological Inc.，cat#10428-H08H)稀释至1μg/ml浓度，以100μl/孔的体积加入96孔高亲和力酶标板中，于4℃冰箱孵育过夜(16-20小时)。用PBST(pH7.4PBS含0.05％Tween-20)洗板4次后，加入用PBST稀释的3％牛血清白蛋白(BSA)封闭液150μl/孔，室温孵育1小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板4次。Dilute BCMA His protein (Sino Biological Inc., cat#10428-H08H) with pH7.4 PBS to a concentration of 1μg/ml, add 100μl/well to a 96-well high-affinity ELISA plate, and incubate in a refrigerator at 4°C Overnight (16-20 hours). After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), add 150 μl/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate for 1 hour at room temperature for blocking. After the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer.
用含3％BSA的PBST稀释待测抗体；以1μM起始，按照10倍的梯度进行稀释，而获得10个浓度梯度；以100μl/孔加到酶标板中，放于室温孵育1小时。孵育结束后用PBST洗板4次，加入100μl/孔用含3％BSA的PBST稀释的HRP标记的羊抗人二抗(Abcam，cat#ab97225)，室温孵育1小时。用PBST洗板4次后，加入100μl/孔TMB显色底物(Cell Signaling Technology，cat#7004S)，于室温避光孵育1分钟，加入100μl/孔终止液(Cell Signaling Technology，cat#7002S)终止反应；用酶标仪(BioTek，型号Synergy H1)在450nm处读取吸收值，分析数据。做浓度-信号值曲线，以分析结果。如下表所示：Dilute the antibody to be tested with PBST containing 3% BSA; start with 1 μM and dilute with a 10-fold gradient to obtain 10 concentration gradients; add 100 μl/well to the microtiter plate and incubate at room temperature for 1 hour. After the incubation, the plate was washed 4 times with PBST, 100 μl/well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted with 3% BSA in PBST was added, and incubated for 1 hour at room temperature. After washing the plate 4 times with PBST, add 100μl/well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature and dark for 1 minute, add 100μl/well of stop solution (Cell Signaling Technology, cat#7002S) Terminate the reaction; use a microplate reader (BioTek, Model Synergy H1) to read the absorbance value at 450nm and analyze the data. Make a concentration-signal value curve to analyze the results. As shown in the following table:
表3.鼠抗体对人BCMA抗原的亲和力(EC 50值) Table 3. Affinity of mouse antibodies to human BCMA antigen (EC 50 value)
鼠抗体Mouse antibody 与人BCMA His抗原的结合EC 50(nM) Binding EC 50 with human BCMA His antigen (nM)
M1M1 0.1110.111
2.体外细胞结合实验：2. In vitro cell binding experiment:
收集高表达BCMA的细胞(过表达BCMA的HEK-293T细胞和表达BCMA的肿瘤细胞，NCI-H929骨髓瘤细胞系)；调节细胞密度后，将细胞分铺于96孔U底板，每孔1×10 5至2×10 5个细胞。以1200g离心5min，去上清；添加100μl稀释的抗体溶液或小鼠免疫血清，4℃度孵育60min；以1200g离心5min，去上清；PBS洗细胞2次后，添加荧光标记二抗(PE-GAM或PE-GAH)100μl每孔，4℃度孵育60min。以1200g离心5min，去上清。细胞用PBS洗2次后，重悬于PBS；使用流式细胞计数仪检测信号，并作浓度曲线，以分析结果。 Collect cells with high BCMA expression (HEK-293T cells overexpressing BCMA and tumor cells expressing BCMA, NCI-H929 myeloma cell line); after adjusting the cell density, spread the cells on a 96-well U bottom plate, 1× per well 10 5 to 2×10 5 cells. Centrifuge at 1200g for 5min and remove the supernatant; add 100μl of diluted antibody solution or mouse immune serum and incubate at 4°C for 60min; centrifuge at 1200g for 5min to remove the supernatant; after washing the cells twice with PBS, add a fluorescently labeled secondary antibody (PE -GAM or PE-GAH) 100μl per well, incubate at 4°C for 60min. Centrifuge at 1200g for 5 min and remove the supernatant. After the cells were washed twice with PBS, they were resuspended in PBS; the signal was detected by a flow cytometer and the concentration curve was drawn to analyze the results.
表4.鼠抗体对表达BCMA的细胞的亲和力(EC 50值) Table 4. Affinity (EC 50 value) of murine antibodies to cells expressing BCMA
Figure PCTCN2021088092-appb-000004
Figure PCTCN2021088092-appb-000004
实施例4：小鼠抗体的人源化Example 4: Humanization of mouse antibodies
如本领域许多文献公示的方法进行鼠源抗人BCMA单克隆抗体的人源化。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种抗体序列。本发明将鼠源抗体M1进行人源化。Humanization of murine anti-human BCMA monoclonal antibodies is carried out as disclosed in many documents in the field. In short, a human constant domain is used instead of the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the murine antibody and the human antibody. The present invention humanizes the murine antibody M1.
在所获得的鼠源抗体VH/VL CDR典型结构的基础上，将重、轻链可变区序列与人源抗体种系数据库比较，获得同源性高的人种系模板。On the basis of the obtained typical structure of mouse antibody VH/VL CDR, the heavy and light chain variable region sequences are compared with the human antibody germline database to obtain a human germline template with high homology.
将鼠源抗体M1的CDR区移植到选择的人源化模板上。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并对CDR区化学不稳定氨基酸残基优化。序列如下：The CDR regions of the murine antibody M1 were grafted onto the selected humanized template. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH are back-mutated, and the CDR region Optimization of chemically unstable amino acid residues. The sequence is as follows:
表5.CDR2区突变Table 5. Mutations in the CDR2 region
名称name 序列sequence 编号serial number
HCDR1HCDR1 GYTFTNYVMHGYTFTNYVMH SEQ ID NO：3SEQ ID NO: 3
HCDR2HCDR2 YIIPYNDGTKYNEKFKGRVYIIPYNDGTKYNEKFKGRV SEQ ID NO：9SEQ ID NO: 9
HCDR3HCDR3 TRLIFDGYYFDYTRLIFDGYYFDY SEQ ID NO：5SEQ ID NO: 5
LCDR1LCDR1 RASKSVSTSGFSYMHRASKSVSTSGFSYMH SEQ ID NO：6SEQ ID NO: 6
LCDR2LCDR2 SLASNLESSLASNLES SEQ ID NO：7SEQ ID NO: 7
LCDR3LCDR3 QHSRELPWTQHSRELPWT SEQ ID NO：8SEQ ID NO: 8
表6.CDR2区突变Table 6. Mutations in the CDR2 region
名称name 序列sequence 编号serial number
HCDR1HCDR1 GYTFTNYVMHGYTFTNYVMH SEQ ID NO：3SEQ ID NO: 3
HCDR2HCDR2 YIIPYNDGTKYNEKFKGKAYIIPYNDGTKYNEKFKGKA SEQ ID NO：4SEQ ID NO: 4
HCDR3HCDR3 TRLIFDGYYFDYTRLIFDGYYFDY SEQ ID NO：5SEQ ID NO: 5
LCDR1LCDR1 RASKSVSTSGFSYMHRASKSVSTSGFSYMH SEQ ID NO：6SEQ ID NO: 6
LCDR2LCDR2 YLASNLESYLASNLES SEQ ID NO：10SEQ ID NO: 10
LCDR3LCDR3 QHSRELPWTQHSRELPWT SEQ ID NO：8SEQ ID NO: 8
经表达测试和回复突变数量的对比，选择出人源化重链可变区HCVR的序列，序列如下：After the expression test and the comparison of the number of back mutations, the sequence of the humanized heavy chain variable region HCVR was selected. The sequence is as follows:
HCVR1HCVR1
Figure PCTCN2021088092-appb-000005
Figure PCTCN2021088092-appb-000005
HCVR2HCVR2
Figure PCTCN2021088092-appb-000006
Figure PCTCN2021088092-appb-000006
HCVR3HCVR3
Figure PCTCN2021088092-appb-000007
Figure PCTCN2021088092-appb-000007
选择出人源化轻链可变区LCVR的序列，序列如下：The sequence of the humanized light chain variable region LCVR was selected, and the sequence is as follows:
LCVR1LCVR1
Figure PCTCN2021088092-appb-000008
Figure PCTCN2021088092-appb-000008
LCVR2LCVR2
Figure PCTCN2021088092-appb-000009
Figure PCTCN2021088092-appb-000009
LCVR3LCVR3
Figure PCTCN2021088092-appb-000010
Figure PCTCN2021088092-appb-000010
将设计的重链和轻链可变区序列分别与人抗体的重链恒定区和轻链恒定区序列连接。示例性地，与人IgG1重链恒定区(SEQ ID NO：31)、人IgG1重链恒定区变体(SEQ ID NO：42)和人抗体κ链恒定区序列(SEQ ID NO：32)连接。示例性得到重链和轻链序列如下：The designed heavy chain and light chain variable region sequences are respectively connected with the heavy chain constant region and light chain constant region sequences of the human antibody. Exemplarily, it is connected to the human IgG1 heavy chain constant region (SEQ ID NO: 31), the human IgG1 heavy chain constant region variant (SEQ ID NO: 42) and the human antibody kappa chain constant region sequence (SEQ ID NO: 32) . Exemplary obtained heavy chain and light chain sequences are as follows:
Ab1 HCAb1 HC
Figure PCTCN2021088092-appb-000011
Figure PCTCN2021088092-appb-000011
Ab2 HCAb2 HC
Figure PCTCN2021088092-appb-000012
Figure PCTCN2021088092-appb-000012
Figure PCTCN2021088092-appb-000013
Figure PCTCN2021088092-appb-000013
Ab3 HCAb3 HC
Figure PCTCN2021088092-appb-000014
Figure PCTCN2021088092-appb-000014
Ab1-1 HCAb1-1 HC
Figure PCTCN2021088092-appb-000015
Figure PCTCN2021088092-appb-000015
Ab1-2 HCAb1-2 HC
Figure PCTCN2021088092-appb-000016
Figure PCTCN2021088092-appb-000016
Ab1 LCAb1 LC
Figure PCTCN2021088092-appb-000017
Figure PCTCN2021088092-appb-000017
Figure PCTCN2021088092-appb-000018
Figure PCTCN2021088092-appb-000018
Ab2 LCAb2 LC
Figure PCTCN2021088092-appb-000019
Figure PCTCN2021088092-appb-000019
Ab3 LCAb3 LC
Figure PCTCN2021088092-appb-000020
Figure PCTCN2021088092-appb-000020
Ab1-1 LCAb1-1 LC
Figure PCTCN2021088092-appb-000021
Figure PCTCN2021088092-appb-000021
Ab1-2 LCAb1-2 LC
Figure PCTCN2021088092-appb-000022
Figure PCTCN2021088092-appb-000022
示例性的恒定区序列如下所示：An exemplary constant region sequence is shown below:
人IgG1重链恒定区Human IgG1 heavy chain constant region
Figure PCTCN2021088092-appb-000023
Figure PCTCN2021088092-appb-000023
人IgG1重链恒定区变体Human IgG1 heavy chain constant region variants
Figure PCTCN2021088092-appb-000024
Figure PCTCN2021088092-appb-000024
Figure PCTCN2021088092-appb-000025
Figure PCTCN2021088092-appb-000025
人抗体κ链恒定区Human antibody kappa chain constant region
Figure PCTCN2021088092-appb-000026
Figure PCTCN2021088092-appb-000026
表7.重链、轻链、可变区的序列编号Table 7. Sequence numbering of heavy chain, light chain and variable region
Figure PCTCN2021088092-appb-000027
Figure PCTCN2021088092-appb-000027
根据以上各人源化抗体的轻链和重链的氨基酸序列合成cDNA片段，插入到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences，Inc.Cat.No.23966)以1：2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。收取细胞培养液，离心过滤后，上样到抗体纯化亲和柱，经磷酸缓冲液洗柱、甘氨酸盐酸缓冲液(pH2.7 0.1M Gly-HCl)洗脱、1M Tris盐酸pH 9.0中和、以及磷酸缓冲液透析，得到本发明的人源化抗体。 CDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of the above humanized antibodies, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days. Collect the cell culture fluid, after centrifugal filtration, load the sample to the antibody purification affinity column, wash the column with phosphate buffer, elution with glycine hydrochloric acid buffer (pH 2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH 9.0, And phosphate buffer dialysis to obtain the humanized antibody of the present invention.
实施例5：体外结合亲和力和动力学实验Example 5: In vitro binding affinity and kinetic experiments
使用实施例3的体外间接ELISA结合实验，测定各人源化抗体对人BCMA抗原的亲和力(EC 50)，如下表所示： Using the in vitro indirect ELISA binding experiment of Example 3, the affinity (EC 50 ) of each humanized antibody to the human BCMA antigen was determined, as shown in the following table:
表8.人源化抗体对人BCMA抗原的亲和力(EC 50) Table 8. The humanized antibody to human BCMA antigen affinity (EC 50)
Figure PCTCN2021088092-appb-000028
Figure PCTCN2021088092-appb-000028
使用实施例3的体外细胞结合实验，测定各人源化抗体对NCI-H929肿瘤细胞的亲和 力(EC 50)，如下表所示： Using the in vitro cell binding experiment of Example 3, the affinity (EC 50 ) of each humanized antibody to NCI-H929 tumor cells was determined, as shown in the following table:
表9.人源化抗体对NCI-H929肿瘤细胞的亲和力(EC 50) Table 9. The affinity of humanized antibody NCI-H929 tumor cells (EC 50)
Figure PCTCN2021088092-appb-000029
Figure PCTCN2021088092-appb-000029
实施例6：双特异性抗体的构建Example 6: Construction of bispecific antibodies
特异性结合CD3的抗原结合片段：将如下序列所示的CD3HCVR和CD3LCVR，通过连接序列连接成单链片段；连接序列是本领域内公知的接头，示例性的接头可以是(GGGGS) n，n选自1、2、3、4或5。 An antigen-binding fragment that specifically binds to CD3: The CD3HCVR and CD3LCVR shown in the following sequence are connected into a single-stranded fragment through a linking sequence; the linking sequence is a well-known linker in the art, and an exemplary linker can be (GGGGS) n , n Selected from 1, 2, 3, 4, or 5.
CD3 HCVRCD3 HCVR
Figure PCTCN2021088092-appb-000030
Figure PCTCN2021088092-appb-000030
CD3 LCVRCD3 LCVR
Figure PCTCN2021088092-appb-000031
Figure PCTCN2021088092-appb-000031
CD3 scFvCD3 scFv
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSS GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL(其中，下划线部分代表接头) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSS GGGGSGGGGSGGGGS QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL (wherein the underlined portions represent linker)
                                       SEQ ID NO：41。SEQ ID NO: 41.
抗CD3抗体或其抗原结合片段的CDR序列如下：The CDR sequence of the anti-CD3 antibody or its antigen-binding fragment is as follows:
表10.CDR序列Table 10. CDR sequences
名称name 序列sequence 编号serial number
HCDR1HCDR1 KYAMNKYAMN SEQ ID NO：35SEQ ID NO: 35
HCDR2HCDR2 RIRSKYNNYATYYADSVKDRIRSKYNNYATYYADSVKD SEQ ID NO：36SEQ ID NO: 36
HCDR3HCDR3 HGNFGNEYISYWAYHGNFGNEYISYWAY SEQ ID NO：37SEQ ID NO: 37
LCDR1LCDR1 GSSTGAVTSGNYPNGSSTGAVTSGNYPN SEQ ID NO：38SEQ ID NO: 38
LCDR2LCDR2 GTKFLAPGTKFLAP SEQ ID NO：39SEQ ID NO: 39
LCDR3LCDR3 VLWYSNRWVVLWYSNRWV SEQ ID NO：40SEQ ID NO: 40
将特异性结合BCMA的抗原结合片段(第一结合区)与特异性结合CD3的抗原结合片段(第二结合区)通过不同的方式连接，获得含有如下序列的双特异性抗体：The antigen-binding fragment (first binding region) that specifically binds to BCMA and the antigen-binding fragment (second binding region) that specifically bind to CD3 are connected in different ways to obtain a bispecific antibody containing the following sequence:
Ab4抗体的构建如下(排列顺序从N端到C端)(结构如图1所示)：The construction of Ab4 antibody is as follows (arrangement order from N-terminal to C-terminal) (the structure is shown in Figure 1):
第一链，第一结合区的重链可变区和重链恒定区；The first chain, the heavy chain variable region and the heavy chain constant region of the first binding region;
第二链，第一结合区的轻链可变区、轻链恒定区和肽接头，以及第二结合区的重链可变区、肽接头和轻链可变区。The second chain, the light chain variable region, light chain constant region and peptide linker of the first binding region, and the heavy chain variable region, peptide linker and light chain variable region of the second binding region.
Ab5抗体的构建如下(排列顺序从N端到C端)(结构如图2所示)：The construction of Ab5 antibody is as follows (arrangement order from N-terminal to C-terminal) (the structure is shown in Figure 2):
第一多肽，从N端到C端包含第一结合区的重链可变区和重链恒定区；The first polypeptide includes the heavy chain variable region and the heavy chain constant region of the first binding region from the N-terminus to the C-terminus;
第二多肽，从N端到C端包含第一结合区的轻链可变区和轻链恒定区；The second polypeptide includes the light chain variable region and the light chain constant region of the first binding region from the N-terminus to the C-terminus;
第三多肽，从N端到C端包含第二结合区的重链可变区、肽接头、第二结合区的轻链可变区、肽接头和重链恒定区。The third polypeptide includes the heavy chain variable region of the second binding region, the peptide linker, the light chain variable region of the second binding region, the peptide linker and the heavy chain constant region from the N-terminus to the C-terminus.
以上构建体中，肽接头为用于连接抗原结合结构域的肽接头，示例性的接头序列可以是(GGGGS)n，n选自1、2、3、4或5。In the above construct, the peptide linker is a peptide linker used to connect the antigen binding domain, and an exemplary linker sequence may be (GGGGS)n, where n is selected from 1, 2, 3, 4, or 5.
以上构建体中，重链恒定区可以选自人抗体重链恒定区(如人IgG1重链恒定区)或其变体(如降低ADCC活性的变体)。重链恒定区可以为相同序列的恒定区(或恒定区结构域)，也可以为包含杵(Knob)和臼(Hole)空间结构的恒定区(或恒定区结构域)，还可以在包含Knob和Hole恒定区(或恒定区结构域)序列的基础上，进一步引入二硫键以促进二聚体的形成。In the above constructs, the heavy chain constant region can be selected from a human antibody heavy chain constant region (such as a human IgG1 heavy chain constant region) or a variant thereof (such as a variant that reduces ADCC activity). The constant region of the heavy chain can be a constant region (or a constant region domain) of the same sequence, or a constant region (or a constant region domain) containing a Knob and a hole (Hole) spatial structure. Based on the sequence of the Hole constant region (or constant region domain), disulfide bonds are further introduced to promote the formation of dimers.
本发明示例性的双特异性抗体包含的重链恒定区序列如下：The sequence of the heavy chain constant region contained in the exemplary bispecific antibody of the present invention is as follows:
CH Knob CH Knob
Figure PCTCN2021088092-appb-000032
Figure PCTCN2021088092-appb-000032
CH Hole CH Hole
Figure PCTCN2021088092-appb-000033
Figure PCTCN2021088092-appb-000033
构建得到的双特异性抗体的序列如下：The sequence of the constructed bispecific antibody is as follows:
Ab4-1Ab4-1
Figure PCTCN2021088092-appb-000034
Figure PCTCN2021088092-appb-000034
Figure PCTCN2021088092-appb-000035
Figure PCTCN2021088092-appb-000035
Ab4-2Ab4-2
EIVLTQSPATLSLSPGERATLSCRASKSVSTSGFSYMHWYQQKPGQAPRLLIYLASNLESGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQHSRELPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGS EVQLVESGGGLVQP GGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVL(斜体是Ab1 LC；下划线是CD3 scFv) EIVLTQSPATLSLSPGERATLSCRASKSVSTSGFSYMHWYQQKPGQAPRLLIYLASNLESGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQHSRELPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGS EVQLVESGGGLVQP GGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYWAYWGQGTLVTVSSG GGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVL (italics Ab1 LC; is underlined CD3 scFv)
                                 SEQ ID NO：27。SEQ ID NO: 27.
Ab5-1Ab5-1
Figure PCTCN2021088092-appb-000036
Figure PCTCN2021088092-appb-000036
Ab5-3Ab5-3
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYN NYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYW AYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAV TSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNRWVFGGGTKLTVLGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(下划线是CD3 scFv；斜体是CH Hole) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYN NYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNEYISYW AYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAV TSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNRWVFGGGTKLTVL GGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (underlined is CD3 scFv; italics CH Hole)
                                   SEQ ID NO：29。SEQ ID NO: 29.
Ab5-2Ab5-2
Figure PCTCN2021088092-appb-000037
Figure PCTCN2021088092-appb-000037
Figure PCTCN2021088092-appb-000038
Figure PCTCN2021088092-appb-000038
表11.抗体及其重链、轻链、可变区的序列编号Table 11. Sequence numbers of antibodies and their heavy chains, light chains and variable regions
Figure PCTCN2021088092-appb-000039
Figure PCTCN2021088092-appb-000039
根据以上双特异性抗体氨基酸序列合成cDNA片段，插入到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences，Inc.Cat.No.23966)以1：2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。收取细胞培养液，离心过滤后上样到抗体纯化亲和柱，经磷酸缓冲液洗柱、甘氨酸盐酸缓冲液(pH2.7 0.1M Gly-HCl)洗脱、1M Tris盐酸pH 9.0中和、以及磷酸缓冲液透析，得到本发明的双特异性抗体。制备得到的双特异性抗体浓度与SEC纯度如下： A cDNA fragment was synthesized based on the amino acid sequence of the above bispecific antibody and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days. Collect the cell culture fluid, centrifuge and filter, load the sample to the antibody purification affinity column, wash the column with phosphate buffer, elution with glycine hydrochloric acid buffer (pH 2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH 9.0, and The phosphate buffer solution was dialyzed to obtain the bispecific antibody of the present invention. The prepared bispecific antibody concentration and SEC purity are as follows:
表12.双特异性抗体的浓度与SEC纯度Table 12. Concentration and SEC purity of bispecific antibodies
双特异性抗体Bispecific antibody 浓度(mg/ml)Concentration (mg/ml) SEC纯度SEC purity
Ab4Ab4 0.5060.506 100％100%
Ab5Ab5 2.332.33 90.12％90.12%
实施例7：体外结合亲和力实验Example 7: In vitro binding affinity experiment
使用实施例3的体外间接ELISA结合实验，测定双特异性抗体对人BCMA抗原的亲和力(EC 50)，如下表13所示： Using the in vitro indirect ELISA binding experiment of Example 3, the affinity (EC 50 ) of the bispecific antibody to the human BCMA antigen was determined, as shown in Table 13 below:
表13.双特异性抗体对人BCMA抗原的亲和力(EC 50) Table 13. bispecific antibody affinity for antigen, human BCMA (EC 50)
Figure PCTCN2021088092-appb-000040
Figure PCTCN2021088092-appb-000040
用pH7.4的PBS将CD3D/CD3E异二聚体蛋白(Acrobiosystems，cat#CDD-H82W0)稀释至1μg/ml浓度，以100μl/孔的体积加入96孔高亲和力酶标板中，于4℃冰箱孵育过夜(16-20小时)。用PBST(pH7.4 PBS含0.05％Tween-20)洗板4次后，加入用PBST稀释的3％牛血清白蛋白(BSA)封闭液150μl/孔，室温孵育1小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板4次。用含3％BSA的PBST稀释待测抗体；以1μM起始，按照10倍的梯度进行稀释，而获得10个浓度梯度；以100μl/孔加到酶标板中，放于室温孵育1小时。孵育结束后用PBST洗板4次，加入100μl/孔用含3％BSA的PBST稀释的HRP标记的羊抗人二抗(Abcam，cat#ab97225)，室温孵育1小时。用PBST洗板4次后，加入100μl/孔TMB显色底物(Cell Signaling Technology，cat#7004S)，于室温避光孵育1分钟，加入100μl/孔终止液(Cell Signaling Technology，cat#7002S)终止反应，用酶标仪(BioTek，型号Synergy H1)在450nm处读取吸收值，分析数据。做浓度信号值曲线，以分析结果，如下表所示：Dilute the CD3D/CD3E heterodimer protein (Acrobiosystems, cat#CDD-H82W0) to a concentration of 1μg/ml with pH7.4 PBS, add 100μl/well to a 96-well high-affinity ELISA plate at 4°C Incubate overnight in the refrigerator (16-20 hours). After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), add 150 μl/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate for 1 hour at room temperature for blocking. After the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer. Dilute the antibody to be tested with PBST containing 3% BSA; start with 1 μM and dilute with a 10-fold gradient to obtain 10 concentration gradients; add 100 μl/well to the microtiter plate and incubate at room temperature for 1 hour. After the incubation, the plate was washed 4 times with PBST, 100 μl/well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted with 3% BSA in PBST was added, and incubated for 1 hour at room temperature. After washing the plate 4 times with PBST, add 100μl/well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature and dark for 1 minute, add 100μl/well of stop solution (Cell Signaling Technology, cat#7002S) The reaction was terminated, and the absorbance value was read at 450nm with a microplate reader (BioTek, model Synergy H1), and the data was analyzed. Make a concentration signal value curve to analyze the results, as shown in the following table:
表14.双特异性抗体对人CD3的亲和力(EC 50) Table 14. The affinity of the bispecific antibodies to human CD3 (EC 50)
Figure PCTCN2021088092-appb-000041
Figure PCTCN2021088092-appb-000041
实施例8：双特异性抗体介导的肿瘤细胞杀伤作用Example 8: Tumor cell killing effect mediated by bispecific antibody
双特异性抗体的BCMA结合部分结合于表达BCMA的肿瘤细胞，CD3结合部分结合于效应T细胞表面的TCR受体，在双特异性抗体引导下形成免疫突触，T细胞通过颗粒酶，穿孔素等一系列细胞因子将肿瘤细胞裂解杀死。The BCMA binding part of the bispecific antibody binds to tumor cells expressing BCMA, and the CD3 binding part binds to the TCR receptor on the surface of effector T cells, and forms an immune synapse under the guidance of the bispecific antibody. T cells pass through granzyme and perforin. A series of cytokines will lyse and kill tumor cells.
将冻存的PBMC于37℃水浴迅速融化，转移细胞悬液至细胞完全培养基(90％RPMI-1640含有10％FBS，100U/100ug/mL青霉素/链霉素，2mM谷氨酰胺)，重悬细胞，于室温2000rpm/min离心5min，弃上清；加入新鲜完全培养基，重悬细胞沉淀；计数，调整细胞密度至1.5x10 5/mL，利用EasySep Human T Cell Iso Kit试剂盒(Stemcell,cat#：17951)分离人总T细胞群；于全透明96-孔U-型底板(Corning，Cat#：3799)中加入33.3μl T细胞悬液5x10 3细胞/孔。将NCI-H929-LUC(Cobioer货号CBP30061L)悬浮于细胞完全培养基，计数；调整细胞密度至1.5x10 6/mL。加入33.3μl NCI-H929-LUC悬液5x10 4/孔于含有人总T细胞群实验板内。将双特异性抗体用完全培养基从最高浓度3000nM，5倍稀释，9个浓度梯度。将各浓度的抗体悬液以33.3μl/孔加入实验板中，并混匀细胞，于37℃，5％CO2培养箱内共孵育48小时后，将实验板置于室温平衡10分钟；同时将分装的ONE-Glo TM Luciferase Assay(Promega，Cat#：E6120)检测试剂以100μl/孔加入实验板中孵育5分钟，将细胞裂解液转移至实验板(Corning,Cat#：3610)中(180μl/孔)；利用酶标仪(Bio-Tek,Synergy H1)读取信号值。 The frozen PBMC was quickly thawed in a 37°C water bath, and the cell suspension was transferred to complete cell culture medium (90% RPMI-1640 containing 10% FBS, 100U/100ug/mL penicillin/streptomycin, 2mM glutamine), Suspend the cells, centrifuge at 2000rpm/min for 5min at room temperature, discard the supernatant; add fresh complete medium, resuspend the cell pellet; count, adjust the cell density to 1.5x10 5 /mL, use the EasySep Human T Cell Iso Kit (Stemcell, cat#: 17951) Isolate human total T cell population; add 33.3 μl T cell suspension 5×10 3 cells/well to a fully transparent 96-well U-shaped bottom plate (Corning, Cat#: 3799). Suspend NCI-H929-LUC (Cobioer catalog number CBP30061L) in cell complete medium and count; adjust the cell density to 1.5x10 6 /mL. Add 33.3μl of NCI-H929-LUC suspension 5x10 4 /well to the experimental plate containing human total T cell population. Dilute the bispecific antibody in complete medium from the highest concentration of 3000 nM, 5-fold dilution, and 9 concentration gradients. Add 33.3μl/well of antibody suspension to the experiment plate, and mix the cells. After incubating for 48 hours in a 37°C, 5% CO2 incubator, put the experiment plate at room temperature to equilibrate for 10 minutes; The aliquoted ONE-Glo TM Luciferase Assay (Promega, Cat#: E6120) detection reagent was added to the experimental plate at 100 μl/well and incubated for 5 minutes. The cell lysate was transferred to the experimental plate (Corning, Cat#: 3610) (180 μl). /Well); Use a microplate reader (Bio-Tek, Synergy H1) to read the signal value.
按照如下公式计算杀伤效率：Calculate the killing efficiency according to the following formula:
细胞杀伤效率％＝(1-实验组的信号值/对照组的信号值)×100％。Cell killing efficiency%=(1-signal value of experimental group/signal value of control group)×100%.
利用GraphPad Prism软件，通过杀伤效率值计算EC 50值，如下表15所示： Using GraphPad Prism software, calculate the EC 50 value through the killing efficiency value, as shown in Table 15 below:
表15.双特异性抗体介导T细胞对NCI-H929-LUC细胞的杀伤(EC 50) Table 15. Bispecific antibody-mediated T cell NCI-H929-LUC cell killing (EC 50)
Figure PCTCN2021088092-appb-000042
Figure PCTCN2021088092-appb-000042
实施例9：双特异性抗体介导的肿瘤杀伤Example 9: Bispecific antibody-mediated tumor killing
NCG小鼠是一种缺少T、B、NK免疫细胞的免疫缺陷小鼠。选取8-9周龄，体重为18-22g的NCG雌性小鼠。通过腹腔注射，在每只小鼠体内注入4x10 6人PBMC细胞。于第3天将1x10 7NCI-H929细胞注入小鼠背部皮下。待肿瘤体积生长至170mm 3左右，将小鼠分为3组，每组8只。通过腹腔注射的方式按照20μg/kg体重的剂量每3天一次给予小鼠IgG1对照抗体或双特异性抗体。14天后测量每只小鼠的瘤体积并计算肿瘤抑制率TGI如下表。 NCG mice are immunodeficient mice lacking T, B, and NK immune cells. Select 8-9 weeks old NCG female mice weighing 18-22 g. By intraperitoneal injection, 4× 10 6 human PBMC cells were injected into each mouse. On the 3rd day, 1 ×10 7 NCI-H929 cells were injected into the back of the mouse subcutaneously. When the tumor volume grows to about 170 mm 3 , the mice are divided into 3 groups, 8 mice in each group. The mouse IgG1 control antibody or bispecific antibody was administered by intraperitoneal injection at a dose of 20 μg/kg body weight once every 3 days. After 14 days, the tumor volume of each mouse was measured and the tumor inhibition rate TGI was calculated as shown in the table below.
抑瘤率TGI＝100％-(第14天给药组的肿瘤体积-第0天给药组的肿瘤体积)/(第14天对照组的肿瘤体积-第0天对照组的肿瘤体积)×100％。Tumor inhibition rate TGI=100%-(tumor volume of the administration group on day 14-tumor volume of the administration group on day 0)/(tumor volume of the control group on day 14-tumor volume of the control group on day 0)× 100%.
表16.双特异性抗体对肿瘤生长的抑制Table 16. Inhibition of tumor growth by bispecific antibodies
给药组G 抑瘤率TGITumor inhibition rate TGI
Ab4Ab4 90％90%
Ab5Ab5 20％20%
选取8-9周龄，体重为18-22g的NCG雌性小鼠。通过腹腔注射，在每只小鼠体内注入5x10 6人PBMC细胞,同时将1x10 7RPMI-8226细胞注入小鼠背部皮下。待肿瘤体积生长至95mm3左右，将小鼠分为3组，每组8只。通过腹腔注射的方式按照20μg/kg体重的剂量每3天一次给予小鼠IgG1对照抗体或双特异性抗体。21天后测量每只小鼠的瘤体积并计算肿瘤抑制率TGI如下表。 Select 8-9 weeks old NCG female mice weighing 18-22 g. Through intraperitoneal injection, 5× 10 6 human PBMC cells were injected into each mouse, and 1×10 7 RPMI-8226 cells were injected into the subcutaneous back of the mouse. After the tumor volume grows to about 95mm3, the mice are divided into 3 groups with 8 mice in each group. The mouse IgG1 control antibody or bispecific antibody was administered by intraperitoneal injection at a dose of 20 μg/kg body weight once every 3 days. After 21 days, the tumor volume of each mouse was measured and the tumor inhibition rate TGI was calculated as shown in the table below.
抑瘤率TGI＝100％-(第21天给药组的肿瘤体积-第0天给药组的肿瘤体积)/(第21天对照组的肿瘤体积-第0天对照组的肿瘤体积)×100％。Tumor inhibition rate TGI=100%-(tumor volume of the administration group on day 21-tumor volume of the administration group on day 0)/(tumor volume of the control group on day 21-tumor volume of the control group on day 0)× 100%.
表17.双特异性抗体对肿瘤生长的抑制Table 17. Inhibition of tumor growth by bispecific antibodies
给药组G 抑瘤率TGITumor inhibition rate TGI
Ab4Ab4 46％46%
Ab5Ab5 22％twenty two%
实施例10：BCMA/CD3双特异性抗体在细胞水平的亲和力测试Example 10: Affinity test of BCMA/CD3 bispecific antibody at the cellular level
本实施例的目的在于评价候选BCMA/CD3双特异性抗体的BCMA结合部分对肿瘤细胞表面的BCMA抗原在亲和水平方面的差异。The purpose of this example is to evaluate the difference in the affinity of the BCMA binding part of the candidate BCMA/CD3 bispecific antibody to the BCMA antigen on the surface of tumor cells.
将处于对数生长期的NCI-H929(ATCC，
Figure PCTCN2021088092-appb-000043
CRL-9068 TM)细胞悬液以300g，离心5min；加入5ml 2％FBS缓冲液重悬细胞，调整细胞密度为1x10 6细胞/ml。于100μL/孔均分至96孔V型底板，300g×5min，4℃离心，弃上清，于100μL/孔加入10个浓度梯度(2500nM至0.064nM，5倍稀释梯度)稀释的候选抗体溶液，4℃孵育1h；300g×5min，4℃离心，洗涤2次，于100μL/孔加入以5μL/10 6细胞比例稀释好的山羊抗人IgG Fc，FITC标记二抗(abcam,cat：97224)溶液，4℃孵育1h；300g×5min，4℃离心，洗涤2次，加入70μl 2％FBS溶液重悬细胞，于流式细胞仪(Bio-Rad，ZE5)检测PE通道的平均荧光强度(MFI)，并作曲线分析抗体结合细胞的EC 50浓度。 NCI-H929 (ATCC,
Figure PCTCN2021088092-appb-000043
CRL-9068 TM ) The cell suspension was centrifuged at 300 g for 5 min; 5 ml of 2% FBS buffer was added to resuspend the cells, and the cell density was adjusted to 1×10 6 cells/ml. Dilute 100μL/well to a 96-well V-bottom plate, 300g×5min, centrifuge at 4℃, discard the supernatant, add 10 concentration gradients (2500nM to 0.064nM, 5-fold dilution gradient) diluted candidate antibody solution to 100μL/well Incubate at 4℃ for 1h; 300g×5min, centrifuge at 4℃, wash twice, add goat anti-human IgG Fc diluted with 5μL/10 6 cells to 100μL/well, FITC-labeled secondary antibody (abcam, cat: 97224) Incubate the solution at 4°C for 1h; 300g×5min, centrifuge at 4°C, wash twice, add 70μl 2% FBS solution to resuspend the cells, and detect the average fluorescence intensity (MFI) of the PE channel on a flow cytometer (Bio-Rad, ZE5) ), and draw a curve to analyze the EC 50 concentration of antibody-bound cells.
表18.双特异性抗体与细胞表面抗原的亲和力Table 18. Affinity of bispecific antibodies to cell surface antigens
Figure PCTCN2021088092-appb-000044
Figure PCTCN2021088092-appb-000044
实施例11：BCMA/CD3双特异性抗体介导的体外肿瘤细胞杀伤作用Example 11: In vitro tumor cell killing effect mediated by BCMA/CD3 bispecific antibody
效应细胞人总T细胞群的准备：将冻存的PBMC于37℃水浴迅速融化，转移细胞悬液至细胞完全培养基(90％RPMI-1640含有10％FBS，100U/100μg/mL青霉素/链霉素，2mM L-谷氨酰胺)，重悬细胞，于室温400g/min离心5min；弃上清，加入新鲜完全培养基；重悬细胞沉淀，计数，调整细胞密度至1x10 7/mL；利用EasySep Human T Cell Iso Kit(Stemcell,cat#：17951)按照操作说明分离人总T细胞群，细胞计数，调整细胞密度至1.2x10 6/mL。 Preparation of human total T cell population of effector cells: quickly thaw the frozen PBMC in a 37°C water bath, and transfer the cell suspension to complete cell culture medium (90% RPMI-1640 contains 10% FBS, 100U/100μg/mL penicillin/chain Tetracycline, 2mM L-glutamine), resuspend the cells and centrifuge at 400g/min for 5min at room temperature; discard the supernatant and add fresh complete medium; resuspend the cell pellet, count, and adjust the cell density to 1x10 7 /mL; use EasySep Human T Cell Iso Kit (Stemcell, cat#: 17951) Follow the instructions to separate the total human T cell population, count the cells, and adjust the cell density to 1.2x10 6 /mL.
制备靶细胞人RPMI-8226(ATCC，cat：CCL-155)悬液，调整细胞密度至1.2x10 5/mL；于全透明96-孔U-型底板(Corning，Cat#：3799)每孔分别各加入50μl两种细胞悬液。将BCMA/CD3双特异性抗体用完全培养基按照10倍稀释，获得9个浓度梯度(以300nM的最高浓度开始)；完全培养基作为阴性对照；加入抗体悬液50μl/孔，并混匀细胞，于37℃，5％CO 2培养箱内共孵育48h后，将实验板置于室温平衡10min；以75μl/孔加入
Figure PCTCN2021088092-appb-000045
细胞活力检测试剂(Promega，Cat#：G7573)，避光震荡裂解350rpm/min，5min；将细胞裂解物转移至白色底部透明实验板(Corning,Cat#：3610)180μL/孔，210g/min离心1min，利用酶标仪(Bio-Tek,Synergy H1)读取信号值。 Prepare the target cell human RPMI-8226 (ATCC, cat: CCL-155) suspension, adjust the cell density to 1.2x10 5 /mL; in each well of a fully transparent 96-well U-shaped bottom plate (Corning, Cat#: 3799) Add 50μl of each of the two cell suspensions. Dilute the BCMA/CD3 bispecific antibody 10-fold with complete medium to obtain 9 concentration gradients (starting with the highest concentration of 300nM); complete medium as a negative control; add antibody suspension 50μl/well, and mix the cells evenly After co-incubating in a 37°C, 5% CO 2 incubator for 48 hours, put the experimental plate at room temperature for 10 minutes; add 75μl/well
Figure PCTCN2021088092-appb-000045
Cell viability detection reagent (Promega, Cat#: G7573), lysate at 350 rpm/min, 5 min, protected from light; transfer the cell lysate to a white bottom transparent experiment plate (Corning, Cat#: 3610) 180 μL/well, 210 g/min centrifugation 1min, read the signal value with a microplate reader (Bio-Tek, Synergy H1).
按照以下公式计算杀伤效率：Calculate the killing efficiency according to the following formula:
细胞杀伤效率％＝(1-实验组的信号值/对照组的信号值)×100Cell killing efficiency%=(1-signal value of experimental group/signal value of control group)×100
利用GraphPad Prism软件，针对剂量-效应抑制公式：Using GraphPad Prism software, for the dose-effect suppression formula:
log(抑制剂)vs效应—变量斜率(四参数)log (inhibitor) vs effect-variable slope (four parameters)
(log(inhibitor)vs.response--Variable slope(four parameters))(log(inhibitor)vs.response--Variableslope(four parameters))
进行曲线拟合，得到IC 50值。结果如下表所示。 Curve fitting is performed to obtain the IC 50 value. The results are shown in the table below.
表19.BCMA/CD3双特异性抗体介导T细胞对RPMI-8226细胞的杀伤Table 19. BCMA/CD3 bispecific antibody mediates the killing of RPMI-8226 cells by T cells
Figure PCTCN2021088092-appb-000046
Figure PCTCN2021088092-appb-000046

Claims (27)

  1. 一种双特异性抗体或其抗原结合片段，其包含：A bispecific antibody or antigen-binding fragment thereof, which comprises:
    特异性结合BCMA的第一结合区、和The first binding region that specifically binds to BCMA, and
    特异性结合CD3的第二结合区；The second binding region that specifically binds to CD3;
    所述第一结合区是抗BCMA抗体或其抗原结合片段，The first binding region is an anti-BCMA antibody or an antigen-binding fragment thereof,
    第二结合区是抗CD3抗体或其抗原结合片段；The second binding region is an anti-CD3 antibody or an antigen-binding fragment thereof;
    其中，in,
    所述抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：4所示的HCDR2和SEQ ID NO：5所示的HCDR3；所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2和SEQ ID NO：8所示的LCDR3；或，The anti-BCMA antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3 and HCDR2 shown in SEQ ID NO: 4 And HCDR3 shown in SEQ ID NO: 5; the light chain variable region includes LCDR1 shown in SEQ ID NO: 6 and LCDR2 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8; or ,
    所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：9所示的HCDR2和SEQ ID NO：5所示的HCDR3；所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2和SEQ ID NO：8所示的LCDR3；或，The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9 and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes SEQ ID NO: LCDR1 shown in 6, LCDR1 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8; or,
    所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：9所示的HCDR2和SEQ ID NO：5所示的HCDR3；所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：10所示的LCDR2和SEQ ID NO：8所示的LCDR3；或，The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9 and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes SEQ ID NO: LCDR1 shown in 6, LCDR1 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8; or,
    所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：4所示的HCDR2和SEQ ID NO：5所示的HCDR3；所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：10所示的LCDR2和SEQ ID NO：8所示的LCDR3。The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes SEQ ID NO: LCDR1 shown in 6, LCDR1 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8.
  2. 如权利要求1所述的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段包含：The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises:
    SEQ ID NO：11所示的重链可变区和SEQ ID NO：14所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14; or,
    SEQ ID NO：12所示的重链可变区和SEQ ID NO：15所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 12 and the light chain variable region shown in SEQ ID NO: 15; or,
    SEQ ID NO：11所示的重链可变区和SEQ ID NO：16所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 16; or,
    SEQ ID NO：13所示的重链可变区和SEQ ID NO：16所示的轻链可变区。The heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 16.
  3. 如权利要求2所述的双特异性抗体或其抗原结合片段，其特征在于，The bispecific antibody or antigen-binding fragment thereof according to claim 2, wherein:
    所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1、IgG2、IgG3、IgG4的重链恒定区或其变体，The anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof,
    优选地，所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1的重链恒定区，或者人IgG1的重链恒定区变体，其中，所述人IgG1的重链恒定区变体与人IgG1重链恒定区相比具有降低的ADCC毒性，Preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, or a heavy chain constant region variant of human IgG1, wherein the heavy chain constant region variant of human IgG1 is similar to that of human IgG1. The heavy chain constant region has reduced ADCC toxicity compared to
    进一步优选地，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO：31所示重链恒定区，或如SEQ ID NO：42所示的重链恒定区变体，或如SEQ ID NO：33所示的重链恒定区变体，或如SEQ ID NO：34所示的重链恒定区变体；Further preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 31, or a heavy chain constant region variant as shown in SEQ ID NO: 42, or as shown in SEQ ID The heavy chain constant region variant shown in NO: 33, or the heavy chain constant region variant shown in SEQ ID NO: 34;
    最优选地，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO：42所示的重链恒定区变体；Most preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region variant as shown in SEQ ID NO: 42;
    任选地，所述抗BCMA抗体或其抗原结合片段进一步包含人抗体κ链、λ链的轻链恒定区或其变体，Optionally, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a human antibody kappa chain, a light chain constant region of a lambda chain or a variant thereof,
    优选地，所述抗BCMA抗体或其抗原结合片段进一步包含人抗体κ链的轻链恒定区；Preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region of a human antibody kappa chain;
    更优选地，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO：32所示的轻链恒定区。More preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO: 32.
  4. 如权利要求3所述的双特异性抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段包含：The bispecific antibody or antigen-binding fragment thereof according to claim 3, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises:
    SEQ ID NO：17所示的重链和SEQ ID NO：20所示的轻链；或，The heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20; or,
    SEQ ID NO：18所示的重链和SEQ ID NO：21所示的轻链；或，The heavy chain shown in SEQ ID NO: 18 and the light chain shown in SEQ ID NO: 21; or,
    SEQ ID NO：19所示的重链和SEQ ID NO：22所示的轻链；或，The heavy chain shown in SEQ ID NO: 19 and the light chain shown in SEQ ID NO: 22; or,
    SEQ ID NO：43所示的重链和SEQ ID NO：22所示的轻链；或，The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 22; or,
    SEQ ID NO：43所示的重链和SEQ ID NO：20所示的轻链。The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 20.
  5. 如权利要求1-4任一项所述的双特异性抗体或其抗原结合片段，所述抗CD3抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein,
    所述第二结合区的重链可变区包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2和SEQ ID NO：37所示的HCDR3；The heavy chain variable region of the second binding region includes HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, and HCDR3 shown in SEQ ID NO: 37;
    所述第二结合区的轻链可变区包含SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2和SEQ ID NO：40所示的LCDR3；The light chain variable region of the second binding region includes LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40;
    优选地，所述第二结合区包含如SEQ ID NO：24所示的重链可变区和如SEQ ID NO：25所示的轻链可变区；Preferably, the second binding region comprises a heavy chain variable region as shown in SEQ ID NO: 24 and a light chain variable region as shown in SEQ ID NO: 25;
    进一步优选地，所述第二结合区的重链可变区和轻链可变区通过肽接头连接；Further preferably, the heavy chain variable region and the light chain variable region of the second binding region are connected by a peptide linker;
    最优选地，所述第二结合区包含如SEQ ID NO：41所示的序列。Most preferably, the second binding region comprises the sequence shown in SEQ ID NO:41.
  6. 如权利要求5所述的双特异性抗体或其抗原结合片段，所述第二结合区的重链可变区的N端连接于第一结合区的轻链恒定区的C端；优选地，所述第二结合区的重链可变区的N端通过肽接头连接于第一结合区的轻链恒定区的C端。The bispecific antibody or antigen-binding fragment thereof according to claim 5, wherein the N-terminus of the heavy chain variable region of the second binding region is connected to the C-terminus of the light chain constant region of the first binding region; preferably, The N terminal of the heavy chain variable region of the second binding region is connected to the C terminal of the light chain constant region of the first binding region through a peptide linker.
  7. 如权利要求5所述的双特异性抗体或其抗原结合片段，其包含2条第一链和2条第二链，其中：The bispecific antibody or antigen-binding fragment thereof according to claim 5, which comprises two first chains and two second chains, wherein:
    所述第一链，从N端到C端包含第一结合区的重链可变区和第一结合区的重链恒定区；The first chain includes the heavy chain variable region of the first binding region and the heavy chain constant region of the first binding region from N-terminus to C-terminus;
    所述第二链，从N端到C端包含第一结合区的轻链可变区、第一结合区的轻链恒定区和第一肽接头，以及第二结合区的重链可变区、第二肽接头和第二结合区的轻链可变区；The second chain includes the light chain variable region of the first binding region, the light chain constant region of the first binding region and the first peptide linker from the N-terminus to the C-terminus, and the heavy chain variable region of the second binding region , The second peptide linker and the light chain variable region of the second binding region;
    第一肽接头和所述第二肽接头是相同的或不同的。The first peptide linker and the second peptide linker are the same or different.
  8. 如权利要求5所述的双特异性抗体或其抗原结合片段，其包含：The bispecific antibody or antigen-binding fragment thereof according to claim 5, which comprises:
    第一多肽，从N端到C端包含第一结合区的重链可变区和重链恒定区；The first polypeptide includes the heavy chain variable region and the heavy chain constant region of the first binding region from the N-terminus to the C-terminus;
    第二多肽，从N端到C端包含第一结合区的轻链可变区和第一结合区的轻链恒定区；The second polypeptide includes the light chain variable region of the first binding region and the light chain constant region of the first binding region from the N-terminus to the C-terminus;
    第三多肽，从N端到C端包含第二结合区的重链可变区、第一肽接头、第二结合区的轻链可变区、第二肽接头和重链恒定区；The third polypeptide includes the heavy chain variable region of the second binding region, the first peptide linker, the light chain variable region of the second binding region, the second peptide linker and the heavy chain constant region from the N-terminus to the C-terminus;
    第一肽接头和所述第二肽接头是相同的或不同的。The first peptide linker and the second peptide linker are the same or different.
  9. 如权利要求8所述的双特异性抗体或其抗原结合片段，其特征在于，The bispecific antibody or antigen-binding fragment thereof according to claim 8, wherein:
    所述第一多肽的重链恒定区包含人IgG的重链恒定区，The heavy chain constant region of the first polypeptide comprises the heavy chain constant region of human IgG,
    所述第三多肽的重链恒定区包含人IgG的重链恒定区结构域，优选地，所述人IgG的重链恒定区结构域从N端到C端包含：铰链区、CH2、和CH3；The heavy chain constant region of the third polypeptide comprises the heavy chain constant region domain of human IgG. Preferably, the heavy chain constant region domain of the human IgG comprises from the N-terminus to the C-terminus: hinge region, CH2, and CH3;
    任选地，所述第一多肽和所述第三多肽在铰链区外进一步包含一个或多个半胱氨酸残基，优选地，所述半胱氨酸残基位于选自以下的位点或组合：349、354或等同位点，进一步优选地，所述第一多肽和所述第三多肽通过所述半胱氨酸残基形成一个或多个二硫桥；Optionally, the first polypeptide and the third polypeptide further comprise one or more cysteine residues outside the hinge region. Preferably, the cysteine residues are located in the group selected from Positions or combinations: 349, 354 or equivalent positions, further preferably, the first polypeptide and the third polypeptide form one or more disulfide bridges through the cysteine residues;
    任选地，所述第一多肽和所述第三多肽的重链恒定区分别包含杵结构域和臼结构域，优选地，所述杵结构域和臼结构域形成异二聚体，进一步优选地，所述杵结构域在以下的位点包含色氨酸残基：366或等同位点，所述臼结构域包含以下任一项或组合：位于366或等同位点的丝氨酸残基、位于368或等同位点的丙氨酸残基、位于407或等同位点的缬氨酸残基；Optionally, the heavy chain constant regions of the first polypeptide and the third polypeptide respectively comprise a knob domain and a socket domain. Preferably, the knob domain and the socket domain form a heterodimer, Further preferably, the knob domain comprises a tryptophan residue at the following position: 366 or an equivalent position, and the hole domain comprises any one or a combination of the following: a serine residue located at the 366 or equivalent position , Alanine residues at 368 or equivalent positions, valine residues at 407 or equivalent positions;
    优选地，所述第一多肽的重链恒定区包含如SEQ ID NO：33所示的序列，Preferably, the heavy chain constant region of the first polypeptide comprises the sequence shown in SEQ ID NO: 33,
    优选地，所述第三多肽的重链恒定区包含如SEQ ID NO：34所示的序列。Preferably, the heavy chain constant region of the third polypeptide comprises the sequence shown in SEQ ID NO: 34.
  10. 如权利要求5-8任一项所述的双特异性抗体或其抗原结合片段，其中所述肽接头选自(GGGGS) n，n选自1-5的整数； The bispecific antibody or antigen-binding fragment thereof according to any one of claims 5-8, wherein the peptide linker is selected from (GGGGS) n , and n is selected from an integer of 1-5;
    优选地，n选自2或3；Preferably, n is selected from 2 or 3;
    进一步优选地，当所述肽接头连接第二结合区的轻链可变区和重链恒定区时，n为2；Further preferably, when the peptide linker connects the light chain variable region and the heavy chain constant region of the second binding region, n is 2;
    当所述肽接头连接第二结合区的重链可变区和第二结合区的轻链可变区时，n为3；When the peptide linker connects the heavy chain variable region of the second binding region and the light chain variable region of the second binding region, n is 3;
    当所述肽接头连接第一结合区的轻链恒定区和第二结合区的重链可变区时，n为3。When the peptide linker connects the light chain constant region of the first binding region and the heavy chain variable region of the second binding region, n is 3.
  11. 如权利要求7所述的双特异性抗体或其抗原结合片段，其含有SEQ ID NO：26和SEQ ID NO：27所示的序列；The bispecific antibody or antigen-binding fragment thereof according to claim 7, which contains the sequence shown in SEQ ID NO: 26 and SEQ ID NO: 27;
    优选，包含两条SEQ ID NO：26所示多肽和两条SEQ ID NO：27所示多肽。Preferably, it comprises two polypeptides shown in SEQ ID NO: 26 and two polypeptides shown in SEQ ID NO: 27.
  12. 如权利要求8所述的双特异性抗体或其抗原结合片段，其含有SEQ ID NO：28，SEQ ID NO：29和SEQ ID NO：20所示的序列。The bispecific antibody or antigen-binding fragment thereof according to claim 8, which contains the sequence shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 20.
  13. 一种抗BCMA抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：An anti-BCMA antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein:
    所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：4所示的HCDR2和SEQ ID NO：5所示的HCDR3；所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2和SEQ ID NO：8所示的LCDR3，或，The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes SEQ ID NO: LCDR1 shown in 6, LCDR1 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8, or,
    所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：9所示的HCDR2和SEQ ID NO：5所示的HCDR3；所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：7所示的LCDR2和SEQ ID NO：8所示的LCDR3，或，The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9 and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes SEQ ID NO: LCDR1 shown in 6, LCDR1 shown in SEQ ID NO: 7 and LCDR3 shown in SEQ ID NO: 8, or,
    所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：9所示的HCDR2和SEQ ID NO：5所示的HCDR3；所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：10所示的LCDR2和SEQ ID NO：8所示的LCDR3，或，The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 9 and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes SEQ ID NO: LCDR1 shown in 6, LCDR1 shown in SEQ ID NO: 10 and LCDR3 shown in SEQ ID NO: 8, or,
    所述重链可变区包含SEQ ID NO：3所示的HCDR1、SEQ ID NO：4所示的HCDR2和SEQ ID NO：5所示的HCDR3；所述轻链可变区包含SEQ ID NO：6所示的LCDR1、SEQ ID NO：10所示的LCDR2和SEQ ID NO：8所示的LCDR3。The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes SEQ ID NO: LCDR1 shown in 6, LCDR1 shown in SEQ ID NO: 10, and LCDR3 shown in SEQ ID NO: 8.
  14. 如权利要求13所述的抗BCMA抗体或其抗原结合片段，其选自鼠源抗体或其抗原结合片段，嵌合抗体或其抗原结合片段，人抗体或其抗原结合片段或者人源化抗体或其抗原结合片段。The anti-BCMA antibody or antigen-binding fragment thereof according to claim 13, which is selected from a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a human antibody or an antigen-binding fragment thereof, or a humanized antibody or Its antigen-binding fragment.
  15. 如权利要求14所述的抗BCMA抗体或其抗原结合片段，其进一步包含人IgG1、IgG2、IgG3、IgG4的重链恒定区或其变体；The anti-BCMA antibody or antigen-binding fragment thereof according to claim 14, which further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof;
    优选地，所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1、IgG2、IgG4的重链恒定区或其变体；Preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises the heavy chain constant region of human IgG1, IgG2, IgG4 or a variant thereof;
    更优选地，所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1的重链恒定区，或者人IgG1的重链恒定区变体，其中，所述人IgG1的重链恒定区变体与野生IgG1重链恒定区相比具有降低的ADCC毒性；More preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgG1, or a heavy chain constant region variant of human IgG1, wherein the heavy chain constant region variant of human IgG1 is similar to that of wild Compared with IgG1 heavy chain constant region, it has reduced ADCC toxicity;
    进一步优选地，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO：31所示重链恒定区，或如SEQ ID NO：42所示的重链恒定区变体。Further preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 31, or a heavy chain constant region variant as shown in SEQ ID NO: 42.
  16. 根据权利要求14所述的抗BCMA抗体或其抗原结合片段，其进一步包含人抗体κ链、λ链的轻链恒定区或其变体；The anti-BCMA antibody or antigen-binding fragment thereof according to claim 14, which further comprises a human antibody kappa chain, a light chain constant region of a lambda chain or a variant thereof;
    优选地，所述抗BCMA抗体或其抗原结合片段进一步包含人抗体κ链的轻链恒定区；Preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region of a human antibody kappa chain;
    更优选地，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO：32所示的轻链恒定区。More preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO: 32.
  17. 如权利要求14所述的抗BCMA抗体或其抗原结合片段，其包含：The anti-BCMA antibody or antigen-binding fragment thereof according to claim 14, which comprises:
    选自以下序列所示的重链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区：SEQ ID NO：11、SEQ ID NO：12或SEQ ID NO：13；和/或，It is selected from the heavy chain variable region shown in the following sequence, or the heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; and/or,
    选自以下序列所示的轻链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区：SEQ ID NO：14、SEQ ID NO：15或SEQ ID NO：16。It is selected from the light chain variable region shown in the following sequence, or the light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.
  18. 如权利要求17所述的抗BCMA抗体或其抗原结合片段，其含有：The anti-BCMA antibody or antigen-binding fragment thereof according to claim 17, which contains:
    选自如下序列所示的重链或与以下序列相比具有至少80％，85％，90％，95％或99％同一性的重链：SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：19或SEQ ID NO：43；和/或，It is selected from the heavy chain shown in the following sequence or the heavy chain with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 43; and/or,
    选自如下序列所示的轻链，或与以下序列相比具有至少80％，85％，90％，95％或99％同一性的轻链：SEQ ID NO：20、SEQ ID NO：21或SEQ ID NO：22。It is selected from the light chain shown in the following sequence, or a light chain with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.
  19. 如权利要求17所述的抗BCMA抗体或其抗原结合片段，其包含：The anti-BCMA antibody or antigen-binding fragment thereof according to claim 17, which comprises:
    SEQ ID NO：11所示的重链可变区和SEQ ID NO：14所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14; or,
    SEQ ID NO：12所示的重链可变区和SEQ ID NO：15所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 12 and the light chain variable region shown in SEQ ID NO: 15; or,
    SEQ ID NO：11所示的重链可变区和SEQ ID NO：16所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 16; or,
    SEQ ID NO：13所示的重链可变区和SEQ ID NO：16所示的轻链可变区。The heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 16.
  20. 如权利要求18所述的抗BCMA抗体或其抗原结合片段，其包含：The anti-BCMA antibody or antigen-binding fragment thereof according to claim 18, which comprises:
    SEQ ID NO：17所示的重链和SEQ ID NO：20所示的轻链；或，The heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20; or,
    SEQ ID NO：18所示的重链和SEQ ID NO：21所示的轻链；或，The heavy chain shown in SEQ ID NO: 18 and the light chain shown in SEQ ID NO: 21; or,
    SEQ ID NO：19所示的重链和SEQ ID NO：22所示的轻链；或，The heavy chain shown in SEQ ID NO: 19 and the light chain shown in SEQ ID NO: 22; or,
    SEQ ID NO：43所示的重链和SEQ ID NO：22所示的轻链；或，The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 22; or,
    SEQ ID NO：43所示的重链和SEQ ID NO：20所示的轻链。The heavy chain shown in SEQ ID NO: 43 and the light chain shown in SEQ ID NO: 20.
  21. 一种多核苷酸，其编码：A polynucleotide encoding:
    权利要求1-12任一项所述的双特异性抗体或其抗原结合片段，或权利要求13-20任 一项所述的抗BCMA体或其抗原结合片段。The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-12, or the anti-BCMA body or the antigen-binding fragment thereof according to any one of claims 13-20.
  22. 一种表达载体，其含有权利要求21所述的多核苷酸。An expression vector containing the polynucleotide of claim 21.
  23. 一种宿主细胞，其导入有或含有：A host cell introduced with or containing:
    权利要求22所述的表达载体，The expression vector of claim 22,
    所述宿主细胞选自细菌、酵母菌或哺乳动物细胞，The host cell is selected from bacteria, yeast or mammalian cells,
    优选为大肠杆菌、毕赤酵母或CHO细胞或HEK293细胞。Preferably, it is Escherichia coli, Pichia pastoris or CHO cell or HEK293 cell.
  24. 一种生产抗BCMA抗体或其抗原结合片段、双特异性抗体或其抗原结合片段的方法，包括步骤：A method for producing anti-BCMA antibody or its antigen-binding fragment, bispecific antibody or its antigen-binding fragment, comprising the steps:
    培养权利要求23所述的宿主细胞；Culturing the host cell of claim 23;
    从培养物中分离所述抗体或其抗原结合片段；以及Isolating the antibody or antigen-binding fragment thereof from the culture; and
    纯化所述抗体或其抗原结合片段。Purify the antibody or antigen-binding fragment thereof.
  25. 一种药物组合物，其含有：A pharmaceutical composition containing:
    权利要求1-12任一项所述的双特异性抗体或其抗原结合片段，或，The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-12, or,
    权利要求13-20任一项所述的抗BCMA抗体或其抗原结合片段；以及可药用的赋形剂、稀释剂或载体。The anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 13-20; and a pharmaceutically acceptable excipient, diluent or carrier.
  26. 权利要求1-12任一项所述的双特异性抗体或其抗原结合片段，或权利要求13-20任一项所述的抗BCMA抗体或其抗原结合片段，或权利要求25所述的药物组合物在用于制备治疗或预防BCMA介导的疾病或病症的药物中的用途。The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-12, or the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 13-20, or the drug according to claim 25 The composition is used for preparing a medicine for treating or preventing BCMA-mediated diseases or disorders.
  27. 根据权利要求26所述的用途，其特征在于，所述BCMA介导的疾病或病症为癌症或自身免疫疾病，The use according to claim 26, wherein the BCMA-mediated disease or disorder is cancer or autoimmune disease,
    优选地，所述癌症为表达BCMA的癌症，更优选淋巴瘤和骨髓瘤；Preferably, the cancer is a cancer expressing BCMA, more preferably lymphoma and myeloma;
    优选地，所述自身免疫疾病选自红斑狼疮、IgA肾病和风湿性关节炎。Preferably, the autoimmune disease is selected from lupus erythematosus, IgA nephropathy and rheumatoid arthritis.
PCT/CN2021/088092 2020-04-17 2021-04-19 Specific antigen binding molecule, and preparation method and pharmaceutical use therefor WO2021209066A1 (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN105980406A (en) * 2013-03-15 2016-09-28 安进研发（慕尼黑）股份有限公司 Binding molecules for bcma and cd3
CN108350076A (en) * 2015-08-17 2018-07-31 詹森药业有限公司 Anti- BCMA antibody, in conjunction with the bispecific antigen binding molecules and application thereof of BCMA and CD3
WO2020018820A1 (en) * 2018-07-19 2020-01-23 Regeneron Pharmaceuticals, Inc. BISPECIFIC ANTI-BCMA x ANTI-CD3 ANTIBODIES AND USES THEREOF

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105980406A (en) * 2013-03-15 2016-09-28 安进研发（慕尼黑）股份有限公司 Binding molecules for bcma and cd3
CN108350076A (en) * 2015-08-17 2018-07-31 詹森药业有限公司 Anti- BCMA antibody, in conjunction with the bispecific antigen binding molecules and application thereof of BCMA and CD3
WO2020018820A1 (en) * 2018-07-19 2020-01-23 Regeneron Pharmaceuticals, Inc. BISPECIFIC ANTI-BCMA x ANTI-CD3 ANTIBODIES AND USES THEREOF

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