WO2021209066A1 - Molécule spécifique de liaison à l'antigène, procédé de préparation correspondant et utilisation pharmaceutique associée - Google Patents

Molécule spécifique de liaison à l'antigène, procédé de préparation correspondant et utilisation pharmaceutique associée Download PDF

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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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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/zh
Publication of WO2021209066A1 publication Critical patent/WO2021209066A1/fr

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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

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Abstract

L'invention concerne un anticorps BCMA, une molécule bispécifique de liaison à l'antigène qui se lie à BCMA et à CD3 et une composition pharmaceutique associée, ainsi qu'une utilisation correspondante dans la préparation d'un médicament anticancéreux ou d'un médicament pour le traitement de maladies auto-immunes.
PCT/CN2021/088092 2020-04-17 2021-04-19 Molécule spécifique de liaison à l'antigène, procédé de préparation correspondant et utilisation pharmaceutique associée WO2021209066A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105980406A (zh) * 2013-03-15 2016-09-28 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
CN108350076A (zh) * 2015-08-17 2018-07-31 詹森药业有限公司 抗-bcma抗体，结合bcma和cd3的双特异性抗原结合分子及其用途
WO2020018820A1 (fr) * 2018-07-19 2020-01-23 Regeneron Pharmaceuticals, Inc. Anticorps anti-bcma x anti-cd3 bispécifiques et leurs utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105980406A (zh) * 2013-03-15 2016-09-28 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
CN108350076A (zh) * 2015-08-17 2018-07-31 詹森药业有限公司 抗-bcma抗体，结合bcma和cd3的双特异性抗原结合分子及其用途
WO2020018820A1 (fr) * 2018-07-19 2020-01-23 Regeneron Pharmaceuticals, Inc. Anticorps anti-bcma x anti-cd3 bispécifiques et leurs utilisations

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