WO2021018168A1 - 抗bcma抗体、其抗原结合片段及其医药用途 - Google Patents

抗bcma抗体、其抗原结合片段及其医药用途 Download PDF

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WO2021018168A1
WO2021018168A1 PCT/CN2020/105408 CN2020105408W WO2021018168A1 WO 2021018168 A1 WO2021018168 A1 WO 2021018168A1 CN 2020105408 W CN2020105408 W CN 2020105408W WO 2021018168 A1 WO2021018168 A1 WO 2021018168A1
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seq
antigen
antibody
binding fragment
bcma
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PCT/CN2020/105408
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English (en)
French (fr)
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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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Application filed by 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 filed Critical 上海翰森生物医药科技有限公司
Priority to KR1020227005610A priority Critical patent/KR20220068984A/ko
Priority to JP2022504507A priority patent/JP2022542088A/ja
Priority to CN202080004455.4A priority patent/CN112585168B/zh
Priority to US17/597,514 priority patent/US20220251228A1/en
Priority to CA3146394A priority patent/CA3146394A1/en
Priority to AU2020322588A priority patent/AU2020322588A1/en
Priority to BR112022001546A priority patent/BR112022001546A2/pt
Priority to EP20848624.1A priority patent/EP4006055A4/en
Priority to MX2022001065A priority patent/MX2022001065A/es
Publication of WO2021018168A1 publication Critical patent/WO2021018168A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6877—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the antibody being an immunoglobulin containing regions, domains or residues from different species
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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Definitions

  • the present disclosure relates to anti-BCMA antibodies, their antigen-binding fragments and their medical uses. Further, the present disclosure relates to the use of anti-BCMA antibodies, antigen-binding fragments thereof, and pharmaceutical compositions comprising anti-BCMA antibodies or antigen-binding fragments thereof to detect or treat BCMA-mediated diseases or disorders.
  • B cells are lymphocytes, which play an important role in adaptive 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.
  • two or three fragments of DNA are selected from a genomic library, and recombined to produce a combinatorial array of antibody variable domains, through which the variable domains encoded by B cells of different lineages are further The changes resulted in up to 109 unique B cell lineages that produced and uniquely targeted specific antibodies.
  • Many diseases involve B cells. Malignant transformation of B cells leads to cancer, including some lymphomas, such as multiple myeloma and Hodgkin’s lymphoma.
  • B cells including systemic lupus erythematosus (SLE) and IgA nephropathy.
  • SLE systemic lupus erythematosus
  • IgA nephropathy IgA nephropathy.
  • Cancers and autoimmune diseases involving B cells can be considered abnormal B cell function, so a possible strategy to control these diseases is to use antibodies that target pathological B cells.
  • BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily, which is a non-glycosylated inner membrane receptor for ligands BAFF (B cell activating factor) and APRIL (proliferation inducing ligand). BCMA and its corresponding ligands can regulate different functions of humoral immunity, B cell development and homeostasis. BCMA is detected in the spleen, lymph nodes, thymus, adrenal glands and liver. It is expressed by human plasmablasts, plasma cells from tonsils, spleen and bone marrow, tonsil memory B cells and germinal center B cells, and a variety of B cell lines The analysis showed that the expression level of BCMA increased after maturation. BCMA is highly expressed in B-cell lymphoma and multiple myeloma.
  • Antibodies against BCMA are described in, for example, Gras M-P. et al. Int Immunol. 7 (1995) 1093-1106, WO200124811, WO200124812, WO2010104949 and WO2012163805.
  • Antibodies against BCMA and their use for the treatment of lymphoma and multiple myeloma are described in, for example, WO2002066516 and WO2010104949.
  • WO2013154760 relates to a chimeric antigen receptor comprising a BCMA recognition part and a T-cell activation part.
  • an antigen binding protein capable of internalization that specifically binds BCMA and inhibits the binding of BAFF and/or APRIL to BCMA is provided, and a conjugate comprising the antigen binding protein and a cytotoxic agent is provided.
  • the present disclosure provides anti-BCMA antibodies with high affinity, high specificity, low immunogenicity, high biological activity, significant anti-tumor effect, and enhanced endocytosis, as well as cytotoxic conjugates containing the antibodies, and their usefulness Use in inhibiting tumors.
  • an anti-BCMA antibody or antigen-binding fragment thereof, or a pharmaceutically acceptable salt thereof which comprises:
  • An antibody heavy chain variable region which comprises at least one HCDR selected from the following sequences: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and
  • An antibody light chain variable region comprising at least one LCDR selected from the following sequences: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
  • the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises:
  • the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises:
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises:
  • the light chain variable region includes:
  • an anti-BCMA antibody or an antigen-binding fragment thereof or a pharmaceutically acceptable salt thereof.
  • the anti-BCMA antibody or an antigen-binding fragment thereof comprises at least one sequence selected from the following sequences or HCDR of mutant sequence:
  • SEQ ID NO: 3 SEQ ID NO: 4 or SEQ ID NO: 5;
  • an anti-BCMA antibody or an antigen-binding fragment thereof or a pharmaceutically acceptable salt thereof.
  • the anti-BCMA antibody or an antigen-binding fragment thereof comprises at least one sequence selected from the following sequences or LCDR of the mutation sequence: SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the anti-BCMA antibody or antigen-binding fragment or pharmaceutically acceptable salt thereof according to the present disclosure comprises a heavy chain variable region, and
  • the chain variable region contains:
  • the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises:
  • LCDR3 shown in the sequence of SEQ ID NO: 8 or its mutant sequence.
  • the anti-BCMA antibody or antigen-binding fragment thereof or a pharmaceutically acceptable salt thereof according to the present disclosure comprises an antibody heavy chain variable region and The light chain variable region, the heavy chain variable region comprising:
  • the light chain variable region includes:
  • LCDR3 shown in the mutation sequence of SEQ ID NO: 8.
  • the anti-BCMA antibody or antigen-binding fragment thereof is a murine antibody.
  • the anti-BCMA antibody or antigen-binding fragment thereof is a chimeric antibody.
  • the anti-BCMA antibody or antigen-binding fragment thereof is a human antibody.
  • the anti-BCMA antibody or antigen-binding fragment thereof is a humanized antibody.
  • the anti-BCMA antibody or antigen-binding fragment thereof or a pharmaceutically acceptable salt thereof according to the present disclosure further comprises a human IgG1 or a variant thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises human IgG1, IgG2, or The constant region of the heavy chain of IgG4.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises an amino acid mutation with enhanced ADCC Toxic IgG1 heavy chain constant region.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises SEQ ID NO: 22 The constant region of the heavy chain shown.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a human kappa chain or A variant thereof, the constant region of the light chain of the lambda chain or a variant thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises SEQ ID NO: 23 The constant region of the light chain shown.
  • the anti-BCMA antibody or antigen-binding fragment or pharmaceutically acceptable salt thereof according to the present disclosure, the anti-BCMA antibody or antigen-binding fragment or pharmaceutically acceptable salt thereof
  • the salt of contains a heavy chain variable region selected from the following sequences: SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises an antibody selected from SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 has a heavy chain variable region with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a sequence selected from The light chain variable region shown: SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises an antibody selected from SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 has a light chain variable region with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof contains a heavy chain of the following sequence : SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises an antibody selected from SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 has a heavy chain with at least 80%, 85%, 90%, 95% or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof contains a light chain of the following sequence : SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises an antibody selected from SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20 has a light chain with at least 80%, 85%, 90%, 95%, or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 9 and a light chain variable region shown in SEQ ID NO: 12 .
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region is selected from SEQ ID NO: 9 , Or at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 9
  • the light chain variable region is selected from SEQ ID NO: 12 or with SEQ ID NO: 12 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO: 10 and the light chain variable region shown in SEQ ID NO: 13 Area.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region is selected from SEQ ID NO: 10 , Or at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 10
  • the light chain variable region is selected from SEQ ID NO: 13 or with SEQ ID NO: 13 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 14 Area.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region is selected from SEQ ID NO: 11 , Or a heavy chain variable region with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 11, the light chain variable region is selected from SEQ ID NO: 14 or SEQ ID NO: 14 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain shown in SEQ ID NO: 15 and a light chain shown in SEQ ID NO: 18.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain
  • the heavy chain is selected from SEQ ID NO: 15, or has a combination with SEQ ID NO: 15.
  • the light chain is selected from SEQ ID NO: 18, or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 18 % Or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain shown in SEQ ID NO: 16 and a light chain shown in SEQ ID NO: 19.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain
  • the heavy chain is selected from SEQ ID NO: 16, or has a combination with SEQ ID NO: 16.
  • the light chain is selected from SEQ ID NO: 19, or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 19 % Or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain shown in SEQ ID NO: 17 and a light chain shown in SEQ ID NO: 20.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain
  • the heavy chain is selected from SEQ ID NO: 17, or has a combination with SEQ ID NO: 17.
  • the light chain is selected from SEQ ID NO: 20, or has at least 80%, 85%, 90%, 95% with SEQ ID NO: 20 % Or 99% identity.
  • a polynucleotide which encodes the anti-BCMA antibody or antigen-binding fragment thereof of the present disclosure.
  • an expression vector which contains the polynucleotide described in the present disclosure.
  • a host cell transformed with the expression vector of the present disclosure.
  • the host cell is bacteria, preferably Escherichia coli.
  • the host cell is yeast, preferably Pichia pastoris.
  • the host cell is a mammalian cell, preferably a CHO cell or HEK293 cell.
  • the present disclosure provides a method for producing an anti-BCMA antibody, including the steps:
  • the antibody is purified.
  • the antibody is purified by affinity chromatography, preferably by a chromatographic method.
  • a pharmaceutical composition which contains the anti-BCMA antibody or antigen-binding fragment thereof described in the present disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient , Diluent or carrier.
  • a detection or diagnostic reagent which contains the anti-BCMA antibody or antigen-binding fragment thereof described in the present disclosure or a pharmaceutically acceptable salt thereof.
  • a detection or diagnosis kit which contains the anti-BCMA antibody or antigen-binding fragment or pharmaceutically acceptable salt thereof described in the present disclosure, and can be used for detection or diagnosis
  • the labeled secondary antibody, buffer and substrate is provided.
  • a detection or diagnosis kit which contains the anti-BCMA antibody or antigen-binding fragment or pharmaceutically acceptable salt thereof described in the present disclosure, and one or more A reagent capable of detecting the binding of the BCMA antibody or its antigen-binding fragment to BCMA.
  • the present disclosure provides that the anti-BCMA antibody or antigen-binding fragment or pharmaceutically acceptable salt thereof described in the present disclosure, or a pharmaceutical composition containing the same, is used in the treatment or prevention of BCMA-mediated diseases or disorders. the use of.
  • the present disclosure provides that the anti-BCMA antibody or antigen-binding fragment or pharmaceutically acceptable salt thereof described in the present disclosure, or the pharmaceutical composition containing the same, is used for the treatment or prevention of BCMA-mediated diseases. Or the use of disease in medicine.
  • the present disclosure provides an anti-BCMA antibody or an antigen-binding fragment thereof or a pharmaceutically acceptable salt thereof according to the present disclosure, or a detection or diagnostic reagent containing the same for use in the detection, diagnosis, and prognosis of BCMA mediation. Leading disease or condition.
  • the present disclosure provides the anti-BCMA antibodies or antigen-binding fragments or pharmaceutically acceptable salts thereof described in the present disclosure, or detection or diagnostic reagents containing them for use in the preparation of BCMA-mediated detection, diagnosis, and prognosis. Use in kits for diseases or conditions.
  • the BCMA-mediated disease or disorder is cancer; preferably BCMA-expressing cancer; more preferably lymphoma, leukemia or myeloma, most preferably multiple myeloma.
  • the BCMA-mediated disease or condition is an autoimmune disease; preferably lupus erythematosus, IgA nephropathy and rheumatoid arthritis.
  • the present disclosure provides an antibody-drug conjugate comprising the anti-BCMA antibody or antigen-binding fragment thereof of the present disclosure coupled with a cytotoxic agent; preferably, the cytotoxic agent is selected from MMAF , SN-38 and Exotecan. Further preferably, the antibody drug conjugate according to the present disclosure has a structural formula selected from the following:
  • the Ab is the anti-BCMA antibody or antigen-binding fragment thereof as described above in the disclosure.
  • the present disclosure provides the use of the antibody drug conjugate according to the present disclosure in the preparation of a medicament for the treatment of BCMA-mediated diseases or disorders, the BCMA-mediated diseases or disorders selected from cancer and self For immune diseases, preferably, the cancer is a cancer expressing BCMA; more preferably lymphoma, leukemia or myeloma, and most preferably multiple myeloma.
  • the autoimmune disease is selected from lupus erythematosus, IgA nephropathy and rheumatoid arthritis.
  • the present disclosure provides a method of treating a BCMA-mediated disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-BCMA antibody according to the present disclosure Or its antigen-binding fragment or its pharmaceutically acceptable salt, its pharmaceutical composition or the antibody drug conjugate of the present disclosure.
  • the anti-BCMA antibody or antigen-binding fragment thereof provided by the present disclosure has high affinity, high specificity, low immunogenicity, high biological activity, and significant anti-tumor effect.
  • the antibody of the present disclosure can be rapidly internalized after binding to the BCMA antigen , Indicating that they are suitable for therapeutic applications in the form of ADCs or other applications for rapid internalization.
  • the ADC forms of the antibodies of the present disclosure can efficiently kill tumor cells expressing BCMA.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of immunoglobulin heavy chains 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 ⁇ chain or ⁇ 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 described in the present disclosure may further comprise a light chain constant region, and the light chain constant region comprises human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain described in the present disclosure may further include a heavy chain constant region, and the heavy chain constant region includes 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 relatively conserved framework regions (FR). 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) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal 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 and VH regions of the antibodies or antigen-binding fragments described in the present disclosure comply with the known Kabat numbering rules and Kabat or AbM or IMGT definition rules (http://bioinf.org.uk /abs/).
  • APC antigen presenting cell
  • DC dendritic cells
  • PBMC peripheral blood mononuclear cells
  • monocytes B lymphoblasts
  • monocyte-derived dendritic cells monocyte-derived dendritic cells
  • antigen presentation refers to the process by which APC captures antigens and enables them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.
  • BCMA includes any variant or isoform of BCMA that is naturally expressed by the cell.
  • the antibodies of the present disclosure can cross-react with BCMA derived from non-human species.
  • the antibody may also be specific for human BCMA, and may not show cross-reactivity with 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 those techniques commonly used in the art and those described herein.
  • the anti-BCMA antibody targets human BCMA with a normal glycosylation pattern.
  • 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 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 that occur during antibody maturation.
  • murine antibody in the present disclosure is a monoclonal antibody to human BCMA prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with BCMA antigen, 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 chain or a variant thereof, or further comprise murine IgG1, IgG2 , IgG3 or IgG4 or its variant heavy chain constant region.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present disclosure may include amino acid residues 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 mouse CDR sequences into a human antibody variable region framework.
  • Humanized antibodies can overcome the shortcomings of strong immune responses induced by chimeric antibodies that carry a large amount of mouse protein components.
  • the variable region of the human antibody can be subjected to minimal reverse mutation to maintain activity.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed, and change the mouse variable region genes.
  • the 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 parent 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.
  • the 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 a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments containing the CH1 and CH2 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.
  • the "Fab' fragment” contains a light chain and a portion of a heavy chain that contains the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, so that it can be between the two heavy chains of the two Fab' fragments The formation of interchain disulfide bonds to form F(ab')2 molecules.
  • the "F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both the heavy and light chains, but lacks the constant region.
  • multispecific antibody is used in its broadest sense to encompass antibodies with polyepitope specificity.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitope specificity; having two or more VL and VH regions Antibodies, each VH-VL unit binds to a different target or a different epitope of the same target; an antibody with two or more single variable regions, each single variable region with a different target Or binding to different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked together Wait.
  • single-chain antibody is a single-chain recombinant protein formed by connecting the heavy chain variable region VH and the light chain variable region VL of an antibody through a connecting peptide. It is the smallest antibody fragment with a complete antigen-binding site.
  • 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 disclosure refers to the ability to interact with human BCMA.
  • antigen-binding site in the present disclosure refers to a three-dimensional site recognized by the antibody or antigen-binding fragment of the present disclosure.
  • 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 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 a 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” used in the present disclosure refer to the binding of an antibody to an epitope on a predetermined antigen.
  • an antibody when measured by surface plasmon resonance (SPR) technology in the instrument, the antibody has an equilibrium dissociation constant of approximately lower than 10 -7 M or even smaller ( K D ) binds to a predetermined antigen, and its binding affinity to the predetermined antigen is at least twice its binding affinity to a non-specific antigen (such as BSA, etc.) other than the predetermined antigen or a closely related antigen.
  • SPR surface plasmon resonance
  • the term "antibody that recognizes an antigen” can be used interchangeably with the term “antibody that specifically binds” herein.
  • cross-reactivity refers to the ability of the antibodies of the present disclosure to bind to BCMA from different species.
  • an antibody of the present disclosure that binds 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.
  • SPR surface plasmon resonance
  • inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • the 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.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a specific antigen.
  • induction for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • 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.
  • mice can be immunized with human BCMA or fragments thereof, and the obtained antibodies can be renatured and purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to the non-human CDR region.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImmunoGeneTics (IMGT), or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • the engineered antibodies or antigen-binding fragments of the present disclosure 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 antibodies of the present disclosure refer to monoclonal antibodies.
  • the monoclonal antibody (mAb) described in the present disclosure refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines.
  • 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, diagnostic agents, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to 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 disclosure, 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 patient or population to be treated in an amount effective to alleviate one or more disease symptoms, 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 can assess whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure 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 they should alleviate the target disease symptoms in a statistically significant number of patients.
  • naturally occurring applied to an object described in the present disclosure refers to the fact that the object can be found in nature.
  • polypeptide sequences or polynucleotide sequences that exist in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified artificially in the laboratory are naturally occurring.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of medical conditions.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may 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.
  • Exogenous refers to substances that are produced outside organisms, cells, or humans depending on the background.
  • Endogenous refers to a substance produced in a cell, organism, or human body according to the background.
  • Identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of 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% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the maximum percent identity.
  • “Pharmaceutical composition” means containing one or more of the antibodies or antigen-binding fragments 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 ingredients and thus the biological activity.
  • DAR Drug Load
  • y is the average number of cytotoxic drugs per antibody in the antibody-drug conjugate.
  • the drug load (DAR) in the antibody-drug conjugate from the coupling reaction can be characterized by conventional means, such as mass spectrometry, HPLC and ELISA, etc., by which the quantitative distribution of the antibody-drug conjugate on the y value can be determined .
  • Example 2 Obtaining mouse hybridoma and antibody sequence
  • the indirect ELISA method as described in Example 3 was used for the immunized mouse serum to evaluate the serum titer and the ability to bind to cell surface antigens.
  • the detection of the titer (larger than 100,000 times dilution) determines the start of cell fusion.
  • the immunized mice with strong serum titer, affinity and FACS binding were selected for one final immunization and then sacrificed. Splenocytes and SP2/0 myeloma cells were fused and plated to obtain hybridomas.
  • the target hybridomas were screened by indirect ELISA, and The strain was established as a monoclonal cell strain 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 a mouse Ig-primer set (Novagen, TB326 Rev. B 0503) and then sequenced to finally obtain the sequence of the mouse antibody M1.
  • the heavy chain and light chain variable region sequences of murine monoclonal antibody M1 are as follows:
  • BCMA His protein (Sino Biological Inc., cat#10620-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 with PBST (pH 7.4 PBS containing 0.05% Tween-20) 4 times, adding 150 ⁇ l/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubating for 1 hour at room temperature for blocking. After the blocking, discard the blocking solution, and wash the plate 4 times with PBST buffer.
  • BSA bovine serum albumin
  • Collect the cultured BCMA highly expressing cells (HEK-293T cells overexpressing BCMA; tumor cells expressing BCMA, NCI-H929, ATCC deposit number CRL-9068), adjust the cell density and spread them on a 96-well U bottom plate, each well 1 ⁇ 10 5 to 2 ⁇ 10 5 cells. Centrifuge at 1200g for 5min, remove the supernatant, add 100ul of serially diluted antibody solution or mouse immune serum, incubate at 4°C for 60min; centrifuge at 1200g for 5min, remove the supernatant, and wash the cells twice with PBS, add a fluorescently labeled secondary antibody (PE-GAM or PE-GAH) 100ul per well, incubate at 4°C for 60min. Centrifuge at 1200g for 5min to remove the supernatant. After washing the cells twice with PBS, resuspend them in PBS, use flow cytometry to detect the signal, and make the concentration curve analysis result.
  • PE-GAM or PE-GAH
  • the humanization of the murine anti-human BCMA monoclonal antibody is carried out according to the methods published in many documents in the field. In short, human constant domains are used to replace parental (murine antibody) constant domains, and human antibody sequences are selected based on the identity of the murine antibody and the human antibody.
  • the present disclosure 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 identity.
  • the CDR region of the murine antibody M1 was transplanted to the selected corresponding 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 backmutated, and the CDR region Chemically unstable amino acid residues were optimized. After expression testing and comparison of the number of back mutations, the sequence of the humanized heavy chain variable region HCVR was selected and designed. The sequence is as follows:
  • the sequence of the humanized light chain variable region LCVR was selected and designed as follows:
  • CDNA fragments were synthesized according to 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 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.
  • NCI-H929 ATCC deposit number CRL-9068
  • NCI-H929 cells were trypsinized (washed with PBS first, 37°C, about 2min), collected and resuspended in pre-cooled FACS buffer to adjust the cell concentration to 1 ⁇ 10 6 cells/mL.
  • the EP tube was taken out at the corresponding time point for 30min and 2h, and placed on ice to pre-cool for 5min. All treatment groups were centrifuged to discard the supernatant (4°C, 1500rpm ⁇ 5min), wash once with FACS buffer and remove the supernatant. Add 250 ⁇ L strip buffer to EP tubes of all treatment groups except the 0min group, incubate at room temperature for 8min, centrifuge to discard the supernatant (4°C, 1500rpm ⁇ 5min), wash twice with FACS buffer, and remove the supernatant. All treatment groups were added with 100 ⁇ L of immunostaining fixative, placed at 4°C for more than 30 minutes, and detected by flow cytometer DxFlex.
  • Percentage of BCMA antibody endocytosis (fluorescence intensity value at each time point—average fluorescence intensity value of blank group)/average fluorescence intensity value at zero point—average fluorescence intensity value of blank group.
  • the antibodies of the present disclosure have cell affinity activity and endocytosis activity, making the antibodies of the present disclosure suitable for coupling with drugs to form antibody-drug conjugates for the treatment of BCMA-mediated diseases.
  • the antibody of the present disclosure is conjugated with MC-MMAF to form an antibody-drug conjugate.
  • the coupling process is shown in the following formula, where Ab stands for Ab2 or Ab3 antibody:
  • the compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile, and 2f solution (concentration 6.17 mg/mL, 3.0 mL) was added to 25 After shaking for 4 hours at °C, the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5), and filtered under sterile conditions with a filter to obtain the product Ab2-MC -MMAF.
  • the DAR average value y of the product Ab2-MC-MMAF was determined by HIC-HPLC to be 4, and the PBS buffer (3.7 mg/mL, 4.7 mL) of the antibody-drug conjugate was refrigerated at 4°C.
  • the product Ab3-MC-MMAF was prepared by the above method.
  • the average DAR value y of the product Ab3-MC-MMAF was determined by HIC-HPLC to be 4.1.
  • the antibody-drug conjugate PBS buffer (3.5mg/mL, 5.0mL ) Refrigerate at 4°C.
  • S-(3-hydroxypropyl) thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile solution for later use.
  • Add the pre-prepared acetonitrile solution of S-(3-hydroxypropyl) thioacetate to the acetic acid/sodium acetate buffer (10.35mg/mL, 9.0mL, 0.97mol) of antibody pH 4.3, and then add 1.0mL dropwise
  • An aqueous solution of sodium cyanoborohydride (14.1mg, 224mol) was shaken and reacted at 25°C for 2 hours.
  • the compound MC-SN-38 (1.3mg, 1.2mol) was dissolved in 0.3ml of acetonitrile, added to the 2h solution (concentration 6.2mg/mL, 3.0mL), and the reaction was shaken at 25°C for 4 hours.
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution at pH 6.5)
  • the product was filtered under sterile conditions with a filter to obtain the product Ab-SN-38 antibody-drug coupling PBS buffer (3.7mg/mL, 4.7mL) of the substance, refrigerated at 4°C.
  • the average value y was determined by the ultraviolet method.
  • the average drug load y CDrug/Cmab.
  • the DAR average value y of the Ab2-SN-38 antibody-drug conjugate determined by the above method is 3.9.
  • 2a (2g, 17.2mmol) was dissolved in 75mL of acetonitrile, and potassium carbonate (9.27g, 67.2mmol), benzyl bromide (20mL, 167.2mmol) and tetrabutylammonium iodide (620mg, 1.68mmol) were added in sequence. ).
  • the reaction solution was stirred at room temperature for 48 hours, filtered through celite, the filter cake was rinsed with ethyl acetate (20ml), the combined filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with a developing solvent system C to obtain Product 5a (3.2g, yield: 90.1%).
  • reaction solution was filtered with diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude product 5c 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo- 2,9-dioxa-4,7-diazaundec-11-acid (20mg), the product was directly subjected to the next reaction without purification.
  • 5d (30 mg, 35.7 ⁇ mol) was dissolved in 3 mL of dichloromethane, 1.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours.
  • the reaction solution was concentrated under reduced pressure, 1.5 mL of toluene was added and concentrated under reduced pressure, repeated twice.
  • the crude product 5e (20mg, 32.3 ⁇ mol) was dissolved in 1mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5°C in an ice water bath, and 4g (31.8mg, 67.3 ⁇ mol) was added.
  • 4g 31.8mg, 67.3 ⁇ mol
  • 0.5mL N,N-dimethylformamide solution add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine salt ( 27.8 mg, 94.3 ⁇ mol), stirred in an ice bath for 10 minutes, removed the ice bath, warmed to room temperature and stirred for 1 hour, the reaction yielded compound 5.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min), collect the corresponding components and concentrate under reduced pressure to obtain products 5-A and 5-B (3.6 mg, 2.6 mg).
  • the average value y was determined by the ultraviolet method. After placing the cuvette containing sodium succinate buffer in the reference absorption cell and the sample determination absorption cell, after deducting the solvent blank, place the cuvette containing the test solution in the sample determination absorption cell Measure the absorbance at 280nm and 370nm.
  • the average drug load y CDrug/Cmab.
  • the Exemplary Product Ab2-Isinotecan was determined to be 7.6 by the above method.
  • Human IgG1 protein was used as the control, and the dose was 1 mg/kg.
  • Tumor inhibition rate 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).
  • the experimental results are shown in Table 9.
  • Human IgG1 protein was used as the control, and the dose was 3 mg/kg.
  • Tumor inhibition rate TGI 100%-(Tumor volume in the administration group on day 14-tumor volume in the administration group on day 0)/(tumor volume in the control group on day 14-tumor volume in the control group on day 0).

Abstract

提供了抗BCMA抗体、其抗原结合片段及其医药用途。进一步地，提供了包含所述抗BCMA抗体CDR区的嵌合抗体、人源化抗体，以及包含抗BCMA抗体或其抗原结合片段的药物组合物，以及其作为抗癌药物和治疗自身免疫疾病的用途。尤其地，提供了一种人源化的抗BCMA抗体，及其在制备用于治疗BCMA介导的疾病或病症的药物中的用途和用于疾病检测和诊断中的用途。

Description

抗BCMA抗体、其抗原结合片段及其医药用途 技术领域
本公开涉及抗BCMA抗体、其抗原结合片段及其医药用途。进一步地，本公开涉及使用抗BCMA抗体、其抗原结合片段以及包含抗BCMA抗体或其抗原结合片段的药物组合物来检测或治疗BCMA介导的疾病或病症。
背景技术
B细胞是淋巴细胞，其在自适应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。在VDJ重组的过程中，其中DNA的两种或三种片段选自基因组文库，并且重组以产生抗体可变结构域的组合阵列，通过其由不同谱系的B细胞编码的可变结构域进一步的改变导致至多10 9种独特的B细胞谱系，其产生和独特靶向具有特异性的抗体。多种疾病涉及B细胞。B细胞的恶性转化导致癌症，包括一些淋巴瘤，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可被认为B细胞功能异常，因此控制这类疾病的可能的策略是使用靶向病理B细胞的抗体。
BCMA(CD269或TNFRSF17)是TNF受体超家族的成员，其是配体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细胞发育和稳态的不同作用。BCMA在脾脏、淋巴结、胸腺、肾上腺和肝脏检测到，由人浆母细胞、来自扁桃体，脾和骨髓的浆细胞，还由扁桃体记忆B细胞和由生发中心B细胞表达，并且多种B细胞系的分析表明在BCMA在成熟后的表达水平增加。BCMA在B细胞淋巴瘤和多发性骨髓瘤中高度表达。
针对BCMA的抗体描述于例如Gras M-P.等Int Immunol.7(1995)1093-1106、WO200124811、WO200124812、WO2010104949和WO2012163805中。针对BCMA的抗体及其用于治疗淋巴瘤和多发性骨髓瘤的用途例如叙述于WO2002066516和WO2010104949中。WO2013154760涉及包含BCMA识别部分和T-细胞激活部分的嵌合抗原受体。
在US9273141提供了一种能够内化的特异性结合BCMA和抑制BAFF和/或APRIL对BCMA的结合的抗原结合蛋白，并提供了包含所述抗原结合蛋白与细胞毒性剂的缀合物。
本公开提供了具有高亲和力、高特异性、低免疫原性、高生物活性、显著抗肿瘤效果、增强的内吞作用的抗BCMA抗体，以及包含所述抗体的细胞毒性偶联物以及其在抑制肿瘤方面的用途。
发明内容
根据本公开的一些实施方案，提供了一种抗BCMA抗体或其抗原结合片段，或其药学上可接受的盐，其包含：
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：SEQ ID NO:3，SEQ ID NO:4和SEQ ID NO:5；和
抗体轻链可变区，所述的抗体轻链可变区包含至少1个选自以下序列所示的LCDR：SEQ ID NO:6，SEQ ID NO:7和SEQ ID NO:8。
在本公开一个优选的实施方案中，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所示的HCDR2和
SEQ ID NO:5所示的HCDR3。
在本公开一个优选的实施方案中，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所示的LCDR2和
SEQ ID NO:8所示的LCDR3。
在本公开一个优选的实施方案中，所述的抗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；以及
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。
根据本公开的一些实施方案，提供了一种抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段包含至少1个选自以下序列或其突变序列的HCDR：
SEQ ID NO:3，SEQ ID NO:4或SEQ ID NO:5；
根据本公开的一些实施方案，提供了一种抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段包含至少1个选自以下序列或其突变序列的LCDR：SEQ ID NO:6，SEQ ID NO:7或SEQ ID NO:8。
在本公开的一个优选方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述抗BCMA抗体或其抗原结合片段包含重链可变区， 所述重链可变区包含：
SEQ ID NO:3的序列或其突变序列所示的HCDR1、
SEQ ID NO:4的序列或其突变序列所示的HCDR2和
SEQ ID NO:5的序列或其突变序列所示的HCDR3。
在本公开的一个优选方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述抗BCMA抗体或其抗原结合片段的轻链可变区包含：
SEQ ID NO:6的序列或其突变序列所示的LCDR1、
SEQ ID NO:7的序列或其突变序列所示的LCDR2和
SEQ ID NO:8的序列或其突变序列所示的LCDR3。
在本公开的一个优选实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段包含抗体重链可变区和轻链可变区，所述重链可变区包含：
SEQ ID NO:3的突变序列所示的HCDR1、
SEQ ID NO:4的突变序列所示的HCDR2和
SEQ ID NO:5的突变序列所示的HCDR3；以及
所述轻链可变区包含：
SEQ ID NO:6的突变序列所示的LCDR1、
SEQ ID NO:7的突变序列所示的LCDR2和
SEQ ID NO:8的突变序列所示的LCDR3。
在本公开的一个优选实施方案中，提供一种如上所述的抗BCMA抗体或其抗原结合片段，或其药学上可接受的盐，其中所述的突变序列为在CDR区发生1-3个优化抗体活性、抗体稳定性或降低免疫原性的氨基酸突变。
在本公开的一个优选实施方案中，所述的抗BCMA抗体或其抗原结合片段为鼠源抗体。
在本公开的一个优选实施方案中，所述的抗BCMA抗体或其抗原结合片段为嵌合抗体。
在本公开的一个优选实施方案中，所述的抗BCMA抗体或其抗原结合片段为人抗体。
在本公开的一个优选实施方案中，所述的抗BCMA抗体或其抗原结合片段为人源化抗体。
在本公开的一个优选实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述抗BCMA抗体或其抗原结合片段进一步包含源自人IgG1或其变体、IgG2或其变体、IgG3或其变体，或IgG4或其变体的重链恒定区。
在本公开的一个优选实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段及其药学上可接受的盐，所述抗BCMA抗体或其抗原结合片段进一步包含源 自人IgG1、IgG2或IgG4的重链恒定区。
在本公开的一个优选实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段及其药学上可接受的盐，所述抗BCMA抗体或其抗原结合片段进一步包含氨基酸突变后具有增强的ADCC毒性的IgG1重链恒定区。
在本公开的一个优选实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段及其药学上可接受的盐，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO:22所示的重链恒定区。
在本公开的一个优选实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段进一步包含源自人κ链或其变体，λ链或其变体的轻链恒定区。
在本公开的一个优选实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO:23所示的轻链恒定区。
在本公开的一个进一步优选的实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐包含选自以下序列所示的重链可变区：SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11。
在本公开的一个进一步优选的实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段包含选自与SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区。
在本公开的一个进一步优选的实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，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：SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14。
在本公开的一个进一步优选的实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段包含选自与SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区。
在本公开的一个进一步优选的实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段含有如下序列的重链：SEQ ID NO：15、SEQ ID NO：16或SEQ ID NO：17。
在本公开的一个进一步优选的实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段包含选自与SEQ ID NO：15、SEQ ID NO：16或SEQ ID NO：17具有至少80％，85％，90％，95％或99％同一性的重链。
在本公开的一个进一步优选的实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段含有如下序列的轻链：SEQ ID NO：18、SEQ ID NO：19或SEQ ID NO：20。
在本公开的一个进一步优选的实施方案中，根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，所述的抗BCMA抗体或其抗原结合片段包含选自与SEQ ID NO：18、SEQ ID NO：19或SEQ ID NO：20具有至少80％，85％，90％，95％或99％同一性的轻链。
在本公开的一个更优选实施方案中，所述的抗BCMA抗体或其抗原结合片段包含SEQ ID NO：9所示的重链可变区和SEQ ID NO：12所示的轻链可变区。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区选自SEQ ID NO：9，或与SEQ ID NO：9具有至少70％，75％，80％，85％，90％，95％或99％的同一性，所述轻链可变区选自SEQ ID NO：12或与SEQ ID NO：12具有至少70％，75％，80％，85％，90％，95％或99％的同一性。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含SEQ ID NO：10所示的重链可变区和SEQ ID NO：13所示的轻链可变区。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区选自SEQ ID NO：10，或与SEQ ID NO：10具有至少70％，75％，80％，85％，90％，95％或99％的同一性，所述轻链可变区选自SEQ ID NO：13或与SEQ ID NO：13具有至少70％，75％，80％，85％，90％，95％或99％的同一性。
在本公开的一个更优选的实施方案中，所述的抗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。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区选自SEQ ID NO：11，或与SEQ ID NO：11具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，所述轻链可变区选自SEQ ID NO：14或与SEQ ID NO：14具有至少70％，75％，80％，85％，90％，95％或99％同一性。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含SEQ ID NO：15所示的重链和SEQ ID NO：18所示的轻链。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含重链和轻链，所述重链选自SEQ ID NO：15，或与SEQ ID NO：15具有至少80％，85％，90％，95％或99％的同一性，所述轻链选自SEQ ID NO：18，或与SEQ ID NO：18具有至少80％，85％，90％，95％或99％的同一性。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含SEQ ID NO：16所示的重链和SEQ ID NO：19所示的轻链。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含重链和轻链，所述重链选自SEQ ID NO：16，或与SEQ ID NO：16具有至少80％，85％，90％，95％或99％的同一性，所述轻链选自SEQ ID NO：19，或与SEQ ID NO：19具有至少80％，85％，90％，95％或99％的同一性。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含SEQ ID NO：17所示的重链和SEQ ID NO：20所示的轻链。
在本公开的一个更优选的实施方案中，所述的抗BCMA抗体或其抗原结合片段包含重链和轻链，所述重链选自SEQ ID NO：17，或与SEQ ID NO：17具有至少80％，85％，90％，95％或99％的同一性，所述轻链选自SEQ ID NO：20，或与SEQ ID NO：20具有至少80％，85％，90％，95％或99％的同一性。
在本公开的又一个实施方案中，提供了一种多核苷酸，其编码本公开的抗BCMA抗体或其抗原结合片段。
在本公开的又一个实施方案中，提供了一种表达载体，其含有本公开所述的多核苷酸。
在本公开的又一个实施方案中，提供了一种宿主细胞，其导入或含有本公开所述的表达载体。
在本公开的又一个实施方案中，提供了一种宿主细胞，其转化有本公开所述的表达载体。
在本公开一个优选的方案中，所述的宿主细胞为细菌，优选为大肠杆菌。
在本公开一个优选的方案中，所述的宿主细胞为酵母菌，优选为毕赤酵母。
在本公开一个优选的方案中，所述的宿主细胞为哺乳动物细胞，优选为CHO细胞或HEK293细胞。
另一方面，本公开提供了一种生产抗BCMA抗体的方法，包括步骤：
培养本公开所述的宿主细胞；
从培养物中分离抗体；以及
对所述抗体进行纯化。
根据本公开提供的生产抗BCMA抗体的方法，其中：
所述培养本公开所述的宿主细胞，优选HEK293细胞；
所述从培养物中分离抗体，优选细胞培养液；以及
所述对所述抗体进行亲和层析纯化，优选以层析方法纯化抗体。
在本公开的又一个实施方案中，提供了一种药物组合物，其含有本公开所述的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，以及可药用的赋形剂、稀释剂或载体。
在本公开的又一个实施方案中，提供了一种检测或诊断试剂，其含有本公开所述的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐。
在本公开的又一个实施方案中，提供了一种检测或诊断试剂盒，其含有本公 开所述的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐、以及可用于检测或诊断的标记的第二抗体、缓冲液和底物。
在本公开的进一步的实施方案中，提供了一种检测或诊断试剂盒，其含有本公开所述的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，和一种或多种能检测该BCMA抗体或其抗原结合片段与BCMA结合的试剂。
另一方面，本公开提供了本公开所述的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，或包含其的药物组合物在用于治疗或预防BCMA介导的疾病或病症的用途。
另一方面，本公开提供了本公开所述的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，或包含其的其药物组合物在制备用于治疗或预防BCMA介导的疾病或病症的药物中的用途。
另一方面，本公开提供了一种本公开所述的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，或包含其的检测或诊断试剂在用于检测、诊断、预后BCMA介导的疾病或病症。
另一方面，本公开提供了本公开所述的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐，或包含其的检测或诊断试剂在用于制备检测、诊断、预后BCMA介导的疾病或病症的试剂盒中的用途。
在具体的实施方案中，所述的BCMA介导的疾病或病症为癌症；优选为表达BCMA的癌症；更优选为淋巴瘤、白血病或骨髓瘤，最优选为多发性骨髓瘤。
在具体的实施方案中，所述的BCMA介导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。
在另一方面，本公开提供了一种包含与细胞毒性剂偶联的本公开所述的抗BCMA抗体或其抗原结合片段的抗体药物偶联物；优选地，所述细胞毒性剂选自MMAF、SN-38和依喜替康。进一步优选地，根据本公开的抗体药物偶联物具有选自以下的结构式：
Figure PCTCN2020105408-appb-000001
Figure PCTCN2020105408-appb-000002
其中所述Ab为本公开如上所述的抗BCMA抗体或其抗原结合片段。
进一步地，本公开提供了根据本公开所述的抗体药物偶联物在制备用于治疗BCMA介导的疾病或病症的药物中的用途，所述BCMA介导的疾病或病症选自癌症和自身免疫疾病，优选地，所述癌症为表达BCMA的癌症；更优选为淋巴瘤、白血病或骨髓瘤，最优选为多发性骨髓瘤。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。
在另一方面，本公开提供了在有需要的受试者中治疗BCMA介导的疾病或病症的方法，所述方法包括向所述受试者施用治疗有效量的根据本公开的抗BCMA抗体或其抗原结合片段或其药学上可接受的盐、其药物组合物或本公开的抗体药物偶联物。
本公开提供的抗BCMA抗体或其抗原结合片段，具有高亲和力，高特异性，低免疫原性，高生物活性，抗肿瘤效果显著，同时，本公开抗体与BCMA抗原结合后能被快速内化，表明它们适合以ADC的形式用于治疗应用，或快速内化的其它应用，本公开抗体ADC的形式可以高效杀伤表达BCMA的肿瘤细胞。
发明详述
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本文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公开所属领域的一般技术人员通常理解的含义。
本公开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。
本公开所述的术语“抗体”指免疫球蛋白，其是由两条相同的重链和两条相同的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。
在本公开中，本公开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。
在本公开中，本公开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。
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个序列相对保守的框架区(FR)。3个高变区决定抗体的特异性，又称为互补决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区和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。本公开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或IMGT定义规则(http://bioinf.org.uk/abs/)。
术语“抗原呈递细胞”或“APC”是在其表面上展示与MHC复合的外来抗原的细胞。T细胞利用T细胞受体(TCR)识别这种复合物。APC的实例包括但不限于树突细胞(DC)、外周血单个核细胞(PBMC)、单核细胞、B淋巴母细胞和单核细胞衍生的树突细胞。
术语“抗原呈递”是指APC捕获抗原和使它们能够被T细胞识别的过程，例如作为MHC-I/MHC-II偶联物的组分。
术语“BCMA”包括由细胞天然表达的BCMA的任何变体或同种型。本公开的抗体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，可不表现出与其他物种的交叉反应性。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。优选地，抗BCMA抗体靶向具有正常糖基化模式的人源BCMA。
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：
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；
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；
3.从重组组合人抗体文库中分离的抗体；以及
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。
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。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人BCMA的单克隆抗体。制备时用BCMA抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开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，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变区和恒定区的抗体。本公开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”)。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的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小鼠蛋白成分，从而诱导的强烈的免疫应答的缺点。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最少反向突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答。建立嵌合抗体，要先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。
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。
“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。
“Fc”区含有包含抗体的CH1和CH2结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。
“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’)2分子。
“F(ab’)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区VH和轻链可变区VL的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双抗体(diabodies)、双特异性双抗体和三抗体(triabodies)、己共价或非共价连接在一起的抗体片段等。
术语“单链抗体”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。
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。
本公开的术语“与BCMA结合”，指能与人BCMA相互作用。
本公开的术语“抗原结合位点”指本公开抗体或抗原结合片段识别的三维空间位点。
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。
本公开所用的术语“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。
术语“交叉反应”是指本公开的抗体与来自不同物种的BCMA结合的能力。例如，结合人BCMA的本公开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。
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。
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。
术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的剌激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指剌激特定的直接细胞杀伤机制。
本公开中所述的“ADCC”，即抗体依赖性细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity)，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。
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且能找到，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)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开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。
本公开的抗体指单克隆抗体。本公开所述的单克隆抗体(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。
“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、诊断剂、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、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，诸如包含本公开的任一种抗体，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。尽本公开的实施方案(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。
整个说明书和权利要求书中使用的术语“基本上由……组成”或其变形表示包括所有所述元件或元件组，并且任选包括与所述元件类似或不同性质的其它元件，所述其它元件非显著改变指定给药方案、方法或组合物的基本性质或新性质。
本公开所述的应用于某个对象的术语“天然存在的”是指这样的事实，即该对象可在自然界中发现。例如存在于可从自然界来源分离得到的生物体(包括病 毒)、且未经人工在实验室中有意修饰的多肽序列或多核苷酸序列即是天然存在的。
“有效量”包含足以改善或预防医字病症的症状或病症的量。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。
“外源性”指要据背景在生物、细胞或人体外产生的物质。
“内源性”指根据背景在细胞、生物或人体内产生的物质。
“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％同源。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。
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。在意指不同名称的情况下，其由上下文清楚可见。
“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。
“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。
“药物载荷”(DAR)由y表示，即抗体-药物偶联物中每个抗体的平均细胞毒性药物数。来自偶联反应的抗体-药物偶联物中的药物载荷(DAR)可通过常规手段表征，诸如质谱，HPLC和ELISA等，通过这些手段可以测定抗体-药物偶联物在y值上的定量分布。
具体实施方式
以下结合实施例用于进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开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。
实施例1：抗原准备
编码带His标签的人BCMA胞外区(BCMA-His)蛋白由SinoBiologics公司合成(Cat No.:10620-H08H)。
BCMA-His序列：
Figure PCTCN2020105408-appb-000003
实施例2：鼠杂交瘤及抗体序列的获得
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。
表1.免疫方案
第1天 第一次免疫，完全弗氏佐剂
第21天 第二次免疫，不完全弗氏佐剂
第35天 第三次免疫，不完全弗氏佐剂
第42天 采血和血清效价检测(3免血)
第49天 第四次免疫，不完全弗氏佐剂
第56天 采血和血清效价检测(4免血)
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)决定启动细胞融合。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骨髓瘤细胞融合后铺板获得杂交瘤，通过间接ELISA筛选到目标杂交瘤，并通过有限稀释法建株为单克隆细胞株。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采用小鼠Ig-引物组(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。
鼠单抗M1的重链和轻链可变区序列如下：
M1 HCVR
Figure PCTCN2020105408-appb-000004
Figure PCTCN2020105408-appb-000005
M1 LCVR
Figure PCTCN2020105408-appb-000006
表2.鼠单抗M1的重链和轻链可变区CDR序列
名称 序列 编号
HCDR1 GYSFSDYEMH SEQ ID NO：3
HCDR2 GIHPGSGGSAYNQKFKG SEQ ID NO：4
HCDR3 TRLDYGYSWAWFPY SEQ ID NO：5
LCDR1 SASSSVIYMN SEQ ID NO：6
LCDR2 GISNLAS SEQ ID NO：7
LCDR3 QQRSSYPLT SEQ ID NO：8
实施例3：抗体的体外结合活性检测方法
(1)体外间接ELISA结合实验：
用pH7.4的PBS将BCMA His蛋白(Sino Biological Inc.，cat#10620-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。
用含3％BSA的PBST稀释待测抗体，1μM起始，10倍梯度，10个剂量，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/孔终止溶液(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。做浓度信号值曲线分析结果，如下表所示：
表3.鼠抗体对人BCMA抗原的亲和力(EC 50值)
鼠抗体 与人BCMA His抗原结合EC 50(nM)
M1 0.53
(2)体外细胞结合实验：
收集培养好的BCMA高表达细胞(过表达BCMA的HEK-293T细胞；表达BCMA的肿瘤细胞，NCI-H929，ATCC保藏号CRL-9068)，调节细胞密度后分铺于96孔U底板，每孔1×10 5至2×10 5个细胞。1200g，5min离心，去上清，添加100ul已梯度稀释的抗体溶液或小鼠免疫血清，4℃度孵育60min；1200g，5min离心，去上清，PBS洗细胞2次后，添加荧光标记二抗(PE-GAM或PE-GAH)100ul每孔，4℃度孵育60min。1200g，5min离心去上清。PBS洗细胞2次后，再重悬于PBS，使用流式细胞仪检测信号，并作浓度曲线分析结果。
表4.鼠抗体对表达BCMA的细胞的亲和力(EC 50值)
Figure PCTCN2020105408-appb-000007
实施例4：小鼠抗体人源化实验
鼠源抗人BCMA单克隆抗体人源化如本领域许多文献公示的方法进行。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一性选择人种抗体序列，本公开将鼠源抗体M1进行人源化。
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，获得同一性高的人种系模板。
将鼠源抗体M1的CDR区移植到选择好的相应人源化模板上。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，经表达测试和回复突变数量对比，选择和设计了人源化重链可变区HCVR的序列，序列如下：
HCVR1
Figure PCTCN2020105408-appb-000008
HCVR2
Figure PCTCN2020105408-appb-000009
HCVR3
Figure PCTCN2020105408-appb-000010
Figure PCTCN2020105408-appb-000011
选择和设计了人源化轻链可变区LCVR的序列，序列如下：
LCVR1
Figure PCTCN2020105408-appb-000012
LCVR2
Figure PCTCN2020105408-appb-000013
LCVR3
Figure PCTCN2020105408-appb-000014
将设计的重链和轻链可变区序列分别与IgG1重链和轻链恒定区序列连接，示例性的重链和轻链恒定区序列分别如下所示：
IgG1 C
Figure PCTCN2020105408-appb-000015
Ig kappa C
Figure PCTCN2020105408-appb-000016
连接后，得到的示例性重链和轻链序列如下：
Ab1 HC
Figure PCTCN2020105408-appb-000017
Figure PCTCN2020105408-appb-000018
Ab2 HC
Figure PCTCN2020105408-appb-000019
Ab3 HC
Figure PCTCN2020105408-appb-000020
Ab1 LC
Figure PCTCN2020105408-appb-000021
Ab2 LC
Figure PCTCN2020105408-appb-000022
Figure PCTCN2020105408-appb-000023
Ab3 LC
Figure PCTCN2020105408-appb-000024
表5.抗体及其重链、轻链、可变区的序列编号
Figure PCTCN2020105408-appb-000025
根据以上各人源化抗体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，得到本公开的人源化抗体蛋白。
实施例5：体外结合亲和力和动力学实验
使用实施例3(1)中所述的体外间接ELISA结合实验测定的各人源化抗体对人BCMA抗原的亲和力(EC 50)如下表6所示：
表6.各人源化抗体对人BCMA抗原的亲和力(EC 50)
Figure PCTCN2020105408-appb-000026
使用实施例3(2)中所述的体外细胞结合实验测定的各人源化抗体对NCI-H929肿瘤细胞的亲和力(EC 50)如下表7所示：
表7.各人源化抗体对NCI-H929肿瘤细胞的亲和力(EC 50)
Figure PCTCN2020105408-appb-000027
实施例6：抗体的内吞作用
检测本公开抗体结合BCMA后是否能够和人BCMA共同内吞入细胞内，用NCI-H929(ATCC保藏号CRL-9068)进行评估。NCI-H929细胞使用胰酶消化(先用PBS清洗一遍，37℃、2min左右)，收集细胞并用预冷的FACS缓冲液重悬，调整细胞浓度为1×10 6个/mL。取EP管，加入1mL细胞悬液，1500rpm离心5分钟后去上清，加入1mL已经配制好的待测抗体重悬细胞，抗体的终浓度均为20μg/ml，4度摇床孵育1h，离心弃上清(4℃、1500rpm×5min)，FACS缓冲液洗涤两次，去上清。每管加入100μL荧光二抗工作液重悬细胞，4℃摇床孵育30min，离心弃上清(4℃、1500rpm×5min)，FACS缓冲液洗涤两次，去上清。每管加入1.0mL预热的NCI-H929细胞完全培养基重悬细胞并混匀，分装为4管，每管200μL，分别为0min组，空白组，30min组和2h组，取出0min及空白组置于冰上，其余放置于37℃培养箱，分别内吞30min、2h，在相应时间点取出EP管，置于冰上预冷5min，所有处理组离心弃上清(4℃、1500rpm×5min)，用FACS缓冲液洗涤一次，去上清。除0min组外所有处理组EP管中加入250μL剥离缓冲液(strip buffer)，室温孵育8min，离心弃上清(4℃、1500rpm×5min)，FACS缓冲液洗涤两次，去上清。所有处理组加入100μL免疫染色固定液，4℃放置30min以上，用流式细胞仪DxFlex进行检测。BCMA抗体内吞百分比＝(各个时间点荧光强度值—空白组平均荧光强度值)/零点时的平均荧光轻度值—空白组平均荧光强度值。结果见下表8：
表8.抗体在NCI-H929肿瘤细胞中的内吞作用(EC 50)
Figure PCTCN2020105408-appb-000028
ND＝未确定
结果显示，与抗BCMA抗体J6M0(描述于美国专利9,273,141)相比，本公开抗体具有更高的内吞效率。
实施例7：抗体偶联MC-MMAF
本公开抗体具有细胞亲和活性且具有细胞内吞活性，使得本公开抗体适合与药物偶联形成抗体-药物偶联物用于治疗BCMA介导的疾病。将本公开的抗体与MC-MMAF偶联，形成抗体-药物偶联物。偶联过程见下式，其中Ab代表Ab2或Ab3抗体：
Figure PCTCN2020105408-appb-000029
第一步，将硫代乙酸S-(3-羟基丙基)酯(0.7mg，5.3mol)溶解于0.9mL乙腈溶液备用。向抗体pH＝4.3的乙酸/乙酸钠缓冲液(10.35mg/mL,9.0mL，0.97mol)加入上述预制的硫代乙酸S-(3-羟基丙基)酯的乙腈溶液，然后滴加1.0mL的氰基硼氢化钠(14.1mg，224mol)的水溶液，于25℃下振荡反应2小时。反应结束后，用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，得产物1f溶液，浓缩到10mg/mL后直接进行下一步反应。
第二步，向1f溶液(11.0mL)中加入0.35mL的2.0M盐酸羧胺溶液，于25℃下振荡反应30分钟后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，得到产物2f溶液(浓度6.17mg/mL，14.7mL)。
第三步，将化合物MC-MMAF(1.1mg，1.2mol，采用PCT专利WO2005081711公开的方法制备得到)溶解于0.3mL乙腈中，加入2f溶液(浓度6.17mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，在无菌条件下用滤器过滤后得到产物Ab2-MC-MMAF。使用HIC-HPLC测定产物Ab2-MC-MMAF的DAR平均值y为4，将抗体-药物偶联物的PBS缓冲液(3.7mg/mL，4.7mL)于4℃冷藏。采用上述 方法制备得到产物Ab3-MC-MMAF，使用HIC-HPLC测定产物Ab3-MC-MMAF的DAR平均值y为4.1，将抗体-药物偶联物的PBS缓冲液(3.5mg/mL，5.0mL)于4℃冷藏。
实施例8：抗体偶联SN-38
通过以下偶联过程制备抗体偶联药物，其中Ab代表Ab2：
Figure PCTCN2020105408-appb-000030
第一步，将硫代乙酸S-(3-羟基丙基)酯(0.7mg，5.3mol)溶解于0.9mL乙腈溶液备用。向抗体pH＝4.3的乙酸/乙酸钠缓冲液(10.35mg/mL,9.0mL，0.97mol)加入上述预制的硫代乙酸S-(3-羟基丙基)酯的乙腈溶液，然后滴加1.0mL的氰基硼氢化钠(14.1mg，224mol)的水溶液，于25℃下振荡反应2小时。反应结束后，用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，得产物1h溶液，浓缩到10mg/mL后直接进行下一步反应。
第二步，向1h溶液(11.0mL)中加入0.35mL的2.0M盐酸羧胺溶液，于25℃下振荡反应30分钟后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，得到产物2h溶液(浓度6.2mg/mL，15.0mL)，浓缩到约10mg/ml后用于下一步反应。
第三步，将化合物MC-SN-38(1.3mg，1.2mol)溶解于0.3ml的乙腈中，加入2h溶液(浓度6.2mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，在无菌条件下用滤器过滤后得到产物Ab-SN-38抗体-药物偶联物的PBS缓冲液(3.7mg/mL，4.7mL)，于4℃冷藏。采用紫外法测定平均值y。将装有琥珀酸钠缓冲液的比色皿分别置于参比吸收池和样品测定吸收池中后，扣除溶剂空白后， 再将装有供试品溶液的比色皿置于样品测定吸收池中，测定280nm和370nm处吸光度。
数据处理：
通过建立标准曲线，测定280nm波长下的吸收，确定抗体含量Cmab，测定370nm波长下的吸收，确定小分子含量CDrug。
药物载量平均值y＝CDrug/Cmab。
通过上述方法测定Ab2-SN-38抗体-药物偶联物的DAR平均值y为3.9。
实施例9：抗体偶联依喜替康
Figure PCTCN2020105408-appb-000031
第一步，将2a(2g，17.2mmol溶于75mL乙腈中，依次加入碳酸钾(9.27g，67.2mmol)、溴化苄(20mL，167.2mmol)和四丁基碘化铵(620mg，1.68mmol)。将反应液室温搅拌48小时，通过硅藻土过滤，滤饼用乙酸乙酯(20ml)淋洗，合并滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到产物5a(3.2g，产率：90.1％)。
第二步，将5a(181.3mg,0.879mmol)和4b(270mg，0.733mmol)加入反应瓶，加入6mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(164mg，1.46mmol)，撤去冰浴，升至室温搅拌40分钟，加入15mL冰水，用乙酸乙酯(40mL×2)和氯仿(20mL×5)萃取，合并有机相并浓缩。所得残余物溶于6mL二氧六环中，加入3mL水，加入碳酸氢钠(73.8mg，0.879mmol)和氯甲酸-9-芴甲酯(190mg，0.734mmol)，室温搅拌2小时。加入30mL水，用乙酸乙酯(20mL×3)萃取，有机相用饱和氯化钠溶液(30mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到产物5b 10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯(73mg，产率：19.4％)。
MS m/z(ESI):515.0[M+1]。
第三步，将5b(30mg，0.058mmol)溶于6.75mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(18mg，含量10％，干型)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液浓缩，得到粗品产物5c 10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸(20mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):424.9[M+1]。
第四步，将1b(15mg，28.2μmol)加入反应瓶，加入1.5mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入粗品5c(20mg，47.1μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(25.4mg，86.2μmol)，冰浴搅拌反应40分钟。加入15mL水，用乙酸乙酯(20mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(20mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物5d(9H-芴-9-基)甲基(2-(((1-环丙基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-2-氧代乙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯(23.7mg，产率：78.9％)。
MS m/z(ESI):842.1[M+1]。
第五步，将5d(30mg，35.7μmol)溶于3mL二氯甲烷中，加入1.5mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入1.5mL甲苯并减压浓缩，重复两次。向残余物中加入4.5mL正己烷打浆，静置后倾倒出上层清液，保留固体。将固体残余物减压浓缩，油泵拉干得到粗品产物5e 2-((2-氨基乙酰氨基)甲氧基)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺(23mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):638.0[M+18]。
第六步，将粗品5e(20mg，32.3μmol)溶于1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入4g(31.8mg，67.3μmol)的0.5mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(27.8mg，94.3μmol)，冰浴搅拌反应10分钟，撤去冰浴，升至室温搅拌1小时，反应生成化合物5。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um 19*250mm；流动相：A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到产物5-A和5-B(3.6mg，2.6mg)。
MS m/z(ESI):1074.4[M+1]。
单一构型化合物5-A(较短保留时间)：
UPLC分析:保留时间1.14分钟，纯度：85％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,2H),4.54-4.40(m,2H),3.76-3.64(m，4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,2H),2.80-2.62(m,2H),2.45-2.30(m,3H),2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H)。
单一构型化合物5-B(较长保留时间)：
UPLC分析:保留时间1.16分钟，纯度：89％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.13(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.60-5.50(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.78-4.68(m,1H),4.60-4.40(m,2H),3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H),3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13(m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.91-0.79(m,3H),0.53-0.34(m,4H)。
其他中间体的制备方法参考中间体5。
在37℃条件下，向抗体Ab2的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；7.3ml，13.8mg/ml，0.681μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.347mL，3.47μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至14.0ml，并取出3.3ml溶液往下反应。
将化合物5-A(3.0mg，3.72μmol)溶解于0.15mL DMSO中，加入到上述3.3ml溶液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到Ab-依喜替康的示例性产物Ab2-依喜替康的PBS缓冲液(1.35mg/mL，13mL)，于4℃冷冻储存。
采用紫外法测定平均值y。将装有琥珀酸钠缓冲液的比色皿分别置于参比吸收池和样品测定吸收池中后，扣除溶剂空白后，再将装有供试品溶液的比色皿置于样品测定吸收池中，测定280nm和370nm处吸光度。
数据处理：
通过建立标准曲线，测定280nm波长下的吸收，确定抗体含量Cmab，测定370nm波长下的吸收，确定小分子含量CDrug。
药物载量平均值y＝CDrug/Cmab。
通过以上方法测定示例性产物Ab2-依喜替康为7.6。通过UV-HPLC纯化获得Ab2-依喜替康(y＝8)样品。
实施例10：抗体药物偶联物的肿瘤杀伤活性
为进一步研究抗体-药物偶联物对体内形成的肿瘤的杀伤作用，在小鼠体内用NCI-H929细胞形成移植瘤后，评估本公开抗体-药物偶联物的抗肿瘤效果。
(1)将9x10 6个NCI-929细胞注射到8周龄的免疫缺陷的裸鼠(NOD-SCID)皮下，8天后开始通过静脉注射注射抗体-药物偶联物Ab2-MC-MMAF(实施例7，y＝4)和Ab2-依喜替康(实施例9，y＝8)，每1周注射一次，剂量为1mg/kg。对照采用人IgG1蛋白，剂量为1mg/kg。对照组或给药组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。抑瘤率＝100％-(第14天给药组肿瘤体积-第0天给药组肿瘤体积)/(第14天对照组肿瘤体积-第0天对照组肿瘤体积)。实验结果如表9所示。抗体-药物偶联物Ab2-MC-MMAF(实施例7，y＝4)和Ab2-依喜替康(实施例9，y＝8)均显示抑瘤活性。
表9抗体-药物偶联物对肿瘤的杀伤作用
Figure PCTCN2020105408-appb-000032
(2)将9x10 6个NCI-929细胞注射到8周龄的免疫缺陷的裸鼠(NOD-SCID)皮下，8天后开始通过静脉注射抗体-药物偶联物Ab2-MC-MMAF(实施例7，y＝4)和Ab3-MC-MMAF(实施例7，y＝4.1)，每1周注射2次，剂量为3mg/kg。对照采用人IgG1蛋白，剂量为3mg/kg。对照组或给药组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。抑瘤率TGI＝100％-(第14天给药组肿瘤体积-第0天给药组肿瘤体积)/(第14天对照组肿瘤体积-第0天对照组肿瘤体积)。实验结果如表10所示，Ab2-MC-MMAF(实施例7，y＝4)和Ab3-MC-MMAF(实施例7，y＝4.1)均显示对肿瘤的杀伤作用。
表10抗体-药物偶联物对肿瘤的杀伤作用
Figure PCTCN2020105408-appb-000033

Claims (23)

  1. 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，所述的重链可变区包含至少1个选自以下序列所示的HCDR：SEQ ID NO:3，SEQ ID NO:4和SEQ ID NO:5；和
    所述的轻链可变区包含至少1个选自以下序列所示的LCDR：SEQ ID NO:6，SEQ ID NO:7和SEQ ID NO:8。
  2. 如权利要求1所述的抗BCMA抗体或其抗原结合片段，其中所述的重链可变区包含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。
  3. 如权利要求1所述的抗BCMA抗体或其抗原结合片段，其中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。
  4. 如权利要求1所述的抗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。
  5. 如权利要求1-4中任一项所述的抗BCMA抗体或其抗原结合片段，其中所述的抗BCMA抗体为鼠源抗体，嵌合抗体，人抗体或人源化抗体。
  6. 如权利要求1-5中任一项所述的抗BCMA抗体或其抗原结合片段，其中所述抗BCMA抗体或其抗原结合片段进一步包含源自人IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区；
    优选地，所述抗BCMA抗体或其抗原结合片段进一步包含源自人IgG1、IgG2或IgG4的重链恒定区；
    更优选地，所述抗BCMA抗体或其抗原结合片段进一步包含氨基酸突变后具有增强的ADCC毒性的IgG1重链恒定区；
    或者，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO:22所示的IgG1重链恒定区。
  7. 如权利要求1-6中任一项所述的抗BCMA抗体或其抗原结合片段，其中所述抗BCMA抗体或其抗原结合片段进一步包含源自人κ链、λ链或其变体的轻链恒定区，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:23所示的轻链恒定区。
  8. 如权利要求1-7中任一项所述的抗BCMA抗体或其抗原结合片段，其中所述抗BCMA抗体或其抗原结合片段包含选自以下序列的重链可变区：SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11，或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区。
  9. 如权利要求1-8中任一项所述的抗BCMA抗体或其抗原结合片段，其中所述抗BCMA抗体或其抗原结合片段包含选自以下序列的轻链可变区：SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14，或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区。
  10. 如权利要求8或9所述的抗BCMA抗体或其抗原结合片段，其中所述抗BCMA抗体或其抗原结合片段包含选自如下序列的重链：SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17，或与其具有至少80％，85％，90％，95％或99％同一性的重链。
  11. 如权利要求8或9所述的抗BCMA抗体或其抗原结合片段，其中所述抗BCMA抗体或其抗原结合片段包含选自如下序列的轻链：SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20，或与其具有至少80％，85％，90％，95％或99％同一性的轻链。
  12. 如权利要求1-11中任一项所述的抗BCMA抗体或其抗原结合片段，其中：
    所述抗BCMA抗体或其抗原结合片段包含SEQ ID NO：9所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：12所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区；或，
    所述抗BCMA抗体或其抗原结合片段包含SEQ ID NO：10所示的重链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：13所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区；或，
    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、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区，和SEQ ID NO：14所示的轻链可变区、或与其具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区。
  13. 如权利要求1-12中任一项所述的抗BCMA抗体或其抗原结合片段，其中，
    所述抗BCMA抗体包含SEQ ID NO：15所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：18所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
    所述抗BCMA抗体包含SEQ ID NO：16所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：19所示的轻链、或与其具有至少80％，85％，90％，95％或99％同一性的轻链；或，
    所述抗BCMA抗体包含SEQ ID NO：17所示的重链、或与其具有至少80％，85％，90％，95％或99％同一性的重链，和SEQ ID NO：20所示的轻链；或与其具有至少80％，85％，90％，95％或99％同一性的轻链。
  14. 一种多核苷酸，其编码权利要求1-13中任一项所述的抗BCMA抗体或其抗原结合片段。
  15. 一种表达载体，其含有权利要求14所述的多核苷酸。
  16. 一种宿主细胞，其导入或含有权利要求15所述的表达载体。
  17. 如权利要求16所述的宿主细胞，其中所述的宿主细胞为细菌、酵母菌或哺乳动物细胞，优选为大肠杆菌、毕赤酵母、CHO细胞或HEK293细胞。
  18. 一种生产抗BCMA抗体的方法，包括步骤：
    培养权利要求16-17中任一项所述的宿主细胞，优选HEK293细胞；
    从培养物中分离抗体，优选细胞培养液；以及
    对所述抗体进行纯化，优选地，以层析方法纯化抗体。
  19. 一种药物组合物，其含有如权利要求1-13中任一项所述的抗BCMA抗体或其抗原结合片段，以及可药用的赋形剂或载体。
  20. 一种检测或诊断试剂盒，其含有如权利要求1-13中任一项所述的抗BCMA抗体或其抗原结合片段，任选地，所述检测或诊断试剂盒还包含一种或多种能检测该BCMA抗体或其抗原结合片段与BCMA结合的试剂。
  21. 一种抗体药物偶联物，其包含与细胞毒性剂偶联的如权利要求1-13中任一项所述的抗BCMA抗体或其抗原结合片段，优选地，所述细胞毒性剂选自MMAF、SN-38和依喜替康。
  22. 根据权利要求1-13中任一项所述的抗BCMA抗体或其抗原结合片段、或 根据权利要求19所述的药物组合物、或根据权利要求21所述的抗体药物偶联物在制备用于治疗或预防BCMA介导的疾病或病症的药物中的用途，优选地，所述BCMA介导的疾病或病症为癌症或自身免疫疾病，其中所述癌症优选为表达BCMA的癌症，更优选为淋巴瘤、白血病或骨髓瘤，最优选为多发性骨髓瘤，所述自身免疫疾病优选选自红斑狼疮、IgA肾病和风湿性关节炎。
  23. 根据权利要求1-13中任一项所述的抗BCMA抗体或其抗原结合片段在用于制备检测、诊断、或预后BCMA介导的疾病或病症的试剂盒中的用途，优选地，所述BCMA介导的疾病或病症为癌症或自身免疫疾病，其中所述癌症优选为表达BCMA的癌症，更优选为淋巴瘤、白血病或骨髓瘤，最优选为多发性骨髓瘤，所述自身免疫疾病优选选自红斑狼疮、IgA肾病和风湿性关节炎。
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