WO2023104100A1 - 结合bcma的抗体及其用途 - Google Patents

结合bcma的抗体及其用途 Download PDF

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WO2023104100A1
WO2023104100A1 PCT/CN2022/137266 CN2022137266W WO2023104100A1 WO 2023104100 A1 WO2023104100 A1 WO 2023104100A1 CN 2022137266 W CN2022137266 W CN 2022137266W WO 2023104100 A1 WO2023104100 A1 WO 2023104100A1
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seq
amino acid
antibody
bcma
variable region
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French (fr)
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危华锋
许丹
比尼亚 达洛夫斯基·戴安娜
普里恩斯·比安卡
博兰·奈德赛卡恩
盖根·詹姆斯
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信达细胞制药(苏州)有限公司
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the invention relates to the field of antibody medicine. Specifically, the present invention relates to an affinity-improved antibody specifically binding to B-cell maturation antigen (BCMA), an antibody comprising the P329G mutation thereof, and a composition containing the same. Furthermore, the present invention relates to nucleic acids encoding said antibodies and host cells comprising said nucleic acids, as well as methods for producing said antibodies. The invention also relates to the therapeutic and diagnostic uses of these BCMA-binding antibodies.
  • BCMA B-cell maturation antigen
  • BCMA B cell maturation antigen
  • CD269 CD269, TNFRSF17
  • TNFRSF tumor necrosis factor receptor superfamily
  • BCMA is a type III transmembrane protein with a cysteine-rich domain (CRD) characteristic of TNFR family members in the extracellular domain (ECD), which forms a ligand-binding motif.
  • Ligands of BCMA include B-cell activating factor (BAFF) and B-cell proliferation-inducing ligand (APRIL), wherein B-cell proliferation-inducing ligand (APRIL) binds to BCMA with a higher affinity to promote tumor cell proliferation.
  • BAFF B-cell activating factor
  • APRIL B-cell proliferation-inducing ligand
  • BCMA is mainly expressed on the surface of mature B cells, that is, plasma cells, and is not expressed in normal hematopoietic stem cells and non-blood-derived tissues. BCMA signaling is indispensable for the survival of long-lasting bone marrow plasma cells, but it is not necessary for overall B cell homeostasis. BCMA on the membrane surface can be cleaved by ⁇ -secretase and shed, and the resulting soluble BCMA (sBCMA) may reduce BCMA signal transduction on the membrane surface by blocking BAFF/APRIL ligand binding.
  • BCMA was found to be overexpressed in multiple myeloma (Multiple Myeloma, MM) cells, which up-regulates canonical and non-canonical NF- ⁇ B signaling, promotes the growth, survival, and adhesion of MM cells, and induces osteoclasts BCMA expression has become an important marker for the diagnosis of MM.
  • MM Multiple Myeloma
  • sBCMA in the serum of MM patients increases, which is proportional to the number of MM cells in the bone marrow, and its concentration changes are closely related to the prognosis and treatment response of MM.
  • Multiple myeloma also known as plasmacytoma or Kahler's disease, is an intractable malignancy of the B-cell lineage characterized by abnormal proliferation of plasma cells.
  • BCMA is only expressed in plasma cells and not expressed in natural and memory B cells
  • BCMA has become a popular target for the treatment of B cell malignancies, especially multiple myeloma.
  • the present invention fulfills this need by providing antibodies that bind BCMA with high target specificity and high affinity, especially to BCMA expressed on the surface of tumor cells.
  • the antibody or antigen-binding fragment specifically binding to BCMA of the present invention has one or more of the following properties:
  • BCMA with high affinity such as human BCMA, cynomolgus monkey BCMA and mouse BCMA
  • the K D of the binding between the anti-BCMA antibody or its antigen-binding fragment and BCMA is about 10 ⁇ 9 M to about 10-12 M, as measured by ForteBio Kinetic Binding Assay;
  • the invention provides an antibody or antigen-binding fragment that specifically binds BCMA, comprising
  • amino acid changes are additions, deletions or substitutions of amino acids.
  • an antibody or antigen-binding fragment of the invention that specifically binds BCMA comprises a heavy chain variable region and a light chain variable region, wherein:
  • said heavy chain variable region comprises HCDR1 shown in GSIVSSYYWT (SEQ ID NO: 19) according to Kabat numbering, or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; HCDR2 represented by SISIAGSTYYNPSLKS (SEQ ID NO: 20), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 represented by ARDRGDTILDV (SEQ ID NO: 21), or A variant of no more than 2 amino acid changes or no more than 1 amino acid change of the HCDR3;
  • the light chain variable region comprises LCDR1 shown in RASQSISRYLN (SEQ ID NO: 28) according to Kabat numbering, or the LCDR1 A variant of no more than 2 amino acid changes or no more than 1 amino acid change; the LCDR2 shown in AASSLQS (SEQ ID NO: 29), or no more than 2 amino acid changes or no more than 1 amino acid change of said
  • said heavy chain variable region comprises HCDR1 according to GSIVSSYYWT (SEQ ID NO: 37) of Kabat numbering, or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; HCDR2 set forth in SISIAGSTYYNPSLKS (SEQ ID NO:38), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 set forth in ARDRGDQILDV (SEQ ID NO:39), or A variant of no more than 2 amino acid changes or no more than 1 amino acid change of the HCDR3;
  • the light chain variable region comprises LCDR1 shown in RASQSISRYLN (SEQ ID NO: 46) according to Kabat numbering, or the LCDR1 A variant of no more than 2 amino acid changes or no more than 1 amino acid change; the LCDR2 shown in AASSLQS (SEQ ID NO: 47), or no more than 2 amino acid changes or no more than 1 amino acid change
  • said heavy chain variable region comprises HCDR1 shown in GTFSNDVIS (SEQ ID NO: 73) according to Kabat numbering, or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; HCDR2 represented by VIIPIFGIANYAQKFQG (SEQ ID NO: 74), or a variant of said HCDR2 having no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 represented by ARGRGYYSSWLLDI (SEQ ID NO: 75), or A variant of no more than 2 amino acid changes or no more than 1 amino acid change of the HCDR3;
  • the light chain variable region comprises LCDR1 shown in QASQDITNYLN (SEQ ID NO: 82) according to Kabat numbering, or the LCDR1 A variant of no more than 2 amino acid changes or no more than 1 amino acid change; the LCDR2 shown in DASNLET (SEQ ID NO: 83), or no more than 2 amino acid changes or no more than 1
  • said heavy chain variable region comprises HCDR1 shown in GTFSNDVIS (SEQ ID NO:91) according to Kabat numbering, or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; HCDR2 represented by VIIPIFGIANYAQKFQG (SEQ ID NO: 92), or a variant of said HCDR2 having no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 represented by ARGRGYYSSWLHDI (SEQ ID NO: 93), or A variant of no more than 2 amino acid changes or no more than 1 amino acid change of the HCDR3;
  • the light chain variable region comprises LCDR1 shown in QASQDITNYLN (SEQ ID NO: 100) according to Kabat numbering, or the LCDR1 A variant of no more than 2 amino acid changes or no more than 1 amino acid change; LCDR2 represented by DASNLET (SEQ ID NO: 101), or no more than 2 amino acid changes or no more than 1 amino acid change
  • amino acid changes are additions, deletions or substitutions of amino acids.
  • an antibody or antigen-binding fragment of the invention that specifically binds BCMA comprises a heavy chain variable region and a light chain variable region, wherein
  • the heavy chain variable region comprises HCDR1 shown in GSIVSSYYWT (SEQ ID NO: 19); HCDR2 shown in SISIAGSTYYNPSLKS (SEQ ID NO: 20); and HCDR3 shown in ARDRGDTILDV (SEQ ID NO: 21)
  • the light chain variable region comprises LCDR1 shown in RASQSISRYLN (SEQ ID NO:28); LCDR2 shown in AASSLQS (SEQ ID NO:29); and LCDR3 shown in QQKYFDIT (SEQ ID NO:30);
  • the heavy chain variable region comprises HCDR1 shown in GSIVSSYYWT (SEQ ID NO:37); HCDR2 shown in SISIAGSTYYNPSLKS (SEQ ID NO:38); and HCDR3 shown in ARDRGDQILDV (SEQ ID NO:39)
  • the light chain variable region comprises LCDR1 shown in RASQSISRYLN (SEQ ID NO:46); LCDR2 shown in AASSLQS (SEQ ID NO:47); and LCDR3 shown in QQKYFDIT (SEQ ID NO:48);
  • the heavy chain variable region comprises HCDR1 shown in GTFSNDVIS (SEQ ID NO:73); HCDR2 shown in VIIPIFGIANYAQKFQG (SEQ ID NO:74); and HCDR3 shown in ARGRGYYSSWLLDI (SEQ ID NO:75)
  • the light chain variable region comprises LCDR1 shown in QASQDITNYLN (SEQ ID NO:82); LCDR2 shown in DASNLET (SEQ ID NO:83); and LCDR3 shown in QQAFDLIT (SEQ ID NO:84); or
  • the heavy chain variable region comprises HCDR1 shown in GTFSNDVIS (SEQ ID NO:91); HCDR2 shown in VIIPIFGIANYAQKFQG (SEQ ID NO:92); and HCDR3 shown in ARGRGYYSSWLHDI (SEQ ID NO:93)
  • the light chain variable region comprises LCDR1 shown in QASQDITNYLN (SEQ ID NO: 100); LCDR2 shown in DASNLET (SEQ ID NO: 101); and LCDR3 shown in QQAFDLIT (SEQ ID NO: 102).
  • an antibody or antigen-binding fragment of the invention that specifically binds BCMA comprises a heavy chain variable region and a light chain variable region, wherein
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 27 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 36 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 45 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 54 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 81 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 90 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence
  • the heavy chain variable region comprises a sequence of SEQ ID NO: 99 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% therewith , 98% or 99% identical sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 108 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Sequences that are 97%, 98% or 99% identical.
  • an antibody or antigen-binding fragment of the invention that specifically binds BCMA comprises a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment comprises
  • an antibody or antigen-binding fragment of the invention that specifically binds BCMA is an IgGl, IgG2, IgG3, or IgG4 antibody; optionally, it is an IgGl or IgG4 antibody; optionally, it is an IgGl antibody.
  • the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, single chain Fv, single chain Fab, or diabody.
  • the antibody or antigen-binding fragment of the invention that specifically binds BCMA further comprises a mutant Fc domain, wherein the amino acid at position P329 according to EU numbering is mutated to glycine (G), which is different from the Fc domain of the unmutated parental antibody.
  • the Fc gamma receptor binding of the mutant Fc domain is reduced compared to the Fc gamma receptor binding of the Fc gamma domain; for example, the mutant Fc domain is a mutant Fc domain of an IgG1, IgG2, IgG3 or IgG4 antibody, preferably, the mutation The Fc domain is a mutant Fc domain of an IgG1 or IgG4 antibody; more preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 antibody;
  • the antibody or antigen-binding fragment comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the heavy chain constant region sequence shown in SEQ ID NO: 111 , a sequence of 98% or 99% identity and wherein the amino acid at position P329 according to EU numbering is mutated to G;
  • the antibody or antigen-binding fragment comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the heavy chain constant region sequence shown in SEQ ID NO: 111 , a sequence of 98% or 99% identity and wherein the amino acid at position P329 according to EU numbering is mutated to G; and the light chain constant region sequence shown in SEQ ID NO: 112 or at least 90%, 91%, 92% therewith %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences;
  • the antibody or antigen-binding fragment comprises a heavy chain constant region sequence set forth in SEQ ID NO:111 and a light chain constant region sequence set forth in SEQ ID NO:112.
  • the present invention provides a nucleic acid encoding the antibody of the first aspect of the invention, a vector comprising the nucleic acid encoding the antibody, a host cell comprising the nucleic acid molecule or vector, and a method for producing the antibody, wherein The method includes culturing the antibody encoding the BCMA molecule that specifically binds to the BCMA molecule described in the first aspect, under conditions suitable for expressing the nucleic acid encoding the antibody or antigen-binding fragment that specifically binds to the BCMA molecule described in the first aspect of the present invention.
  • the method further includes recovering the antibody that specifically binds to the BCMA molecule from the host cell Antibodies or antigen-binding fragments.
  • the host cell is prokaryotic or eukaryotic, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof, most preferably, the Host cells are HEK293 cells or CHO cells.
  • the invention relates to a conjugate, fusion or bispecific antibody comprising an anti-BCMA antibody or an antigen-binding fragment thereof of the first aspect of the invention.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-BCMA antibody or antigen-binding fragment thereof of the first aspect of the present invention, or the conjugate, fusion or bispecific antibody of the third aspect of the present invention, and any Optionally a pharmaceutically acceptable carrier.
  • the present invention relates to the anti-BCMA antibody or antigen-binding fragment thereof of the first aspect of the present invention, the conjugate, fusion or bispecific antibody of the third aspect of the present invention, or the pharmaceutical combination of the fourth aspect of the present invention
  • a substance for the preparation of a medicament for preventing or treating a B-cell-related disease in a subject for example, the B-cell-related disease is selected from: B-cell malignancies, plasma cell malignancies, autoimmune diseases, preferably selected from From: Multiple myeloma, Non-Hodgkin's lymphoma, B-cell proliferation of uncertain malignant potential, Lymphomatoid granulomatosis, Post-transplantation lymphoproliferative disorder, Immunomodulatory disorder, Rheumatoid arthritis, Myasthenia gravis, Idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves disease, Wegener's granulomatosis, polyarte
  • the present invention provides a kit for detecting BCMA in a sample, the kit comprising the anti-BCMA antibody or antigen-binding fragment thereof of the first aspect of the present invention, for performing the following steps:
  • Figure 1 shows that the ADI-38497 antibody only binds to H929 cells expressing BCMA antigen, but not to BCMA knockout BCMA-KO-H929 cells.
  • Fig. 2A shows a schematic diagram of a method for measuring antibody affinity by surface plasmon resonance (SPR).
  • Figure 2B shows representative affinity profiles of the ADI-38497 PG antibody to recombinant human, cynomolgus monkey, mouse, rat, and rabbit BCMA proteins measured by SPR.
  • Figure 2C shows the binding ability of P329G BCMA antibody to CHO-GS cells stably expressing human, cynomolgus and mouse BCMA.
  • Figure 2D shows the binding activity of P329G BCMA antibody to BCMA-positive multiple myeloma cell lines MM.1s, RPMI8226, U266, H929, L363 and AMO1.
  • Figure 3A shows the ability of ADI-38497 WT antibody and ADI-38497 PG antibody to mediate ADCC killing.
  • Figure 3B shows the ability of ADI-38497 WT antibody and ADI-38497 PG antibody to mediate ADCP killing.
  • Figure 3C shows the ability of the ADI-38497 PG antibody to mediate lysis of target cells.
  • Figure 4A and Figure 4B show the results of pharmacokinetic experiments of ADI-38497 PG antibody in mice.
  • Figure 5A shows the therapeutic effect of ADI-38497 PG antibody in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 high-expressing BCMA tumor cells.
  • Figure 5B shows the changes in body weight of immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 high-expressing BCMA tumor cells treated with ADI-38497 PG antibody.
  • Figure 6A shows the antitumor efficacy of different doses of ADI-38497 PG antibody in immunodeficient tumor-bearing mice inoculated with human H929-luc tumor cells in the tail vein.
  • Figure 6B shows the changes in body weight of immunodeficient tumor-bearing mice inoculated with human H929-luc tumor cells in the tail vein when treated with ADI-38497 PG antibody.
  • Figure 7A shows the therapeutic effect of ADI-38497 PG antibody in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells.
  • Figure 7B shows the changes in body weight of immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells when treated with ADI-38497 PG antibody.
  • Figure 7C and Figure 7D show the results of mouse hematology and blood biochemical tests in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells when treated with ADI-38497 PG antibody.
  • BCMA and “B cell maturation antigen” are used interchangeably and include variants, isoforms, species homologs and analogs of human BCMA having at least one epitope identical to BCMA (e.g., human BCMA) .
  • BCMA proteins may also include fragments of BCMA, such as the extracellular domain and fragments of the extracellular domain, eg, fragments that retain the ability to bind to any antibody of the invention.
  • BCMA antibody As used herein, the terms “BCMA antibody”, “antibody against BCMA”, “antibody that specifically binds BCMA”, “antibody that specifically targets BCMA”, “antibody that specifically recognizes BCMA” are used interchangeably, Means an antibody capable of specifically binding to B-cell maturation antigen (BCMA).
  • antibody is used herein in the broadest sense to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), single chain antibodies, whole antibodies and antibody fragments.
  • the antibodies of the invention are single domain antibodies or heavy chain antibodies.
  • Antibody fragment or "antigen-binding fragment” are used interchangeably herein to refer to a molecule, other than an intact antibody, that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain Fv, single chain Fab, or diabodies.
  • An antibody exhibiting the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody capable of binding at least 50%, 60%, 70%, 80%, 90% or more than 95% of the reference antibody affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point”).
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of the heavy chain are generally referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including, for example, antibody-based three-dimensional Chothia (Chothia et al.
  • a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplified CDRs of the present invention).
  • a reference CDR sequence eg, any of the exemplified CDRs of the present invention.
  • reference is made to numbering positions according to the Kabat numbering system.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the region of minimal overlap can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • a minimal binding unit may be a subsection of a CDR.
  • the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by conservative amino acid residues.
  • a “humanized” antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • all or substantially all of the CDRs (eg, CDRs) in the humanized antibody correspond to those of a non-human antibody
  • all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.
  • Human antibody refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human Antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • the Fc region of an immunoglobulin comprises two constant domain domains, CH2 and CH3, and in other embodiments, the Fc region of an immunoglobulin comprises three constant domains, CH2, CH3 and CH4.
  • Binding of IgG to Fc ⁇ receptors or C1q is dependent on residues localized in the hinge region and CH2 domain. Two regions of the CH2 domain are critical for Fc ⁇ R and complement C1q binding and have unique sequences in IgG2 and IgG4. Substitution of residues 233-236 in human IgG1 and IgG2 and substitution of residues 327, 330 and 331 in human IgG4 has been shown to substantially reduce ADCC and CDC activity (Armour et al., Eur. J. Immunol. 29(8 ), 1999, 2613-2624; Shields et al., J. Biol. Chem. 276(9), 2001, 6591-6604).
  • Functional Fc region and “functional Fc region” and similar terms may be used interchangeably, referring to an Fc region having effector functions of a wild-type Fc region.
  • Variant Fc region “Fc mutant”, “Fc region carrying a mutation”, “mutant Fc region”, “Fc region variant”, “Fc variant”, “variant Fc region” and “mutated Fc region” Similar terms such as “region” can be used interchangeably, and refer to an Fc region comprising at least one amino acid modification that is distinguished from a native sequence Fc region/wild-type Fc region.
  • a variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by one or more amino acid substitutions, deletions or additions.
  • the variant Fc region has at least one amino acid substitution compared to the Fc region of wild-type IgG, the at least one amino acid substitution being a glycine (G) substitution of the amino acid at position P329 according to EU numbering.
  • Fc receptor refers to a molecule that binds the Fc region of an antibody.
  • the FcR is a native human FcR.
  • the FcR is a receptor that binds an IgG antibody, namely an Fc ⁇ R, including three receptors, Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), and Fc ⁇ RIII (CD16), and allelic variants and variants of these receptors. splice form.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA and Fc ⁇ RIIB
  • Fc ⁇ RIII receptors include Fc ⁇ RIIIA and Fc ⁇ RIIIB.
  • effector functions refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with immunoglobulin isotype.
  • immunoglobulin effector functions include: Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen Uptake of antigen by presenting cells, Clq binding and complement-dependent cytotoxicity (CDC), downregulation of cell surface receptors (eg, B cell receptors), and B cell activation.
  • antibody-dependent cell-mediated cytotoxicity is one of the main mechanisms by which certain cytotoxic effector cells, such as natural killer (NK) cells, mediate killing of target cells and foreign host cells.
  • cytotoxic effector cells such as natural killer (NK) cells
  • NK natural killer
  • the antibodies of the invention provide antibody-dependent cytotoxicity of T lymphocytes, enhance antibody-dependent cytotoxicity of NK cells.
  • ADCP antibody-dependent cellular phagocytosis
  • complement-dependent cytotoxicity refers to the lysis of target cells in the presence of complement.
  • the complement system is part of the innate immune system made up of a series of proteins.
  • the proteins of the complement system are called “complement”, represented by abbreviations C1, C2, C3, etc., which are a group of heat-labile proteins that exist in human or vertebrate serum and interstitial fluid, and have enzymatic activity after activation.
  • CIq is the first component of the complement-dependent cytotoxicity (CDC) pathway, capable of binding six antibodies, but binding to two IgGs is sufficient to activate the complement cascade.
  • Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind the relevant antigen, activating a series of complement cascades that form holes in the target cell membrane, resulting in target cell death.
  • C1q first component of the complement system
  • a CDC assay can be performed, for example, by the method described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).
  • variable region refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen.
  • the variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • bind or “specifically bind” means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm layer interferometry techniques or other conventional binding assays known in the art.
  • a “conjugate” is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.
  • inhibitor refers to the reduction of some parameter (eg, activity) of a given molecule.
  • this term includes substances that inhibit the activity of a given molecule (eg, BCMA) by at least 5%, 10%, 20%, 30%, 40% or more. Therefore, inhibition need not be 100%.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rodents). mouse).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rodents.
  • rodents e.g., mice and rodents.
  • an individual or subject is a human.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • cancer and “cancerous” refer to the physiological disorder of unregulated cell growth in mammals.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancerous and cancerous cells and tissues.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as an antibody, and facilitates detection of the agent to which it is conjugated or fused. Labels can themselves be detectable (eg, radioisotopic or fluorescent labels) or, in the case of enzymatic labels, can catalyze the chemical alteration of a detectable substrate compound or composition.
  • the term is intended to encompass direct labeling of a probe or antibody by conjugating (ie, physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.
  • an “isolated” antibody means that it has been separated from a component of its natural environment.
  • the BCMA antibody is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse determined by HPLC).
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse determined by HPLC.
  • nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • isolated nucleic acid encoding an antibody to BCMA refers to one or more nucleic acid molecules encoding a chain of an antibody to BCMA or fragments thereof, including such nucleic acid molecules in a single vector or in separate vectors, as well as present in a host cell Such nucleic acid molecules at one or more positions.
  • the sequences are aligned for optimal comparison purposes (e.g., a first and second amino acid sequence or nucleic acid sequence may be placed between a first and a second amino acid sequence or nucleic acid sequence for optimal alignment). Gaps may be introduced in one or both or non-homologous sequences may be discarded for comparison purposes).
  • the length of the aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http://www.gcg.com available), use the Blossum 62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 to determine the distance between two amino acid sequences. percent identity.
  • using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and Length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • amino acid change and “amino acid modification” are used interchangeably to refer to additions, deletions, substitutions and other modifications of amino acids. Any combination of amino acid additions, deletions, substitutions and other modifications can be made, provided that the final polypeptide sequence possesses the desired properties.
  • amino acid substitutions to the antibody result in decreased binding of the antibody to an Fc receptor.
  • non-conservative amino acid substitutions ie the substitution of one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred.
  • Amino acid substitutions include non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxy lysine) substitution.
  • Amino acid changes can be made using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. A method of changing amino acid side chain groups by methods other than genetic engineering, such as chemical modification, may be useful.
  • Various names may be used herein to refer to the same amino acid change. For example, a substitution from proline to glycine at position 329 of the Fc domain may be denoted as 329G, G329, G329 , P329G Pro329Gly, or simply "PG".
  • conservative sequence modification refers to an amino acid modification or change that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment comprising an amino acid sequence.
  • conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into antibodies or antibody fragments of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • a conservative substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), ⁇ -side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenyl Alanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • composition refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. ingredients.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, buffer or stabilizer, etc. with which the active substance is administered.
  • BCMA-associated disease refers to any condition caused by, aggravated by, or otherwise associated with increased expression or activity of BCMA.
  • treating means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibody molecules of the invention are used to delay the development of a disease or to slow the progression of a disease.
  • prevention includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
  • an effective amount refers to such an amount or dose of an antibody or composition of the present invention that, after administration to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as: the species of mammal; body weight, age and general health; the particular disease involved; the extent or severity of the disease; the individual The patient's response; the specific antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.
  • a “therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount of an antibody or antibody fragment or composition thereof can vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition thereof are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, relative to an untreated subject. 60% or 70% and still more preferably at least about 80% or 90%.
  • Compounds can be evaluated for their ability to inhibit a measurable parameter (eg, cancer) in animal model systems predictive of efficacy in human tumors.
  • prophylactically effective amount refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • vector refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity in originally transformed cells are included herein.
  • a host cell is any type of cellular system that can be used to produce an antibody molecule of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
  • Host cells include cultured cells as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.
  • Subject/patient sample refers to a collection of cells, tissues or body fluids obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood components; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid ), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject.
  • Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • tumor samples herein include, but are not limited to, tumor biopsy, fine needle aspirate, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimen, circulating tumor cells, serum, plasma, circulating Plasma proteins in ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples, or frozen tumors sample.
  • the invention provides antibodies that bind BCMA with high target specificity and high affinity, comprising a heavy chain variable region and a light chain variable region, wherein:
  • said heavy chain variable region comprises HCDR1 shown in GSIVSSYYWT (SEQ ID NO: 19) according to Kabat numbering, or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; HCDR2 represented by SISIAGSTYYNPSLKS (SEQ ID NO: 20), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 represented by ARDRGDTILDV (SEQ ID NO: 21), or A variant of no more than 2 amino acid changes or no more than 1 amino acid change of the HCDR3;
  • the light chain variable region comprises LCDR1 shown in RASQSISRYLN (SEQ ID NO: 28) according to Kabat numbering, or the LCDR1 A variant of no more than 2 amino acid changes or no more than 1 amino acid change; the LCDR2 shown in AASSLQS (SEQ ID NO: 29), or no more than 2 amino acid changes or no more than 1 amino acid change of said
  • said heavy chain variable region comprises HCDR1 according to GSIVSSYYWT (SEQ ID NO: 37) of Kabat numbering, or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; HCDR2 set forth in SISIAGSTYYNPSLKS (SEQ ID NO:38), or a variant of said HCDR2 with no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 set forth in ARDRGDQILDV (SEQ ID NO:39), or A variant of no more than 2 amino acid changes or no more than 1 amino acid change of the HCDR3;
  • the light chain variable region comprises LCDR1 shown in RASQSISRYLN (SEQ ID NO: 46) according to Kabat numbering, or the LCDR1 A variant of no more than 2 amino acid changes or no more than 1 amino acid change; the LCDR2 shown in AASSLQS (SEQ ID NO: 47), or no more than 2 amino acid changes or no more than 1 amino acid change
  • said heavy chain variable region comprises HCDR1 shown in GTFSNDVIS (SEQ ID NO: 73) according to Kabat numbering, or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; HCDR2 represented by VIIPIFGIANYAQKFQG (SEQ ID NO: 74), or a variant of said HCDR2 having no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 represented by ARGRGYYSSWLLDI (SEQ ID NO: 75), or A variant of no more than 2 amino acid changes or no more than 1 amino acid change of the HCDR3;
  • the light chain variable region comprises LCDR1 shown in QASQDITNYLN (SEQ ID NO: 82) according to Kabat numbering, or the LCDR1 A variant of no more than 2 amino acid changes or no more than 1 amino acid change; the LCDR2 shown in DASNLET (SEQ ID NO: 83), or no more than 2 amino acid changes or no more than 1
  • said heavy chain variable region comprises HCDR1 shown in GTFSNDVIS (SEQ ID NO:91) according to Kabat numbering, or a variant of said HCDR1 with no more than 2 amino acid changes or no more than 1 amino acid change; HCDR2 represented by VIIPIFGIANYAQKFQG (SEQ ID NO: 92), or a variant of said HCDR2 having no more than 2 amino acid changes or no more than 1 amino acid change; and HCDR3 represented by ARGRGYYSSWLHDI (SEQ ID NO: 93), or A variant of no more than 2 amino acid changes or no more than 1 amino acid change of the HCDR3;
  • the light chain variable region comprises LCDR1 shown in QASQDITNYLN (SEQ ID NO: 100) according to Kabat numbering, or the LCDR1 A variant of no more than 2 amino acid changes or no more than 1 amino acid change; LCDR2 represented by DASNLET (SEQ ID NO: 101), or no more than 2 amino acid changes or no more than 1 amino acid change
  • amino acid changes are additions, deletions or substitutions of amino acids.
  • an antibody that binds a BCMA molecule of the invention binds mammalian BCMA, eg, human, cynomolgus, mouse, rat, and rabbit BCMA.
  • an antibody of the invention that binds a BCMA molecule comprises a heavy chain variable region and a light chain variable region that specifically binds BCMA, wherein:
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 27 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 36 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 45 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 54 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence
  • the heavy chain variable region comprises the sequence of SEQ ID NO: 81 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 90 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof % identity sequence
  • the heavy chain variable region comprises a sequence of SEQ ID NO: 99 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% therewith , 98% or 99% identical sequence
  • the light chain variable region comprises the sequence of SEQ ID NO: 108 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sequences;
  • amino acid change in the sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is preferably amino acid substitution, more preferably amino acid conservative substitution , preferably, said amino acid changes do not occur in the CDR regions.
  • an antibody of the invention that binds a BCMA molecule is an IgG1, IgG2, IgG3 or IgG4 antibody; preferably, it is an IgG1 or IgG4 antibody; more preferably, it is an IgG1 antibody, e.g., a human IgG1 antibody.
  • the antibodies provided herein that bind to a BCMA molecule comprise a mutant Fc domain wherein the amino acid at position P329 is mutated to glycine (G) according to EU numbering, the same as the Fc ⁇ of the Fc domain of the parental antibody that is not mutated.
  • a mutant Fc domain has reduced Fc ⁇ receptor binding compared to receptor binding; for example, the mutant Fc domain is a mutant Fc domain of an IgG1, IgG2, IgG3 or IgG4 antibody, preferably, the mutant Fc domain is A mutant Fc domain of an IgG1 or IgG4 antibody; more preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 antibody, for example, the mutant Fc domain is a mutant Fc domain of a human IgG1 antibody.
  • Antibodies that bind to BCMA molecules containing the P329G mutant Fc domain cannot exert antibody-dependent cellular cytotoxicity by binding to Fc ⁇ receptors, nor can they exert antibody-dependent cellular phagocytosis (ADCP).
  • an antibody that binds a BCMA molecule of the invention has one or more of the following properties:
  • BCMA with high affinity such as human BCMA, cynomolgus monkey BCMA and mouse BCMA
  • the K D of the binding between the anti-BCMA antibody or its antigen-binding fragment and BCMA is about 10 ⁇ 9 M to about 10-12 M, as measured by ForteBio Kinetic Binding Assay;
  • the invention provides a nucleic acid encoding any of the above antibodies or fragments thereof or any chain thereof that binds a BCMA molecule.
  • a vector comprising said nucleic acid is provided.
  • the vector is an expression vector.
  • a host cell comprising said nucleic acid or said vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or HEK293 cells), or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • the host cell is prokaryotic.
  • a nucleic acid of the invention comprises a nucleic acid encoding an antibody of the invention that binds a BCMA molecule.
  • one or more vectors comprising the nucleic acid are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • the vector is a pcDNA3.4 expression vector.
  • the expression vector can be transfected or introduced into a suitable host cell.
  • Various techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, biolistic, lipid-based transfection or other conventional techniques.
  • protoplast fusion cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the produced transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the particular expression vector and Mammalian host cell alteration or optimization.
  • cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells.
  • a marker can, for example, confer prototrophy, biocidal (eg, antibiotic) or heavy metal (eg, copper) resistance, etc. to an auxotrophic host.
  • the selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splicing signals, as well as transcriptional promoters, enhancers and termination signals.
  • a host cell comprising a polynucleotide of the invention.
  • host cells comprising an expression vector of the invention are provided.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies.
  • Suitable host cells include prokaryotic microorganisms such as E. coli.
  • the host cells can also be eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as insect cells and the like. Vertebrate cells can also be used as hosts.
  • mammalian cell lines adapted for growth in suspension can be used.
  • Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African Green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2) , Chinese hamster ovary cells (CHO cells), CHO-S cells, NSO cells, myeloma cell lines such as Y0, NSO, P3X63 and Sp2/0, etc.
  • COS-7 SV40 transformed monkey kidney CV1 line
  • HEK293 or 293F cells baby hamster kidney cells
  • BHK baby hamster kidney cells
  • CV1 African Green monkey kidney cells
  • HELA human cervical cancer cells
  • MDCK buffalo rat liver cells
  • W138 human lung cells
  • HepG2 human liver cells
  • CHO cells Chinese
  • the host cells are CHO cells or HEK293 cells.
  • the present invention provides a method for preparing an antibody (including a P329G mutant antibody) that binds to a BCMA molecule, wherein said method comprises a nucleic acid suitable for expressing said antibody (including a P329G mutant antibody) that binds to a BCMA molecule
  • the host cell comprising the nucleic acid encoding the antibody binding to BCMA molecule (including P329G mutant antibody) or the expression vector comprising said nucleic acid is cultivated under the conditions, and optionally the antibody binding to BCMA molecule (including P329G mutant antibody) is isolated ).
  • the method further comprises recovering antibodies (including P329G mutant antibodies) that bind to BCMA molecules from the host cells (or host cell culture medium).
  • Antibodies that bind to BCMA molecules of the present invention can be analyzed by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion, etc. Purification by resistance chromatography, etc. The actual conditions used to purify a particular protein will also depend on such factors as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art.
  • the purity of the BCMA molecule-binding antibodies of the invention, including P329G mutant antibodies can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography Chromatography, etc.
  • Antibodies provided herein that bind to BCMA molecules, including P329G mutant antibodies, can be identified, screened or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • the antigen-binding activity of the BCMA-binding antibody (including the P329G mutant antibody) of the present invention is tested, for example, by known methods such as FACS, ELISA or Western blotting. Binding to BCMA can be assayed using methods known in the art, exemplary methods are disclosed herein.
  • the binding of BCMA molecule-binding antibodies of the invention, including P329G mutant antibodies, to cell surface BCMA is determined using FACS.
  • the invention also provides assays for identifying biologically active antibodies that bind BCMA molecules, including P329G mutant antibodies.
  • Biological activity may include, for example, ADCC action, CDC action, and the like.
  • Cells for use in any of the above in vitro assays include cell lines that either naturally express BCMA or have been engineered to express BCMA.
  • the modified BCMA-expressing cell line is a cell line that does not normally express BCMA and expresses BCMA after the DNA encoding BCMA is transfected into the cells.
  • the invention provides fusions or conjugates comprising an antibody of the invention. Fusions or conjugates can be produced by fusing or conjugating antibodies of the invention to heterologous molecules.
  • antibody polypeptides of the invention may be fused or conjugated to one or more heterologous molecules, including but not limited to proteins/polypeptides/peptides, labels, drugs, and cytotoxic agents .
  • heterologous molecules including but not limited to proteins/polypeptides/peptides, labels, drugs, and cytotoxic agents .
  • an antibody of the invention is recombinantly fused to a heterologous protein or polypeptide or peptide to form a fusion protein.
  • an antibody of the invention is conjugated to a protein molecule or a non-protein molecule to produce a conjugate.
  • antibodies of the invention may be fused or conjugated to heterologous molecules in the form of full-length antibodies or antibody fragments.
  • Linkers can be used to covalently link different entities in fusions and/or conjugates of the invention.
  • Linkers include chemical linkers or single-chain peptide linkers.
  • antibodies of the invention are fused to other peptides or proteins via peptide linkers.
  • antibodies of the invention are conjugated to other molecules such as markers or drug molecules via chemical linkers.
  • the peptide linkers of the present invention include peptides composed of amino acid residues. Such linker peptides are generally flexible, allowing the antigen-binding moiety to which it is attached to move independently.
  • the length of the linker peptide can be easily determined by those skilled in the art according to the actual situation, such as at least 4-15 amino acids, or longer, such as about 20-25 amino acids.
  • the invention provides the use of the anti-BCMA antibody, fusion or conjugate of the invention in diagnosis and detection. Any of the anti-BCMA antibodies, fusions or conjugates provided herein can be used to detect the presence of human BCMA in a biological sample.
  • detection includes quantitative or qualitative detection.
  • Exemplary detection methods include, but are not limited to, immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (eg, RT-PCR).
  • biological samples include bodily fluids, cells, or tissues.
  • the biological sample is blood, serum, or other liquid sample of biological origin.
  • anti-BCMA antibodies, fusions or conjugates are provided for use in diagnostic or detection methods.
  • methods of detecting the presence of BCMA in a biological sample are provided.
  • the method comprises contacting a biological sample with an anti-BCMA antibody, fusion, or conjugate described herein under conditions that permit binding of the anti-BCMA antibody, fusion, or conjugate to BCMA, and detecting the anti-BCMA antibody, fusion, or conjugate. Whether a complex is formed between the BCMA antibody, fusion or conjugate and BCMA.
  • Such methods may be in vitro or in vivo methods.
  • an anti-BCMA antibody, fusion or conjugate is used to select a subject suitable for treatment with an anti-BCMA antibody, eg, when BCMA is a biomarker for patient selection.
  • exemplary disorders that can be diagnosed using the antibodies, fusions or conjugates of the invention include B cell related disorders such as multiple myeloma.
  • a method of stratifying a multiple myeloma (MM) patient with an antibody, fusion or conjugate of the invention comprising determining said patient's B cell, preferably malignant B cell Whether the cells express BCMA protein on the surface of the B cell, wherein the B cell expresses BCMA protein on its surface, the patient will be likely to respond to and use a therapeutic agent targeting BCMA (such as an anti-BCMA antibody) for treatment.
  • a therapeutic agent targeting BCMA such as an anti-BCMA antibody
  • an anti-BCMA antibody can be conjugated to a diagnostic or detectable agent.
  • the invention provides kits for diagnosis or detection comprising any of the anti-BCMA antibodies, fusions or conjugates of the invention.
  • the invention provides a method of treating a B cell-related disorder, comprising administering to said subject an effective amount of an antibody of the invention or an antigen-binding fragment thereof, or a fusion or conjugate of the invention.
  • a B cell-associated disorder is a disorder associated with abnormal B cell activity, including but not limited to B cell malignancies, plasma cell malignancies, autoimmune diseases.
  • Exemplary disorders that can be treated using BCMA antibodies include, for example, multiple myeloma, non-Hodgkin's lymphoma, B cell proliferation of uncertain malignant potential, lymphomatoid granulomatosis, post-transplantation lymphoproliferative disorders, immunomodulatory disorders, Rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves disease, Wegener's granulomatosis, polyarteritis nodosa, Sjögren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, ANCA-associated vasculitis, Goodpasture disease, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease ,
  • the antibodies, fusions and conjugates of the invention are used to treat B-cell related disorders in humans, such as B-cell malignancies, preferably multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).
  • B-cell malignancies preferably multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).
  • the anti-BCMA antibodies, fusions and conjugates of the invention have anti-tumor effects including, but not limited to, for example, reducing tumor volume, reducing tumor cell number, reducing tumor cell proliferation, or reducing tumor cell survival.
  • the BCMA antibodies, fusions and conjugates of the invention may be administered in combination with other therapeutic modalities for the treatment of the above mentioned diseases such as tumors.
  • Such other treatment modalities include therapeutic agents, radiation therapy, chemotherapy, transplantation, immunotherapy, and the like.
  • the antibody molecules, fusions and conjugates of the invention are used in combination with other therapeutic agents.
  • Exemplary therapeutic agents include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies or other active agents and adjuvants, such as antineoplastic drugs.
  • affinity variant design and functional determination were carried out on the BCMA parental antibodies ADI-34861 and ADI-34857.
  • affinity matured Fabs derived from the parental antibodies ADI-34861 and ADI-34857 was performed by phage display using standard protocols (Silacci et al., (2005), Proteomics 5, 2340-50).
  • the affinity matured antibody ADI-38491 which has the VH sequence shown in SEQ ID NO:27 and the VL sequence shown in SEQ ID NO:36
  • the antibody ADI- 38497 which has the VH sequence shown in SEQ ID NO:45 and the VL sequence shown in SEQ ID NO:54
  • the affinity matured antibody ADI-38481 shows Have the VH shown in SEQ ID NO:81, the VL sequence shown in SEQ ID NO:90
  • antibody ADI-38484 it has the VH shown in SEQ ID NO:99, the VL sequence shown in SEQ ID NO:108)
  • the nucleotide sequence (SEQ ID NO: 26) and the nucleotide sequence (SEQ ID NO: 35) of the heavy chain variable region encoding the antibody ADI-38491 heavy chain and the nucleotide sequence (SEQ ID NO: 35) of the heavy chain variable region of the antibody ADI-38491, respectively, and the heavy chain of the antibody ADI-38497 can be The nucleotide sequence (SEQ ID NO:44) of the variable region and the nucleotide sequence (SEQ ID NO:53) of the variable region of the light chain, the nucleotide sequence (SEQ ID NO:53) of the variable region of the heavy chain of the antibody ADI-38481 NO:80) and the nucleotide sequence (SEQ ID NO:89) of the light chain variable region, the nucleotide sequence (SEQ ID NO:98) and the light chain variable region of the antibody ADI-38484 heavy chain variable region
  • the nucleotide sequence (SEQ ID NO: 107) was constructed on the modified
  • the antibody affinity was determined by using the capture antibody (AHC) biological probe of the anti-human antibody Fc segment to capture the Fc segment of the antibody.
  • AHC capture antibody
  • Antibody ADI-34861, antibody ADI-38491, antibody ADI-38497, antibody ADI-34857, antibody ADI-38481, and antibody ADI-38484 were diluted to 4 ⁇ g/ml with PBS buffer, respectively, and flowed through the AHC probe (Cat: 18 -0015, PALL) surface, the time is 120s. Human, cynomolgus monkey, and mouse BCMA (60nM) were used as the mobile phase, the binding time was 180s, and the dissociation time was 180s. After the experiment was completed, the response value of the blank control (PBS buffer) was subtracted, and the software was used to perform 1:1 Langmuir binding model fitting to calculate the kinetic constant of antigen-antibody binding. The kinetic constants are shown in Table 1 below.
  • NCI-H929 cells also referred to as H929 cells in the text
  • H929 cells purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • NCI-H929 cells are a human multiple myeloma cell line that naturally expresses BCMA molecules on the cell surface.
  • BCMA-KO-H929 cells are based on H929 Cell-based, using CRISPR-Cas9 technology to specifically target the BCMA gene, causing frameshift mutations to cause cell lines that cannot normally express BCMA molecules (constructed by Nanjing GenScript Biotechnology Co., Ltd.), human BCMA CHO- S cells were prepared by introducing exogenous human BCMA into CHO-S cells.
  • the preparation method of human BCMA CHO-S cells is as follows: the sequence encoding human BCMA (NP_001183.2, SEQ ID NO: 109) is cloned into the multiple cloning site of the pcDNA3.3 (Invitrogen) vector, and the expression of human BCMA is obtained.
  • Vector then the expression vector expressing human BCMA is introduced into CHO-S cells (ATCC) for eukaryotic expression, and CHO-S cells expressing human BCMA on the cell surface are obtained.
  • Human BCMA CHO-S cells or H929 cells were inoculated into 96-well plates at 1.0 ⁇ 10 5 cells/well, and diluted 10 nM of each antibody (antibody ADI-34861, antibody ADI-38491, antibody ADI-38497, antibody ADI -34857, antibody ADI-38481, antibody ADI-38484).
  • antibody ADI-34861, antibody ADI-38491, and antibody ADI-38497 all bind to BCMA expressed on the cell surface, and the binding affinity of antibody ADI-38497 to BCMA expressed on the cell surface is significantly improved.
  • Antibody ADI-34857, antibody ADI-38481, and antibody ADI-38484 all bind to BCMA expressed on the cell surface, and the binding affinity of antibodies ADI-38481 and ADI-38484 to BCMA expressed on the cell surface is significantly improved.
  • H929 cells and BCMA-KO-H929 cells were seeded into 96-well plates at 3.0 ⁇ 10 5 cells/well, and diluted ADI-38497 antibody (1 ⁇ g/well) was added, and no antibody was added to negative control wells. After incubation at 4°C for 30 minutes, the cells were washed, and 100 ⁇ L of APC-labeled goat anti-human IgG secondary antibody (Jackson ImmunoResearch Inc, catalog number: 109-136-097) was added to all cell wells, and incubated at 4°C for 30 minutes. The cells were then washed and the binding of the antibody ADI-38497 antibody to the BCMA molecules expressed on the surface of each cell was detected by flow cytometry.
  • APC-labeled goat anti-human IgG secondary antibody Jackson ImmunoResearch Inc, catalog number: 109-136-097
  • the ADI-38497 antibody only binds to the H929 cells expressing the BCMA antigen, but not to the BCMA-KO-H929 cells with BCMA gene knockout. Therefore, the ADI-38497 antibody can specifically bind to the BCMA antigen.
  • GSK company BCMA antibody clone J6M0 light and heavy chain variable region sequence Obtained from the US9273141B2 patent, GSK company BCMA antibody clone J6M0 light and heavy chain variable region sequence, as a control antibody (GSK IgG).
  • the light and heavy chain variable region sequences of GSK IgG, ADI-38497, and ADI-38484 antibodies were synthesized from the entire gene, and loaded into human IgG1 heavy chain constant region containing WT (SEQ ID NO: 110) or human IgG1 containing P329G point mutation
  • the heavy chain constant region (SEQ ID NO: 111) and the ⁇ light chain constant region (SEQ ID NO: 112) pcDNA3.4 expression vector purchased from Shanghai Boying.
  • the light and heavy chain expression vectors were co-transfected into HEK293 cells through PEI at a molar ratio of 2:3, and culture supernatants were collected after 5-7 days of culture.
  • the antibody-containing supernatant medium was purified in one step through a Protein A column, and then dialyzed against PBS. The concentration was detected by reading the absorbance value at 280nm with a NanoDrop instrument, and the purity of the sample was detected by SDS-PAGE and SEC-HPLC. Obtained GSK WT antibody, GSK PG antibody; ADI-38497 WT antibody, ADI-38497 PG antibody; ADI-38484 WT antibody, ADI-38484 PG antibody.
  • the specific method is as follows: After coupling anti-human Fc IgG (Ab97221, Abcam) to the surface of a CM5 chip (29149603, Cytiva), the ADI-38497 PG antibody was captured on the chip surface, and the BCMA antigen in the mobile phase was detected by detecting the antibody on the chip surface and the BCMA antigen in the mobile phase. Binding and dissociation between the obtained affinity and kinetic constants.
  • the assay process used 10 ⁇ HBS-EP+(BR-1006-69, Cytiva) diluted 10 times as the experimental buffer. Each cycle in the affinity assay consists of capture of the ADI-38497 PG antibody, binding of one concentration of antigen, and regeneration of the chip.
  • the antigen after gradient dilution (antigen concentration gradient is 1.25-40nM, 2-fold dilution) flows over the surface of the chip from low concentration to high concentration at a flow rate of 30 ⁇ l/min, and the binding time is 180s.
  • Set the appropriate dissociation Time (900s or 600s or 60s).
  • the chip was regenerated using 10 mM glycine-HCl, pH 1.5 (BR-1003-54, Cytiva).
  • Figure 2B shows representative affinity profiles of the ADI-38497 PG antibody to recombinant human, cynomolgus monkey, mouse, rat, and rabbit BCMA proteins measured by SPR.
  • the results showed that the ADI-38497 PG antibody could bind to the above-mentioned BCMA proteins from different species, and the order of binding activity was human BCMA>monkey BCMA>mouse BCMA>rat BCMA>rabbit BCMA.
  • CHO GS cells expressing BCMA antigens from different species were prepared. Specifically, BCMA genes derived from human, mouse, and cynomolgus monkeys were synthesized and cloned into lentiviral vectors, and then lentiviruses containing BCMA genes from different species were packaged, and CHO GS cells were infected with the lentiviruses.
  • CHO GS cell lines expressing BCMA antigens from different species were obtained by flow cytometry sorting, namely hBCMA-CHO GS, mBCMA-CHO GS and cynoBCMA-CHO GS cells.
  • FACS buffer to prepare ADI-38497 PG antibody and GSK-derived BCMA antibody (that is, GSK PG IgG is used as Benchmark) to prepare 10-fold serial dilutions of different concentrations of antibody solutions, respectively, and 1E5 prepared expression different species CHO GS cells belonging to BCMA antigen were incubated at 4°C for 30 minutes, washed with FACS buffer, and then incubated with Fc ⁇ fragment-specific APC-goat anti-human IgG (Jackson ImmunoResearch, 109-136-098) at 4°C for 30 minutes.
  • the P329G antibody bound to the cells was detected by flow cytometry, and the APC channel MFI was analyzed, with the antibody concentration as the X axis and the APC channel MFI as the Y axis for drawing and calculating the binding EC50.
  • Figure 2C shows the binding ability of different concentrations of P329G BCMA antibody to CHO-GS cells stably expressing human, cynomolgus monkey and mouse BCMA. It can be seen from Figure 2C that the ADI-38497 PG IgG antibody can bind to different species of BCMA expressed on the cell surface, while the BCMA antibody (Benchmark) derived from GSK has a high species-specificity for BCMA, and it does not recognize mouse BCMA. The results were consistent with the SPR detection results.
  • Figure 2D shows the relationship between different concentrations of P329G BCMA antibody and positive multiple myeloma cell lines MM.1s, RPMI8226, U266, H929, L363 and AMO1 expressing BCMA
  • MM.1s was purchased from Nanjing Kebai Biotechnology Co., Ltd., CBP60239
  • RPMI8226 was purchased From Nanjing Kebai Biotechnology Co., Ltd., CBP60244
  • U266 was purchased from Wuhan Punuosheng Life Technology Co., Ltd., CL-0510
  • H929 was purchased from Nanjing Kebai Biotechnology Co., Ltd., CBP60243
  • L363 was purchased from Nanjing Kebai Biotechnology Co., Ltd., CBP6024
  • AMO1 was purchased from Nanjing Kebai Biotechnology Co., Ltd., CBP60242) binding activity, ADI-38497 PG antibody, ADI-38484 PG antibody can bind to positive tumor cells expressing BCMA in a concentration
  • PBMC cells Peripheral Blood Mononuclear Cells, peripheral blood mononuclear cells
  • RPMI 1640 medium containing 10% fetal bovine serum
  • the killing effect on the target cells is plotted and analyzed with the antibody concentration as the X-axis and the cell lysis ratio as the Y-axis.
  • the cells were collected, washed twice with FACS buffer, and CD3, CD56, CD16 and CD107a antibodies were added, among which the CD107a antibody should be added in advance, and incubated with the cells at 37°C for 1 hour.
  • the above cell-antibody mixture was stained at 4°C for 30 minutes, washed twice, resuspended in FACS buffer, and detected by flow cytometry.
  • Figure 3A shows the ability of ADI-38497 WT antibody and ADI-38497 PG antibody to mediate ADCC killing. , CD3, CD56, CD16 and CD107a expression), all showed that only the WT antibody mediated the ADCC cytotoxic killing effect on the positive H929 tumor cells expressing BCMA, while the P329G mutant antibody lacked the ability to induce the ADCC effect.
  • ADCP reporter cell line Promega, G9871
  • H929 cells in the logarithmic growth phase
  • mix the ADCP reporter cells and H929 target cells according to the effect-to-target ratio of 2:1, 5:1, and mix with different concentrations of BCMA antibody, 37
  • luciferase detection kit Promega, E2620
  • Figure 3B shows the ability of ADI-38497 WT antibody and ADI-38497 PG antibody to mediate ADCP killing.
  • the results showed that when different effect-to-target ratios (2:1 or 5:1) were tested, only the ADI-38497 WT antibody mediated the ADCP killing effect on BCMA-positive H929 tumor cells, while the P329G mutant antibody lacked the ability to induce ADCP ability to kill.
  • H929 cells and L363 cells in the logarithmic growth phase were taken and spread in a certain number of well plates; a part of the H929 cells and L363 cells in the logarithmic growth phase were treated as target cells and treated with mitomycin C as a positive control. Then add different concentrations of ADI-38497 PG antibody to mix, continue to culture at 37°C for 48 hours, 72 hours and 120 hours respectively, use CellTiter-Glo (Promega, G9242) to detect the proportion of living cells, take the co-incubation time as the X axis, and the fluorescence The readings are plotted and analyzed on the Y-axis.
  • Figure 3C shows whether the ADI-38497 PG antibody has the ability to inhibit the proliferation of tumor cells.
  • the results show that different incubation times (48 hours, 72 hours and 120 hours) and different ADI-38497 PG antibody concentrations (5 ⁇ g/ml, 50 ⁇ g/ ml), all showed that the ADI-38497 PG antibody itself lacked the ability to inhibit tumor cell proliferation.
  • BALB/c mice (age 4-6 weeks, body weight 15-17g, female) were divided into 3 groups, namely ADI-38497 PG antibody, 1mg/kg antibody group; ADI-38497 PG antibody, 10mg/kg antibody group; and ADI-38497 PG antibody, 200mg/kg antibody group, 9 mice in each group; the antibody was diluted to 0.1mg/mL, 1mg/mL and 20mg/mL with 1 ⁇ PBS, and the administration volume of each mouse was 10mL/ kg, that is, the antibody doses are 1mg/kg, 10mg/mL and 200mg/mL respectively; the administration method is intravenous injection, and the administration frequency is single.
  • Coat the 96-well ELISA plate one day in advance Dilute the BCMA antigen to 1 ⁇ g/ml with coating solution (take a pack of carbonate (Thermo, 28382) powder, dissolve in 400 mL ultrapure water, dilute to 500 mL, and mix well to obtain the coating solution), 100 ⁇ L per well , seal the plate with film, and leave overnight at room temperature. Pour off the coating solution, pat dry on absorbent paper, then add 300 ⁇ L of washing solution to each well, shake and mix for 10 seconds, pat dry the washing solution, and repeat washing 3 times. Add blocking solution to each hole with a row gun, 200 ⁇ L, seal the plate with a sealing film, and incubate at room temperature for 2 hours. Then wash the plate once.
  • coating solution take a pack of carbonate (Thermo, 28382) powder, dissolve in 400 mL ultrapure water, dilute to 500 mL, and mix well to obtain the coating solution
  • 100 ⁇ L per well seal the plate with film,
  • diluted standard curve graded with known concentration of BCMA antibody to prepare the standard curve (for example, use the known concentration of ADI-38497 PG antibody to prepare the standard curve), quality control samples and samples to be tested in 100 ⁇ L per well, at room temperature Incubate for 2 hours. Pour off the pre-coating solution, pat dry on absorbent paper, then add 300 ⁇ L of washing solution to each well, shake and mix for 10 seconds, pat dry the washing solution, and repeat washing 3 times. Repeat once. Goat anti-human IgG- Dilute Fc-HRP antibody (BETHYL) 1:100,000, add 100 ⁇ L to each well, incubate at room temperature in the dark for 1 hour. Then wash the plate once.
  • BETHYL Goat anti-human IgG- Dilute Fc-HRP antibody
  • TMB substrate to the 96-well ELISA plate, 100 ⁇ L per well, and incubate at room temperature in the dark. Light color development for 5 minutes. Add 50 ⁇ L ELISA stop solution to each well, shake for 10 seconds, and read OD450nm and OD620nm values within 30 minutes.
  • FIGS 4A and 4B show the results of pharmacokinetic experiments of ADI-38497 PG antibody (hereinafter also referred to as PG Ab in vivo experiments in mice) in mice.
  • ADI-38497 PG antibody 1mg/kg AUC0-inf, Cmax, CL, T1/2 were 2480 ⁇ g ⁇ h/mL, 30ug/ml, 0.40ml/kg/h, 145h;
  • mice were inoculated with tumors and treated. Specifically, H929 cells were resuspended with 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/mL. NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right side of the back, subcutaneously injected with H929 cell suspension, the injection volume was 0.2mL/mouse, that is, the inoculation volume was 1 ⁇ 106 cells/mouse mouse. Seven days after tumor cell inoculation, mice with tumor volumes ranging from 50.82 to 104.36 mm3 were divided into PBS vehicle group and ADI-38497 PG antibody group (hereinafter also referred to as "PG Ab group"). 7 mice.
  • PG Ab group ADI-38497 PG antibody group
  • the antibody was administered on the 7th day, the volume of administration was 10 mL/kg per mouse, the administration frequency was once a week, and the administration method was intraperitoneal injection.
  • the body weight of the mice, the maximum long axis (L) and maximum width axis (W) of the tumor tissue were monitored twice a week.
  • Figure 5A shows the therapeutic effect of ADI-38497 PG antibody in immunodeficient tumor-bearing mice inoculated subcutaneously with human BCMA expression-positive H929 tumor cells. The results showed that in the H929 tumor model with high expression of BCMA, the administration of PG antibody produced a significant anti-tumor effect.
  • Figure 5B shows the body weight changes of the mice in this experiment. The results showed that in the H929 tumor model with high expression of BCMA, there was no significant change in the body weight of the mice after administration of the PG antibody.
  • the ADI-38497 PG antibody has a significant anti-BCMA high-expression tumor effect, and has no obvious toxic and side effects.
  • H929-luc cells were prepared. Specifically, H929 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were used to package the lentivirus containing the GFP-luciferase gene, and the H929 cells were infected with the obtained lentivirus, and then sorted by flow cytometry to obtain H929-luc cell line expressing both GFP and luciferase.
  • H929-luc cells were resuspend with 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 25 ⁇ 10 6 cells/mL.
  • NOG mice (age 4-6 weeks, body weight 15-17g, female) were injected with H929-luc cell suspension through the tail vein, with an injection volume of 0.2 mL/mouse.
  • the substrate D-Luciferin (15mg/mL) was injected intraperitoneally, with an injection volume of 10mL/kg/mouse, and was analyzed by IVIS spectrum imaging 10 minutes after substrate injection.
  • mice with fluorescence signals between 1.17 ⁇ 10 7 and 1.43 ⁇ 10 8 photons/sec were divided into vehicle group, PG Ab, 0.3mg/kg group, PG Ab, 3mg/kg group, 6-7 mice in each group mice.
  • Antibodies with a concentration of 0.03 mg/mL and 0.3 mg/mL were respectively prepared. After the grouping was completed, antibody administration was started on the 14th day. The volume of administration was 10 mL/kg for each mouse, and the administration frequency was once a week. The way of administration is intraperitoneal injection.
  • Figure 6A shows the anti-tumor efficacy of different doses of PG antibody in immunodeficient tumor-bearing mice inoculated with human H929-luc tumor cells in the tail vein.
  • PG antibody began to produce anti-tumor drug effect about 1 week after administration, and showed a dose-dependent effect; the drug effect gradually weakened after 2 weeks.
  • Figure 6B shows the body weight changes of the mice in the above experiments. The results showed that the body weight of mice in each treatment group increased steadily during the treatment period, suggesting that PG antibody treatment did not induce obvious toxicity.
  • mice were inoculated with tumors and treated. Specifically, H929 cells were resuspended with 1 ⁇ PBS to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/mL. NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right back, and injected subcutaneously with 5 ⁇ 10 6 cells/mL of H929 cell suspension, with an injection volume of 0.2 mL/mouse. Six days after tumor cell inoculation, mice with tumor volumes ranging from 38.49 to 104.77mm3 were divided into groups, as shown in Table 7, which were non-tumor-bearing vehicle group, tumor-bearing vehicle group, and PG Ab group. 24 mice per group.
  • Antibodies with a concentration of 1 mg/mL were prepared. After the grouping was completed, the antibodies were administered, with a volume of 10 mL/kg administered to each mouse. The frequency of administration was once a week, and the frequency of administration was 3 times. The administration method was intraperitoneal injection. The body weight of the mice, the maximum long axis (L) and maximum width axis (W) of the tumor tissue were monitored twice a week. Peripheral blood was collected before the first antibody administration, before the third antibody administration, and at the end of the experiment. Four mice in each group were used for hematology and blood biochemical tests.
  • Figure 7A shows the therapeutic effect of PG antibody in immunodeficient tumor-bearing mice inoculated subcutaneously with human H929 tumor cells. The results showed that PG antibody has anti-tumor effect.
  • Figure 7B shows the body weight changes of the mice in this experiment. The results showed that there was no significant change in the body weight of the control mice during the PG antibody treatment, suggesting that the PG antibody did not induce obvious toxic reactions.
  • FIG. 7C and Figure 7D show the results of the mouse hematology and blood biochemical tests in the above experiments. The results showed that the hematological and blood biochemical indexes of the mice during the PG antibody treatment had no significant changes compared with the control mice, indicating that the PG antibody treatment did not produce toxic reactions.

Abstract

提供特异性结合 BCMA的亲和力提高的抗体和包含 P329G突变的抗体、以及包含所述抗体的缀合物、融合物、双特异性抗体或药物组合物。此外，还提供编码所述抗体的核酸及包含所述核酸的宿主细胞，以及制备所述抗体的方法。还涉及这些结合BCMA的抗体的治疗和诊断用途。

Description

结合BCMA的抗体及其用途 技术领域
本发明涉及抗体药物领域。具体而言，本发明涉及特异性结合B细胞成熟抗原(BCMA)的亲和力提高抗体和其包含P329G突变的抗体以及含有所述抗体的组合物。此外，本发明涉及编码所述抗体的核酸及包含所述核酸的宿主细胞，以及制备所述抗体的方法。本发明还涉及这些结合BCMA的抗体的治疗和诊断用途。
背景技术
B细胞成熟抗原(BCMA，即CD269，TNFRSF17)是肿瘤坏死因子受体超家族成员(TNFRSF)。BCMA是III型跨膜蛋白，在胞外结构域(ECD)中具有TNFR家族成员特征性的富半胱氨酸结构域(CRD)，该结构域形成配体结合基序。BCMA的配体包括B细胞激活因子(BAFF)和B细胞增殖诱导配体(APRIL)，其中B细胞增殖诱导配体(APRIL)与BCMA以更高的亲和力结合，促进肿瘤细胞增殖。
BCMA主要表达在成熟B细胞即浆细胞表面，在正常造血干细胞和非血源组织中不表达，BCMA信号对于长效骨髓浆细胞的生存不可或缺，但非总体B细胞稳态所必需。膜表面BCMA能够被γ分泌酶酶切而脱落，产生的可溶性BCMA(sBCMA)可能通过封闭BAFF/APRIL配体结合来降低膜表面BCMA信号传导。临床前模型以及人体肿瘤中发现BCMA在多发性骨髓瘤(Multiple Myeloma,MM)细胞中过表达，其上调经典以及非经典NF-κB信号，促进MM细胞生长、生存、粘附，诱导破骨细胞激活、血管生产、转移及免疫抑制等，BCMA表达已经成为诊断MM的重要标志物。此外，MM患者血清中sBCMA水平升高，与骨髓中MM细胞数量呈正比例相关，且其浓度变化与MM预后及治疗应答密切相关。
多发性骨髓瘤，也称为浆细胞瘤或者卡勒氏病，是一种难治愈的B细胞系的恶性肿瘤，特征是浆细胞异常增生。鉴于BCMA仅限于表达在浆细胞中，在天然和记忆性B细胞中不表达的特性，BCMA成为治疗B细胞恶性肿瘤，尤其是多发性骨髓瘤的热门靶点。本领域仍然需要新的BCMA特异性结合分子。本发明通过提供了以高靶特异性和高亲和力结合BCMA，尤其是与肿瘤细胞表面上表达的BCMA结合的抗体，满足了这方面的需求。
发明概述
本发明人通过研究，开发了一组新型的具有高亲和力结合BCMA的抗BCMA抗体。本发明的特异性结合BCMA的抗体或抗原结合片段具有以下一个或多个特性：
(1)以高亲和力结合BCMA，例如人BCMA、食蟹猴BCMA和小鼠BCMA，例如，所述抗BCMA抗体或其抗原结合片段与BCMA之间结合的K D是约10 -9M至约10 -12M，如通过ForteBio动力学结合测定法所测量；
(2)特异性地与细胞表面表达的BCMA结合；
(3)对表达BCMA的细胞具有ADCC细胞毒杀伤效应；
(4)对表达BCMA的细胞具有ADCP杀伤效应；
(5)阻断、抑制表达人BCMA的细胞(尤其是多发性骨髓瘤细胞)的生长、和/或杀死所述细胞；和
(6)对表达BCMA的肿瘤具有体内抗肿瘤效应，并且无明显毒副效应。
在第一方面，本发明提供了特异性结合BCMA的抗体或抗原结合片段，其包含
(a)SEQ ID NO:27所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:36所示的轻链可变区氨基酸序列中的3个CDR；或者与所述6个CDR区具有单个CDR或者多个CDR不超过每个CDR区2个或1个氨基酸变化的变体；
(b)SEQ ID NO:45所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:54所示的轻链可变区氨基酸序列中的3个CDR；或者与所述6个CDR区具有单个CDR或者多个CDR不超过每个CDR区2个或1个氨基酸变化的变体；
(c)SEQ ID NO:81所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:90所示的轻链可变区氨基酸序列中的3个CDR；或者与所述6个CDR区具有单个CDR或者多个CDR不超过每个CDR区2个或1个氨基酸变化的变体；或
(d)SEQ ID NO:99所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:108所示的轻链可变区氨基酸序列中的3个CDR；或者与所述6个CDR区具有单个CDR或者多个CDR不超过每个CDR区2个或1个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或取代。
在一些实施方案中，本发明的特异性结合BCMA的抗体或抗原结合片段包含重链可变区和轻链可变区，其中：
(a)所述重链可变区包含根据Kabat编号的GSIVSSSYYWT(SEQ ID NO:19)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；SISIAGSTYYNPSLKS(SEQ ID NO:20)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARDRGDTILDV(SEQ ID NO:21)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的RASQSISRYLN(SEQ ID NO:28)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；AASSLQS(SEQ ID NO:29)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQKYFDIT(SEQ ID NO:30)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(b)所述重链可变区包含根据Kabat编号的GSIVSSSYYWT(SEQ ID NO:37)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；SISIAGSTYYNPSLKS(SEQ ID NO:38)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARDRGDQILDV(SEQ ID NO:39)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的RASQSISRYLN(SEQ ID NO:46)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；AASSLQS(SEQ ID NO:47)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和 QQKYFDIT(SEQ ID NO:48)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(c)所述重链可变区包含根据Kabat编号的GTFSNDVIS(SEQ ID NO:73)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；VIIPIFGIANYAQKFQG(SEQ ID NO:74)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARGRGYYSSWLLDI(SEQ ID NO:75)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的QASQDITNYLN(SEQ ID NO:82)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；DASNLET(SEQ ID NO:83)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQAFDLIT(SEQ ID NO:84)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；或
(d)所述重链可变区包含根据Kabat编号的GTFSNDVIS(SEQ ID NO:91)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；VIIPIFGIANYAQKFQG(SEQ ID NO:92)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARGRGYYSSWLHDI(SEQ ID NO:93)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的QASQDITNYLN(SEQ ID NO:100)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；DASNLET(SEQ ID NO:101)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQAFDLIT(SEQ ID NO:102)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或取代。
在一些实施方案中，本发明的特异性结合BCMA的抗体或抗原结合片段包含重链可变区和轻链可变区，其中
(a)所述重链可变区包含GSIVSSSYYWT(SEQ ID NO:19)所示的HCDR1；SISIAGSTYYNPSLKS(SEQ ID NO:20)所示的HCDR2；和ARDRGDTILDV(SEQ ID NO:21)所示的HCDR3；所述轻链可变区包含RASQSISRYLN(SEQ ID NO:28)所示的LCDR1；AASSLQS(SEQ ID NO:29)所示的LCDR2；和QQKYFDIT(SEQ ID NO:30)所示的LCDR3；
(b)所述重链可变区包含GSIVSSSYYWT(SEQ ID NO:37)所示的HCDR1；SISIAGSTYYNPSLKS(SEQ ID NO:38)所示的HCDR2；和ARDRGDQILDV(SEQ ID NO:39)所示的HCDR3；所述轻链可变区包含RASQSISRYLN(SEQ ID NO:46)所示的LCDR1；AASSLQS(SEQ ID NO:47)所示的LCDR2；和QQKYFDIT(SEQ ID NO:48)所示的LCDR3；
(c)所述重链可变区包含GTFSNDVIS(SEQ ID NO:73)所示的HCDR1；VIIPIFGIANYAQKFQG(SEQ ID NO:74)所示的HCDR2；和ARGRGYYSSWLLDI(SEQ ID NO:75)所示的HCDR3；所述轻链可变区包含QASQDITNYLN(SEQ ID NO:82)所示的LCDR1；DASNLET(SEQ ID NO:83)所示的LCDR2；和QQAFDLIT(SEQ ID NO:84)所示的LCDR3； 或
(d)所述重链可变区包含GTFSNDVIS(SEQ ID NO:91)所示的HCDR1；VIIPIFGIANYAQKFQG(SEQ ID NO:92)所示的HCDR2；和ARGRGYYSSWLHDI(SEQ ID NO:93)所示的HCDR3；所述轻链可变区包含QASQDITNYLN(SEQ ID NO:100)所示的LCDR1；DASNLET(SEQ ID NO:101)所示的LCDR2；和QQAFDLIT(SEQ ID NO:102)所示的LCDR3。
在一些实施方案中，本发明的特异性结合BCMA的抗体或抗原结合片段包含重链可变区和轻链可变区，其中
(a)重链可变区包含SEQ ID NO:27的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:36的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
(b)重链可变区包含SEQ ID NO:45的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:54的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
(c)重链可变区包含SEQ ID NO:81的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:90的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；或(d)重链可变区包含SEQ ID NO:99的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:108的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在一些实施方案中，本发明的特异性结合BCMA的抗体或抗原结合片段包含重链可变区和轻链可变区，其中，所述抗体或抗原结合片段包含
(a)SEQ ID NO:27所示的重链可变区和SEQ ID NO:36所示的轻链可变区；
(b)SEQ ID NO:45所示的重链可变区和SEQ ID NO:54所示的轻链可变区；
(c)SEQ ID NO:81所示的重链可变区和SEQ ID NO:90所示的轻链可变区；或
(d)SEQ ID NO:99所示的重链可变区和SEQ ID NO:108所示的轻链可变区。
在一些实施方案中，本发明的特异性结合BCMA的抗体或抗原结合片段是IgG1、IgG2、IgG3或IgG4抗体；任选地，其是IgG1或IgG4抗体；任选地，其是IgG1抗体。在一些实施方案中，所述抗原结合片段是Fab、Fab’、F(ab’)2、Fv、单链Fv、单链Fab或双体抗体(diabody)。
在一些实施方案中，本发明的特异性结合BCMA的抗体或抗原结合片段还包含突变Fc结构域，其中根据EU编号的P329位置处的氨基酸突变为甘氨酸(G)，与未突变的亲本抗体Fc结构域的Fcγ受体结合相比，突变Fc结构域的Fcγ受体结合降低；例如，所述突变Fc结构域是IgG1、IgG2、IgG3或IgG4抗体的突变Fc结构域，优选地，所述突变Fc结构域是IgG1或IgG4抗体的突变Fc结构域；更优选地，所述突变Fc结构域是IgG1抗体的突变Fc结构域；
例如，所述抗体或抗原结合片段包含SEQ ID NO:111所示的重链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列且其中根据EU编号的P329位置处的氨基酸突变为G；
例如，所述抗体或抗原结合片段包含SEQ ID NO:111所示的重链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列且其中根据EU编号的P329位置处的氨基酸突变为G；和SEQ ID NO:112所示的轻链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
例如，所述抗体或抗原结合片段包含SEQ ID NO:111所示的重链恒定区序列和SEQ ID NO:112所示的轻链恒定区序列。
在第二方面，本发明提供了编码本发明第一方面的抗体的核酸、包含编码所述抗体的核酸的载体、包含所述核酸分子或载体的宿主细胞、以及制备所述抗体的方法，所述方法包括在适于表达编码本发明第一方面所述的特异性结合BCMA分子的抗体或抗原结合片段的核酸的条件下，培养导入有编码第一方面所述的特异性结合BCMA分子的抗体或抗原结合片段的核酸的表达载体的宿主细胞，分离所述特异性结合BCMA分子的抗体或抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述特异性结合BCMA分子的抗体或抗原结合片段。优选地，所述宿主细胞是原核的或真核的，更优选的选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞，最优选地，所述宿主细胞是HEK293细胞或CHO细胞。
在第三方面，本发明涉及包含本发明第一方面的抗BCMA抗体或其抗原结合片段的缀合物、融合物或双特异性抗体。
在第四方面，本发明涉及药物组合物，其包含本发明第一方面的抗BCMA抗体或其抗原结合片段、或本发明第三方面的缀合物、融合物或双特异性抗体，以及任选地可药用载体。
在第五方面，本发明涉及本发明第一方面的抗BCMA抗体或其抗原结合片段、本发明第三方面的缀合物、融合物或双特异性抗体、或本发明第四方面的药物组合物的用途，用于制备在受试者中预防或治疗B细胞相关疾病的药物，例如，所述B细胞相关病症选自：B细胞恶性肿瘤、浆细胞恶性肿瘤、自身免疫疾病，优选地选自：多发性骨髓瘤、非霍奇金淋巴瘤、恶性潜能不确定的B细胞增殖、淋巴瘤样肉芽肿病、移植后淋巴增生病症、免疫调节病症、类风湿性关节炎、重症肌无力、特发性血小板减少性紫癜、抗磷脂综合征、恰加斯病、格雷夫斯病、韦格纳肉芽肿、结节性多动脉炎、舍格伦氏综合征、寻常天疱疮、硬皮病、多发性硬化症、ANCA相关血管炎、古德帕斯丘氏病、川崎病、自身免疫性溶血性贫血和急进性肾小球肾炎、重链病、原发性或免疫细胞相关的淀粉样变性、或意义未明的单克隆丙种球蛋白病，优选地，所述B细胞相关病况是B细胞恶性肿瘤，更优选地，多发性骨髓瘤(MM)或非霍奇金淋巴瘤(NHL)。
在第六方面，本发明提供了检测样品中BCMA的试剂盒，所述试剂盒包含本发明第一方面的抗BCMA抗体或其抗原结合片段，用于实施以下步骤：
(a)将样品与本发明第一方面的抗BCMA抗体或其抗原结合片段接触；和
(b)检测所述抗BCMA抗体或其抗原结合片段和BCMA间的复合物的形成；任选地，所述抗BCMA抗体或其抗原结合片段是被可检测地标记的。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1显示了ADI-38497抗体仅与表达BCMA抗原的H929细胞相结合，而与BCMA基因敲除的BCMA-KO-H929细胞不结合。
图2A显示了采用表面等离子共振法(SPR)测定抗体亲和力的方法示意图。
图2B显示了采用SPR测定ADI-38497 PG抗体与重组人、食蟹猴、小鼠、大鼠和兔的BCMA蛋白的代表性亲和力图谱。
图2C显示了P329G BCMA抗体与稳定表达人、食蟹猴及小鼠BCMA的CHO-GS细胞的结合能力。
图2D显示了P329G BCMA抗体与表达BCMA的阳性多发性骨髓瘤细胞系MM.1s、RPMI8226、U266、H929、L363及AMO1的结合活性。
图3A显示了ADI-38497 WT抗体和ADI-38497 PG抗体介导ADCC杀伤的能力。
图3B显示了ADI-38497 WT抗体和ADI-38497 PG抗体介导ADCP杀伤的能力。
图3C显示了ADI-38497 PG抗体介导靶细胞裂解的能力。
图4A和图4B显示了小鼠中ADI-38497 PG抗体的药代动力学实验结果。
图5A显示了在皮下接种人H929高表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中ADI-38497 PG抗体的治疗效应。
图5B显示了在皮下接种人H929高表达BCMA肿瘤细胞的免疫缺陷荷瘤小鼠中使用ADI-38497 PG抗体治疗时，小鼠的体重变化。
图6A显示了在尾静脉接种人H929-luc肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量ADI-38497 PG抗体的抗肿瘤药效。
图6B显示了在尾静脉接种人H929-luc肿瘤细胞的免疫缺陷荷瘤小鼠中使用ADI-38497 PG抗体治疗时，小鼠的体重变化。
图7A显示了在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中ADI-38497 PG抗体的治疗效应。
图7B显示了在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中使用ADI-38497 PG抗体治疗时，小鼠的体重变化。
图7C和图7D显示了在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中使用ADI-38497 PG抗体治疗时，小鼠血液学和血生化检测结果。
发明详述
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
I.定义
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“BCMA”和“B细胞成熟抗原”可互换地使用，其包括人BCMA的变体、同种型、物种同源物和与BCMA(例如人BCMA)具有至少一个相同表位的类似物。BCMA蛋白也可包括BCMA的片段，诸如胞外结构域以及胞外结构域的片段，例如保持与本发明任何抗体的结合能力的片段。
如本文所用的术语“BCMA抗体”、“针对BCMA的抗体”、“特异性结合BCMA的抗体”、“特异性靶向BCMA的抗体”、“特异性识别BCMA的抗体”可互换地使用，意指能够与B细胞成熟抗原(BCMA)特异性结合的抗体。
术语“抗体”在本文中以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体和抗体片段。优选地，本发明的抗体是单结构域抗体或重链抗体。
“抗体片段”或“抗原结合片段”在本文中可互换地使用，指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fab、Fab’、F(ab’)2、Fv、单链Fv、单链Fab或双体抗体(diabody)。
与参照抗体显示相同或相似的结合亲和力和/或特异性的抗体是指这样的抗体，其能够具有参照抗体的至少50％、60％、70％、80％、90％或95％以上的结合亲和力和/或特异性。这可以通过本领域已知的任何测定结合亲和力和/或特异性的方法进行测定。
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。在一个给定的重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College  London)，International ImMunoGeneTics database(IMGT)(http://imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
CDR也可以基于与参考CDR序列(例如本发明示例的CDR之任一)具有相同的Kabat编号位置而确定。在本发明中，当提及抗体可变区和具体CDR序列(包括重链可变区残基)时，是指根据Kabat编号系统的编号位置。
尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM和Contact方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia或AbM定义的其余CDR残基可以被保守氨基酸残基替代。
“人源化”抗体是指包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体中的所有或基本上所有的CDR(例如，CDR)对应于非人抗体的那些，并且所有或基本上所有的FR对应于人抗体的那些。人源化抗体任选可以包含至少一部分的来源于人抗体的抗体恒定区。抗体(例如非人抗体)的“人源化形式”是指已经进行了人源化的抗体。
“人抗体”指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于下述抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。在某些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羰基端。然而，Fc区的C端赖氨酸(Lys447)可以存在或者可以不存在。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。
在某些实施方案中，免疫球蛋白的Fc区包含两个恒定结构域域，即CH2和CH3，在另一些实施方案中，免疫球蛋白的Fc区包含三个恒定结构域，即CH2、CH3和CH4。
IgG与Fcγ受体或C1q的结合依赖于定位在铰链区和CH2结构域中的残基。CH2结构域的两个区域对FcγR和补体C1q结合至关重要，并且在IgG2和IgG4中具有唯一的序列。已显示取代人IgG1和IgG2中233-236位的残基和取代人IgG4中327、330和331位的残基可大幅降低ADCC和CDC活性(Armour等人,Eur.J.Immunol.29(8)，1999，2613-2624；Shields等人,J.Biol.Chem.276(9)，2001，6591-6604)。
“功能性Fc区”与“有功能Fc区”等类似术语可以互换使用，指具有野生型Fc区的效应功能的Fc区。
“变异Fc区”、“Fc突变体”、“携带突变的Fc区”、“突变Fc区”、“Fc区变 体”、“Fc变体”、“变体Fc区”和“突变的Fc区”等类似术语可以互换使用，指包含至少一处氨基酸修饰而区别于天然序列Fc区/野生型Fc区的Fc区。
在一些实施方案中，变异Fc区包含与天然序列Fc区的氨基酸序列相差一处或多处氨基酸取代、缺失或添加的氨基酸序列。在一些实施方案中，变异Fc区与野生型IgG的Fc区相比具有至少一处氨基酸取代，所述至少一处氨基酸取代是将根据EU编号的P329位置处的氨基酸取代为甘氨酸(G)。
“Fc受体”或“FcR”指结合抗体Fc区的分子。在一些实施方案中，FcR是天然人FcR。在一些实施方案中，FcR是结合IgG抗体的受体，即FcγR，包括FcγRI(CD64)、FcγRII(CD32)和FcγRIII(CD16)三种受体，以及这些受体的等位变体和可变剪接形式。FcγRII受体包括FcγRIIA和FcγRIIB，FcγRIII受体包括FcγRIIIA和FcγRIIIB。
术语“效应功能”指随免疫球蛋白同种型变动的归因于免疫球蛋白Fc区的那些生物学活性。免疫球蛋白效应子功能的例子包括：Fc受体结合作用、抗体依赖的细胞介导的细胞毒性(ADCC)、抗体依赖的细胞吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞摄取抗原、C1q结合和补体依赖的细胞毒性(CDC)、下调细胞表面受体(例如B细胞受体)和B细胞活化。
术语“抗体依赖的细胞介导的细胞毒性(ADCC)”是某些细胞毒性效应细胞(例如天然杀伤(NK)细胞)介导对靶细胞和外来宿主细胞杀伤的主要机制之一。在一些实施方案中，本发明的抗体提供T淋巴细胞的抗体依赖性细胞毒作用、增强NK细胞的抗体依赖性细胞毒作用。
术语“抗体依赖的细胞吞噬作用(ADCP)”指一种细胞反应，其中通过结合靶细胞的抗体与巨噬细胞表面的FcγRIIIa结合，诱导激活巨噬细胞，从而使靶细胞内化和被吞噬体酸化降解。ADCP也可由FcγRIIa和FcγRI介导，但是占比较小。
术语“补体依赖的细胞毒性作用(CDC)”是指在补体存在下靶细胞的裂解。补体系统是由一系列蛋白质组成的先天免疫系统的一部分。补体系统的蛋白质称为“补体”，以缩写符号C1、C2、C3等表示，其是存在于人或脊椎动物血清、组织液中的一组不耐热的，经活化后具有酶活性的蛋白质。C1q是依赖补体的细胞毒性(CDC)途径的第一成分，其能够结合六个抗体，但与两个IgG结合就足以活化补体级联。经典补体途径的激活由补体系统的第一组分(C1q)与结合相关抗原的抗体(适当的亚类)结合而启动，活化一系列补体级联反应，在靶细胞膜中形成孔洞，从而导致靶细胞死亡。为了评估补体活化，可以执行CDC测定法，通过例如Gazzano-Santoro等人，J.Immunol.Methods 202:163(1996)中所述的方法。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的构架区(FR)和三个互补决定区(CDR)。(参见，例如，Kindt等Kuby Immunology，6 th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、SPR或生物膜层干涉技术或本领域已知的其他常规结合测定法测定。
“缀合物”是与一个或多个其他物质(包括但不限于细胞毒性剂或标记)缀合的抗体。
术语“抑制”或“阻断”是指使所给出分子的某些参数(例如，活性)降低。例如，这个术语包括使得所给出的分子(例如，BCMA)被抑制至少5％、10％、20％、30％、40％或更多的活性的物质。因此，抑制作用不必是100％。
术语“个体”或“受试者”可互换地使用，包括哺乳动物。哺乳动物包括但不限于驯化动物(例如，牛、羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体或受试者是人。
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。
术语“癌症”和“癌性”是指哺乳动物中细胞生长不受调节的生理疾患。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如，抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体，以及通过与直接被标记的另一种试剂反应来间接标记探针或抗体。间接标记的实例包括使用荧光标记的第二抗体进行的第一抗体的检测和具有生物素的DNA探针的末端标记，使得其可以用荧光标记的链霉抗生素蛋白来检测。
“分离的”抗体是指已经与其天然环境的组分分离。在一些实施方案中，将BCMA抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。对于用于评估抗体纯度的方法的综述，参见，例如，Flatman等，J.Chromatogr.B848：79-87(2007)。
“分离的”核酸是指这样的核酸分子，其已经与其天然环境的组分分离。分离的核酸包括包含在通常包含该核酸分子的细胞中的核酸分子，但是该核酸分子存在于染色体外或在不同于其天然染色体位置的染色体位置处。“分离的编码BCMA抗体的核酸”是指一个或多个核酸分子，其编码BCMA抗体的链或其片段，包括在单一载体或分开的载体中的这样的核酸分子，以及存在于宿主细胞中的一个或多个位置处的这样的核酸分子。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol. Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
术语“氨基酸变化”和“氨基酸修饰”可互换地使用，是指氨基酸的添加、缺失、取代和其他修饰。可以进行氨基酸的添加、缺失、取代和其他修饰的任意组合，条件是最终的多肽序列具有所需的特性。在一些实施方案中，对抗体的氨基酸取代导致抗体与Fc受体的结合降低。为了改变例如Fc区结合特征的目的，特别优选非保守氨基酸取代，即用具有不同结构和/或化学性质的另一种氨基酸取代一种氨基酸。氨基酸取代包括用非天然存在的氨基酸或二十种标准氨基酸的天然存在的氨基酸衍生物(例如、4-羟基脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟基赖氨酸)的取代。可以使用本领域公知的遗传或化学方法产生氨基酸变化。遗传方法可包括定点诱变、PCR、基因合成等。通过除基因工程化之外的方法(如化学修饰)改变氨基酸侧链基团的方法可能是有用的。本文可使用多种名称来表示相同的氨基酸变化。例如，从Fc结构域的329位的脯氨酸到甘氨酸的取代可以表示为329G、G329、G 329、P329G Pro329Gly、或简称为“PG”。
术语“保守序列修饰”、“保守序列变化”指未显著影响或改变含有氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰或变化。这类种保守修饰包括氨基酸取代、添加和缺失。可以通过本领域已知的标准技术，如位点定向诱变和PCR介导的诱变向本发明的抗体或抗体片段引入修饰。保守性取代是氨基酸残基由具有相似侧链的氨基酸残基替换的氨基酸取代。已经在本领域中定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸)、β-侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“可药用载体(carrier)”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、缓冲剂或稳定剂等。
术语“与BCMA相关的疾病”是指由BCMA增加的表达或活性引起、加重或以其它方式与其相关的任何病症。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的抗体分子用来延缓疾病发展或用来减慢疾病的进展。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中于癌症症状发生前的药物施用。
术语“有效量”指本发明的抗体或组合物的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如哺乳动物的物种；体重、年龄和一般健康状况；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。抗体或抗体片段或其组合物的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和抗体或抗体部分在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中抗体或抗体片段或其组合物的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率、肿瘤体积等)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％或90％。可以在预示人肿瘤中的功效的动物模型系统中评价化合物抑制可度量参数(例如，癌症)的能力。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
在本文中当谈及核酸时使用的术语“载体(vector)”是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其有效相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。宿主细胞是可以用来产生本发明抗体分子的任何类型的细胞系统，包括真核细胞，例如，哺乳动物细胞、昆虫细胞、酵母细胞；和原核细胞，例如，大肠杆菌细胞。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。
“受试者/患者样品”指从患者或受试者得到的细胞、组织或体液的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。肿瘤样品的例子在本文中包括但不限于肿瘤活检、细针吸出物、支气管灌洗液、胸膜液(胸水)、痰液、尿液、手术标本、循环中的肿瘤细胞、血清、血浆、循环中的血浆蛋白质、腹水、衍生自肿瘤或展现出肿瘤样特性的原代细胞培养物或细胞系，以及保存的肿瘤样品，诸如福尔马林固定的、石蜡包埋的肿瘤样品或冷冻的肿瘤样品。
II.本发明的特异性结合BCMA分子的抗体和其包含突变Fc结构域的抗体
本发明提供了以高靶特异性和高亲和性结合BCMA的抗体，其包含重链可变区和轻链可变区，其中：
(a)所述重链可变区包含根据Kabat编号的GSIVSSSYYWT(SEQ ID NO:19)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；SISIAGSTYYNPSLKS(SEQ ID NO:20)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARDRGDTILDV(SEQ ID NO:21)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的RASQSISRYLN(SEQ ID NO:28)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；AASSLQS(SEQ ID NO:29)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQKYFDIT(SEQ ID NO:30)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(b)所述重链可变区包含根据Kabat编号的GSIVSSSYYWT(SEQ ID NO:37)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；SISIAGSTYYNPSLKS(SEQ ID NO:38)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARDRGDQILDV(SEQ ID NO:39)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的RASQSISRYLN(SEQ ID NO:46)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；AASSLQS(SEQ ID NO:47)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQKYFDIT(SEQ ID NO:48)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(c)所述重链可变区包含根据Kabat编号的GTFSNDVIS(SEQ ID NO:73)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；VIIPIFGIANYAQKFQG(SEQ ID NO:74)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARGRGYYSSWLLDI(SEQ ID NO:75)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻 链可变区包含根据Kabat编号的QASQDITNYLN(SEQ ID NO:82)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；DASNLET(SEQ ID NO:83)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQAFDLIT(SEQ ID NO:84)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；或
(d)所述重链可变区包含根据Kabat编号的GTFSNDVIS(SEQ ID NO:91)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；VIIPIFGIANYAQKFQG(SEQ ID NO:92)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARGRGYYSSWLHDI(SEQ ID NO:93)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的QASQDITNYLN(SEQ ID NO:100)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；DASNLET(SEQ ID NO:101)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQAFDLIT(SEQ ID NO:102)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或取代。
在一些实施方案中，本发明的结合BCMA分子的抗体结合哺乳动物BCMA，例如人、食蟹猴、小鼠、大鼠和兔的BCMA。
在一些实施方案中，本发明的结合BCMA分子的抗体包含特异性结合BCMA的重链可变区和轻链可变区，其中：
(a)重链可变区包含SEQ ID NO:27的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:36的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
(b)重链可变区包含SEQ ID NO:45的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:54的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
(c)重链可变区包含SEQ ID NO:81的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:90的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；或(d)重链可变区包含SEQ ID NO:99的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:108的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
其中所述至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列中的氨基酸变化优选氨基酸取代，更优选氨基酸保守取代，优选地，所述氨基酸变化不发生在CDR区中。
在一些实施方案中，本发明的结合BCMA分子的抗体是IgG1、IgG2、IgG3或IgG4抗体；优选地，其是IgG1或IgG4抗体；更优选地，其是IgG1抗体，例如，人IgG1抗体。
在一些实施方案中，本文中所提供的结合BCMA分子的抗体包含突变Fc结构域，其中根据EU编号的P329位置处的氨基酸突变为甘氨酸(G)，与未突变的亲本抗体Fc结构域的Fcγ受体结合相比，突变Fc结构域的Fcγ受体结合降低；例如，所述突变Fc结构域是IgG1、IgG2、IgG3或IgG4抗体的突变Fc结构域，优选地，所述突变Fc结构域是IgG1或IgG4抗体的突变Fc结构域；更优选地，所述突变Fc结构域是IgG1抗体的突变Fc结构域，例如，所述突变Fc结构域是人IgG1抗体的突变Fc结构域。
包含P329G突变Fc结构域的结合BCMA分子的抗体不能通过与Fcγ受体结合来发挥抗体依赖性细胞毒性，也不能发挥抗体依赖的细胞吞噬作用(ADCP)。
在一些实施方案中，本发明的结合BCMA分子的抗体具有以下一个或多个特性：
(1)以高亲和力结合BCMA，例如人BCMA、食蟹猴BCMA和小鼠BCMA，例如，所述抗BCMA抗体或其抗原结合片段与BCMA之间结合的K D是约10 -9M至约10 -12M，如通过ForteBio动力学结合测定法所测量；
(2)特异性地与细胞表面表达的BCMA结合；
(3)对表达BCMA的细胞具有ADCC细胞毒杀伤效应；
(4)对表达BCMA的细胞具有ADCP杀伤效应；
(5)阻断、抑制表达人BCMA的细胞(尤其是多发性骨髓瘤细胞)的生长、和/或杀死所述细胞；和
(6)对表达BCMA的肿瘤具有体内抗肿瘤效应，并且无明显毒副效应。
在一些实施方案中，本发明提供了编码以上任何结合BCMA分子的抗体或其片段或其任一条链的核酸。在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体。在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或HEK293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。在另一个实施方案中，宿主细胞是原核的。
例如，本发明的核酸包含编码本发明的结合BCMA分子的抗体的核酸。在一些实施方案中，提供包含所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个实施方案中，载体是pcDNA3.4表达载体。
一旦已经制备了用于表达的表达载体或DNA序列，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质的转染或其他常规技术。在原生质体融合的情况下，将细胞在培养基中培育并且筛选适宜的活性。用于培养所产生的转染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。
另外，可以通过引入允许选择已转染的宿主细胞的一个或多个标记物，选出已经稳定将DNA掺入至其染色体中的细胞。标记物可以例如向营养缺陷型宿主提供原养型、杀生物抗性(例如，抗生素)或重金属(如铜)抗性等。可选择标记基因可以与待表达的DNA序列直接连接 或通过共转化引入相同的细胞中。也可能需要额外元件以便最佳合成mRNA。这些元件可以包括剪接信号，以及转录启动子、增强子和终止信号。
在一个实施方案中，提供了包含本发明多核苷酸的宿主细胞。在一些实施方案中，提供了包含本发明表达载体的宿主细胞。在一些实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体的其它细胞。合适的宿主细胞包括原核微生物，如大肠杆菌。宿主细胞还可以是真核微生物如丝状真菌或酵母，或各种真核细胞，例如昆虫细胞等。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK293或293F细胞)、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、人肝脏细胞(HepG2)、中国仓鼠卵巢细胞(CHO细胞)、CHO-S细胞、NSO细胞、骨髓瘤细胞系如Y0、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编著，Humana Press，Totowa，NJ)，第255-268页(2003)。在一个优选的实施方案中，所述宿主细胞是CHO细胞或HEK293细胞。
在一个实施方案中，本发明提供了制备结合BCMA分子的抗体(包括P329G突变抗体)的方法，其中所述方法包括在适于表达编码所述结合BCMA分子的抗体(包括P329G突变抗体)的核酸的条件下培养包含编码所述结合BCMA分子的抗体(包括P329G突变抗体)的核酸或包含所述核酸的表达载体的宿主细胞，以及任选地分离所述结合BCMA分子的抗体(包括P329G突变抗体)。在某个实施方案中，所述方法还包括从所述宿主细胞(或宿主细胞培养基)回收结合BCMA分子的抗体(包括P329G突变抗体)。
如本文所述制备的本发明的结合BCMA分子的抗体(包括P329G突变抗体)可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的结合BCMA分子的抗体(包括P329G突变抗体)的纯度，所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。
可以通过本领域中已知的多种测定法对本文中提供的结合BCMA分子的抗体(包括P329G突变抗体)鉴定、筛选或表征其物理/化学特性和/或生物学活性。一方面，对本发明的结合BCMA分子的抗体(包括P329G突变抗体)测试其抗原结合活性，例如通过已知的方法诸如FACS、ELISA或Western印迹等来进行。可使用本领域已知方法来测定对BCMA的结合，本文中公开了例示性方法。在一些实施方案中，使用FACS测定本发明的结合BCMA分子的抗体(包括P329G突变抗体)对细胞表面BCMA(例如人BCMA)的结合。
本发明还提供了用于鉴定具有生物学活性的结合BCMA分子的抗体(包括P329G突变抗体)的测定法。生物学活性可以包括例如ADCC作用、CDC作用等。
供任何上述体外测定法使用的细胞包括天然表达BCMA或经改造而表达BCMA细胞系。所述经改造而表达BCMA细胞系是正常情况下不表达BCMA的、将编码BCMA 的DNA转染入细胞之后表达BCMA的细胞系。
III.融合物和缀合物
本发明提供了包含本发明抗体的融合物或缀合物。可以通过将本发明抗体融合或缀合于异源分子而产生融合物或缀合物。
在一些实施方案中，本发明的抗体多肽可以与一个或多个异源分子融合或缀合，其中所述异源分子包括但不限于蛋白/多肽/肽、标记物、药物、和细胞毒性剂。蛋白质、多肽或肽或化学分子与抗体融合或缀合的方法是本领域已知的。参见，例如，US 5,336,603、US 5,622,929和EP 367,166。
在一个实施方案中，本发明抗体与异源蛋白或多肽或肽重组融合而形成融合蛋白。在又一实施方案中，本发明的抗体与蛋白分子或非蛋白分子缀合而产生缀合物。
在一些实施方案中，本发明抗体可以以全长抗体或抗体片段的形式与异源分子融合或缀合。
接头可以用于共价连接本发明融合物和/或缀合物中的不同实体。接头包括化学接头或单链肽接头。在一些实施方案中，本发明的抗体通过肽接头融合到其它肽段或蛋白质上。在一些实施方案中，本发明的抗体通过化学接头缀合到其它分子例如标记物或药物分子上。
本发明的肽接头包括由氨基酸残基组成的肽。这样的接头肽通常是柔性的，允许与之连接的抗原结合部分独立地移动。接头肽的长度可以是由本领域技术人员根据实际情况而容易地确定，例如长至少4-15个氨基酸，或者更长，例如大约20-25个氨基酸。
IV.用于诊断和检测的方法和组合物
本发明提供了本发明的抗BCMA抗体、融合物或缀合物在诊断和检测中的用途。本文中提供的任何抗BCMA抗体、融合物或缀合物均可以用于检测生物样品中人BCMA的存在。
本文中使用的术语“检测”包括定量或定性检测。示例性的检测方法包括但不限于，免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在一些实施方案中，生物样品包括体液、细胞或组织。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。
在一个实施方案中，提供了抗BCMA抗体、融合物或缀合物用于诊断或检测方法。在进一步的方面中，提供了检测生物样品中BCMA存在的方法。在一些实施方案中，该方法包括将生物样品在允许抗BCMA抗体、融合物或缀合物与BCMA结合的条件下接触本文中所述的抗BCMA抗体、融合物或缀合物，并且检测抗BCMA抗体、融合物或缀合物和BCMA之间是否形成了复合物。这样的方法可以是体外或体内方法。
在一个实施方案中，将抗BCMA抗体、融合物或缀合物用于选择适宜使用抗BCMA抗体治疗的受试者，例如，当BCMA是用于患者选择的生物标志物时。可以使用本发明的抗体、融合物或缀合物诊断的示例性病症包括，B细胞相关病症，例如多发性骨髓瘤。在一些实施方案中，提供了用本发明的抗体、融合物或缀合物对多发性骨髓瘤(MM)患者进行分层的方法，所述方法包括确定所述患者的B细胞、优选恶性B细胞是否在所述B细胞的表面上表达BCMA蛋白，其中所述B细胞在其表面上表达BCMA蛋白，则所述患者将可能响应 并使用以BCMA为靶点的治疗剂(例如抗BCMA抗体)进行治疗。在一些实施方案中，抗BCMA抗体可以与诊断剂或可检测剂缀合。在一些实施方案中，本发明提供用于诊断或检测的试剂盒，其包含本发明的任何抗BCMA抗体、融合物或缀合物。
V.用于治疗的方法和组合物
本发明提供了治疗B细胞相关病症的方法，包括向所述受试者施用有效量的本发明的抗体或其抗原结合片段、或本发明的融合物或缀合物。
B细胞相关病症是与异常B细胞活性相关的病症，包括但不限于B细胞恶性肿瘤、浆细胞恶性肿瘤、自身免疫疾病。可以使用BCMA抗体治疗的示例性病症包括例如，多发性骨髓瘤、非霍奇金淋巴瘤、恶性潜能不确定的B细胞增殖、淋巴瘤样肉芽肿病、移植后淋巴增生病症、免疫调节病症、类风湿性关节炎、重症肌无力、特发性血小板减少性紫癜、抗磷脂综合征、恰加斯病、格雷夫斯病、韦格纳肉芽肿、结节性多动脉炎、舍格伦氏综合征、寻常天疱疮、硬皮病、多发性硬化症、ANCA相关血管炎、古德帕斯丘氏病、川崎病、自身免疫性溶血性贫血和急进性肾小球肾炎、重链病、原发性或免疫细胞相关的淀粉样变性、或意义未明的单克隆丙种球蛋白病、全身性红斑狼疮、风湿性关节炎。
在一些实施方案中，本发明的抗体、融合物和缀合物用于治疗人类的B细胞相关病症，如B细胞恶性肿瘤，优选地，多发性骨髓瘤(MM)或非霍奇金淋巴瘤(NHL)。在一些实施方案中，本发明的抗BCMA抗体、融合物和缀合物具有抗肿瘤作用，包括但不限于例如，减少肿瘤体积、减少肿瘤细胞数目、减少肿瘤细胞增殖或减少肿瘤细胞存活。
可以理解，可将本发明的BCMA抗体、融合物和缀合物与其它治疗形式组合施用，用于上述疾病例如肿瘤的治疗。所述其它治疗形式包括治疗剂、放疗、化疗、移植、免疫疗法等。在一些实施方案中，本发明抗体分子、融合物和缀合物与其它治疗剂联合使用。示例性的治疗剂包括细胞因子、生长因子、类固醇、NSAID、DMARD、抗炎剂、化疗剂、放疗剂、治疗性抗体或者其它活性剂和辅助剂，例如抗肿瘤药物。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。
实施例
实施例1 BCMA亲和力改善抗体的产生和表达
从国际申请号PCT/CN2019/074419(BCMA抗体相关专利)获得BCMA亲本抗体ADI-34861的重链可变区和轻链可变区序列(分别为SEQ ID NO:9所示的VH、SEQ ID NO:18所示的VL序列)和BCMA亲本抗体ADI-34857的重链可变区和轻链可变区序列(分别为SEQ ID NO:63所示的VH、SEQ ID NO:72所示的VL序列)。
为了获得对BCMA亲和力改善的抗体，本实施例对BCMA亲本抗体ADI-34861和ADI-34857进行了亲和力变体设计和功能测定。
使用标准方案(Silacci等人,(2005),Proteomics 5,2340-50)，通过噬菌体展示法实施亲本抗体ADI-34861和ADI-34857衍生的亲和力成熟Fab的产生。对亲本抗体ADI-34861，经多轮淘选后，初步获得亲和力成熟抗体ADI-38491(其具有SEQ ID NO:27所示的VH、SEQ ID NO:36所示 的VL序列)和抗体ADI-38497(其具有SEQ ID NO:45所示的VH、SEQ ID NO:54所示的VL序列)；对亲本抗体ADI-34857，经多轮淘选后，初步获得亲和力成熟抗体ADI-38481(其具有SEQ ID NO:81所示的VH、SEQ ID NO:90所示的VL序列)和抗体ADI-38484(其具有SEQ ID NO:99所示的VH、SEQ ID NO:108所示的VL序列)
分别将编码抗体ADI-38491重链可变区的核苷酸序列(SEQ ID NO:26)和轻链可变区的核苷酸序列(SEQ ID NO:35)、抗体ADI-38497重链可变区的核苷酸序列(SEQ ID NO:44)和轻链可变区的核苷酸序列(SEQ ID NO:53)、抗体ADI-38481重链可变区的核苷酸序列(SEQ ID NO:80)和轻链可变区的核苷酸序列(SEQ ID NO:89)、抗体ADI-38484重链可变区的核苷酸序列(SEQ ID NO:98)和轻链可变区的核苷酸序列(SEQ ID NO:107)构建至经过改造的含有轻、重链恒定区片段的真核表达载体质粒pcDNA3.3(Invitrogen)上，按照制造商的说明，使用ExpiCHO瞬转表达系统(Thermo Fisher，A29133)在CHO-K细胞中共表达抗体的全长重链和轻链，然后通过蛋白A亲和层析进行纯化，获得抗体ADI-38491、抗体ADI-38497、抗体ADI-38481、抗体ADI-38484。
实施例2 Fortebio检测抗体亲和力
利用Fortebio公司的Octet QKe system仪器，采用抗人抗体Fc段的捕获抗体(AHC)生物探针捕获抗体Fc段的方法测定抗体亲和力。具体操作如下。
将抗体ADI-34861、抗体ADI-38491、抗体ADI-38497、抗体ADI-34857、抗体ADI-38481、抗体ADI-38484分别用PBS缓冲液稀释至4μg/ml，流经AHC探针(Cat:18-0015，PALL)表面，时间为120s。使用人、食蟹猴、小鼠BCMA(60nM)作为流动相，结合时间为180s，解离时间为180s。实验完毕，扣除空白对照(PBS缓冲液)响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。动力学常数如下表1所示。
结果表明，与相应的亲本抗体ADI-34861相比，突变后的抗体ADI-38491、抗体ADI-38497亲和力显著提高；与相应的亲本抗体ADI-34857相比，突变后的抗体ADI-38481、抗体ADI-38484亲和力显著提高。
表1各抗体与BCMA的结合亲和力
Figure PCTCN2022137266-appb-000001
实施例3基于细胞的抗体功能测定
为了测试抗体对细胞表面上表达的BCMA的结合亲和力，使用三种细胞(NCI-H929细胞、BCMA-KO-H929细胞和人BCMA CHO-S细胞)进行了测定。NCI-H929细胞(文中也简称为H929细胞)(购自南京科佰生物科技有限公司)是一种细胞表面天然表达BCMA分子的人多发性骨髓瘤细胞系，BCMA-KO-H929细胞是以H929细胞为基础，利用CRISPR-Cas9技术，特异性靶向BCMA基因，使其发生移码突变，进而无法正常表达BCMA分子的细胞系(委托南京金斯瑞生物科技有限公司构建)，人BCMA CHO-S细胞是将外源的人BCMA导入CHO-S细胞制备的。人BCMA CHO-S细胞的制备方法如下：将编码人BCMA(NP_001183.2，SEQ ID NO:109)的序列克隆入pcDNA3.3(Invitrogen)载体的多克隆位点，获得了表达人BCMA的表达载体；然后将该表达人BCMA的表达载体导入CHO-S细胞(ATCC)进行真核表达，获得了在细胞表面表达人BCMA的CHO-S细胞。
将人BCMA CHO-S细胞或H929细胞以1.0×10 5个细胞/孔接种到96孔板，并添加稀释的10nM各抗体(抗体ADI-34861、抗体ADI-38491、抗体ADI-38497、抗体ADI-34857、抗体ADI-38481、抗体ADI-38484)。在4℃孵育30分钟后，洗涤细胞，并添加100μL APC标记的山羊抗人IgG第二抗体(Jackson ImmunoResearch Inc，货号：109-136-097，别藻蓝蛋白标记的特异性结合F(ab’) 2片段的山羊抗人IgG第二抗体F(ab’) 2片段(Allophycocyanin(APC)AffiniPure F(ab’) 2Fragment Goat Anti-Human IgG,F(ab’) 2fragment specific))，在4℃孵育30分钟。然后洗涤细胞并通过流式细胞术(Beckman Coulter)检测各抗体与细胞表面表达的BCMA分子的结合。使用CHO-S细胞作为阴性对照。
表2.各抗体与细胞表面表达的BCMA的结合
Figure PCTCN2022137266-appb-000002
由表2可见，抗体ADI-34861、抗体ADI-38491、抗体ADI-38497均与细胞表面表达的BCMA结合，且抗体ADI-38497对细胞表面上表达的BCMA的结合亲和力显著性提高。抗体ADI-34857、抗体ADI-38481、抗体ADI-38484均与细胞表面表达的BCMA结合，且抗体ADI-38481、ADI-38484对细胞表面上表达的BCMA的结合亲和力显著性提高。
进一步地，将H929细胞和BCMA-KO-H929细胞以3.0×10 5个细胞/孔接种到96孔板， 并添加稀释的ADI-38497抗体(1μg/孔)，阴性对照孔不加抗体。在4℃孵育30分钟后，洗涤细胞，并添加100μL APC标记的山羊抗人IgG第二抗体(Jackson ImmunoResearch Inc，货号：109-136-097)至所有细胞孔中，在4℃孵育30分钟。然后洗涤细胞并通过流式细胞术检测抗体ADI-38497抗体与各细胞表面表达的BCMA分子的结合。
由图1可见，ADI-38497抗体仅与表达BCMA抗原的H929细胞相结合，而与BCMA基因敲除的BCMA-KO-H929细胞不结合，因此，ADI-38497抗体能与BCMA抗原特异性结合。
实施例4 ADI-38497 WT抗体、ADI-38497 PG抗体、ADI-38484 WT抗体和ADI-38484 PG抗体的制备和抗原结合活性检测
4.1ADI-38497 WT抗体、ADI-38497 PG抗体、ADI-38484 WT抗体和ADI-38484 PG抗体的制备
从US9273141B2专利中获得GSK公司BCMA抗体克隆J6M0轻重链可变区序列，作为对照抗体(GSK IgG)。
采用全基因合成GSK IgG、ADI-38497、ADI-38484抗体轻重链可变区序列，装入含有WT的人源IgG1重链恒定区(SEQ ID NO:110)或含有P329G点突变的人源IgG1重链恒定区(SEQ ID NO:111)和κ轻链恒定区(SEQ ID NO:112)的pcDNA3.4表达载体(购自上海伯英)上。将轻重链表达载体按照2:3摩尔比通过PEI共转染到HEK293细胞中，培养5～7天后收集培养基上清。含有抗体的上清培养基通过Protein A柱进行一步纯化，之后用PBS透析。采用NanoDrop仪器读取280nm吸光度值检测浓度，并用SDS-PAGE和SEC-HPLC方法检测样品纯度。获得了GSK WT抗体、GSK PG抗体；ADI-38497 WT抗体、ADI-38497 PG抗体；ADI-38484 WT抗体、ADI-38484 PG抗体。
4.2 ADI-38497 PG抗体的亲和力检测
通过Biacore T200测定了ADI-38497 PG抗体与不同种属BCMA的亲和力，图2A显示了采用表面等离子共振法(SPR)测定抗体亲和力的方法示意图。
具体方法如下：将抗人Fc IgG(Ab97221,Abcam)偶联到CM5芯片(29149603,Cytiva)表面后，将ADI-38497 PG抗体捕获在芯片表面，通过检测芯片表面抗体与流动相中的BCMA抗原之间的结合与解离获得亲和力及动力学常数。测定过程使用10倍稀释后的10×HBS-EP+(BR-1006-69,Cytiva)作为实验缓冲液。亲和力检测中的每个循环包括捕获ADI-38497 PG抗体、结合一种浓度抗原及芯片再生。将梯度稀释后的抗原(抗原浓度梯度为1.25-40nM，2倍稀释)，以30μl/分钟的流速流由低浓度到高浓度的顺序流过芯片表面，结合时间180s，设定合适的解离时间(900s或600s或60s)。最后使用10mM甘氨酸-HCl,pH 1.5(BR-1003-54,Cytiva)对芯片进行再生。
数据结果通过Biacore T200分析软件(版本号3.1)，使用1:1结合模型进行分析。
图2B展示了采用SPR测定ADI-38497 PG抗体与重组人、食蟹猴、小鼠、大鼠和兔BCMA蛋白的代表性亲和力图谱。结果显示，ADI-38497 PG抗体与上述不同种属来源的BCMA蛋白均可结合，其中结合活性的高低顺序依次为人BCMA>猴BCMA>小鼠BCMA>大鼠BCMA>兔BCMA。
表3 ADI-38497 PG抗体与来自不同种属的BCMA蛋白的结合亲和力
Figure PCTCN2022137266-appb-000003
4.3 P329G BCMA抗体与不同种属来源BCMA抗原结合活性检测
首先，制备了表达不同种属来源BCMA抗原的CHO GS细胞。具体而言，将人、小鼠、食蟹猴源的BCMA基因合成并克隆至慢病毒载体中，随后包装含不同种属来源BCMA基因的慢病毒，并用此慢病毒感染CHO GS细胞，后续经流式细胞术分选，得到表达不同种属来源BCMA抗原的CHO GS细胞系，即hBCMA-CHO GS、mBCMA-CHO GS和cynoBCMA-CHO GS细胞。
然后，用FACS缓冲液将ADI-38497 PG抗体及GSK来源的BCMA抗体(即，GSK PG IgG用作Benchmark)配制成10倍梯度稀释的不同浓度抗体溶液，分别与1E5个所制备的表达不同种属来源BCMA抗原的CHO GS细胞4℃孵育30分钟，用FACS缓冲液清洗后，再与Fcγ片段特异的APC-山羊抗人IgG(Jackson ImmunoResearch,109-136-098)在4℃孵育30分钟。采用流式细胞术检测与细胞结合的P329G抗体，分析APC通道MFI，以抗体浓度为X轴，APC通道MFI为Y轴进行绘图并计算结合的EC50。
图2C显示了不同浓度P329G BCMA抗体与稳定表达人、食蟹猴及小鼠BCMA的CHO-GS细胞的结合能力。由图2C可见，ADI-38497 PG IgG抗体能够与细胞表面表达的不同种属BCMA结合，而GSK来源的BCMA抗体(Benchmark)具有较高的种属BCMA特异性，其不识别小鼠BCMA，该结果与SPR检测结果一致。
表4 P329G BCMA抗体与表达不同种属BCMA的CHO-GS细胞的结合EC50值
Figure PCTCN2022137266-appb-000004
Figure PCTCN2022137266-appb-000005
4.4 P329G BCMA抗体与肿瘤细胞表面BCMA抗原结合活性检测
取适量处于对数生长期的肿瘤细胞，FACS缓冲液洗涤2次，加入ADI-38497 PG抗体、ADI-38484 PG抗体及用作Benchmark的GSK PG IgG，对作为染色对照的细胞加入同种型hIgG1抗体，4℃染色30分钟，洗涤两次，加入APC-F(ab') 2片段的山羊抗人IgG抗体，4℃染色30分钟，细胞洗涤两次后FACS缓冲液重悬，采用流式细胞仪进行检测。
图2D显示不同浓度P329G BCMA抗体与表达BCMA的阳性多发性骨髓瘤细胞系MM.1s、RPMI8226、U266、H929、L363及AMO1(MM.1s购自南京科佰生物科技有限公司，CBP60239；RPMI8226购自南京科佰生物科技有限公司，CBP60244；U266购自武汉普诺赛生命科技有限公司，CL-0510；H929购自南京科佰生物科技有限公司，CBP60243；L363南京科佰生物科技有限公司，CBP6024；AMO1购自南京科佰生物科技有限公司，CBP60242)的结合活性，ADI-38497 PG抗体、ADI-38484 PG抗体能够与表达BCMA的阳性肿瘤细胞结合并呈现浓度依赖性。在所述表达BCMA的阳性肿瘤细胞中，MM.1s细胞有最高水平BCMA表达，RPMI8226、U266和H929细胞以中等水平表达BCMA，L363、AMO1细胞以低水平表达BCMA。
表5 P329G BCMA抗体与表达BCMA的阳性肿瘤细胞的结合EC50值
Figure PCTCN2022137266-appb-000006
实施例5 ADI-38497 WT抗体和ADI-38497 PG抗体的生物学功能检测
5.1 ADCC效应功能检测
复苏供者3的PBMC细胞(Peripheral Blood Mononuclear Cells，外周血单个核细胞)，用含有10％胎牛血清的RPMI 1640培养基重悬，并在37℃稳定1-2小时。按照效靶比25:1混合PBMC和靶细胞，并与不同浓度的BCMA抗体进行混合，37℃分别继续培养4小时和24小时，用LDH检测试剂盒(Promega，G1780)检测抗体介导的PBMC对靶细胞的杀伤效应，并以抗体浓度为X轴，细胞裂解比例为Y轴进行绘图和分析。同时收集细胞，用FACS缓冲液洗涤2次，加入CD3、CD56、CD16及CD107a抗体，其中CD107a抗体需提前加入，与细胞在37℃共孵育1小时。上述细胞抗体混合液于4℃染色30分钟，洗涤两次，FACS缓冲液重悬，采用流式细胞仪进行检测。
图3A显示ADI-38497 WT抗体和ADI-38497 PG抗体介导ADCC杀伤的能力，结果表明，当测试不同的孵育时间(4小时、24小时)和使用不同的检测指标(对靶细胞的细胞毒性、对CD3、CD56、CD16及CD107a表达的影响)，均显示只有WT抗体介导了对表达BCMA的阳性H929肿瘤细胞的ADCC细胞毒杀伤效应，而P329G突变抗体缺乏诱导ADCC效应的能力。
5.2 ADCP效应功能检测
取对数生长期的ADCP报告细胞系(Promega,G9871)与H929细胞，按照效靶比2:1、5:1混合ADCP报告细胞和H929靶细胞，并与不同浓度的BCMA抗体进行混合，37℃继续培养20小时，用萤光素酶检测试剂盒(Promega，E2620)检测抗体介导的依赖靶细胞的报告细胞激活效应，并以抗体浓度为X轴，荧光读值变化为Y轴进行绘图和分析。
图3B显示ADI-38497 WT抗体和ADI-38497 PG抗体介导ADCP杀伤的能力。结果表明，当测试不同的效靶比(2:1或5:1)，均显示只有ADI-38497 WT抗体介导了对BCMA表达阳性H929肿瘤细胞的ADCP杀伤效应，而P329G突变抗体缺乏诱导ADCP杀伤效应的能力。
5.3 P329G突变抗体的抗增殖功能检测
取对数生长期H929细胞和L363细胞，按一定数量铺于孔板中；并将一部分对数生长期H929细胞和L363细胞作为靶细胞经丝裂霉素C处理，用作阳性对照。随后加入不同浓度的ADI-38497 PG抗体进行混合，37℃继续分别培养48小时、72小时及120小时，用CellTiter-Glo(Promega,G9242)检测活细胞比例，以共孵育时间为X轴，荧光读值为Y轴进行绘图并分析。
图3C显示了ADI-38497 PG抗体是否具有抑制肿瘤细胞增殖能力，结果表明，测试的不同孵育时间(48小时、72小时及120小时)与不同ADI-38497 PG抗体浓度(5μg/ml、50μg/ml)，均显示ADI-38497 PG抗体本身缺乏抑制肿瘤细胞增殖的能力。
实施例6 ADI-38497 PG抗体的体内药代动力学研究
6.1抗体注射及取样
将BALB/c小鼠(年龄4-6周，体重15-17g，雌性)分成3组，即ADI-38497 PG抗体，1mg/kg抗体组；ADI-38497 PG抗体，10mg/kg抗体组；和ADI-38497 PG抗体，200mg/kg抗体组，每组9只小鼠；用1×PBS将抗体分别稀释至0.1mg/mL,1mg/mL和20mg/mL，每只小鼠给药体积10mL/kg，即抗体给药剂量分别为1mg/kg，10mg/mL和200mg/mL；给药方式为静脉注射，给药频率为单次。抗体给药后5分钟、30分钟、2h、6h、24h、48h、96h、168h、336h和504h小鼠眼眶后静脉丛采集血样100μL，3000g离心，吸取上清液用于血药浓度测定。
6.2 ADI-38497 PG抗体检测
提前一天包被96孔酶标板。用包被液(取一包碳酸盐(Thermo,28382)粉末，溶解于400mL超纯水，定容至500mL，混匀即为包被液)将BCMA抗原稀释至1μg/ml，每孔100μL，封板膜封板，室温过夜。倒掉包被溶液，在吸水纸上拍干，然后每孔加入300μL洗液，振荡混匀10秒，拍干洗液后，重复洗涤3次。排枪加入封闭液，每孔200μL，封板膜封板，室温孵育2h。随后洗板1次。将稀释好的标准曲线(由已知浓度的BCMA抗体梯度稀释，制备标准曲线(例如，使用已知浓度的ADI-38497 PG抗体制备标准曲线)、质量控制样品和待测样本每孔100μL，室温孵育2h。倒掉预包被溶液，在吸水纸上拍干，然后每孔加入300μL洗液，振荡混匀10秒，拍干洗液后，重复洗涤3次。重复一次。将山羊抗人IgG-Fc-HRP抗体(BETHYL)1:10万稀释，每孔加入100μL，室温避光孵育1h。随后洗板1次。将TMB底物加入到96孔酶标板中，每孔100μL，室温下避光显色5分钟。每孔加入50μL ELISA终止液，震荡10秒，30分钟内读取OD450nm和OD620nm值。
图4A和4B显示小鼠中ADI-38497 PG抗体(下文小鼠体内实验中也简称为PG Ab)药代动力学实验结果。小鼠静脉注射1mg/kg、10mg/kg、200mg/kg的ADI-38497 PG抗体后，血清中ADI-38497 PG抗体暴露量(Cmax和AUClast)呈现剂量依赖效应，其他药代动力学参数无明显差异，如表6所示，ADI-38497 PG抗体1mg/kg：AUC0-inf，Cmax，CL，T1/2分别为2480μg×h/mL，30ug/ml，0.40ml/kg/h、145h；ADI-38497 PG抗体10mg/kg：AUC0-inf，Cmax，CL，T1/2分别为24720μg×h/mL，187ug/ml，0.32ml/kg/h、219h；ADI-38497 PG抗体200mg/kg：AUC0-inf，Cmax，CL，T1/2分别为397734μg×h/mL，3895ug/ml，0.43ml/kg/h、197h，ADI-38497 PG抗体1mg/kg半衰期略短于ADI-38497 PG抗体10mg/kg和200mg/kg。
表6 ADI-38497 PG抗体药代动力学实验结果
Figure PCTCN2022137266-appb-000007
实施例7 ADI-38497 PG抗体体内抗肿瘤效应
进行了小鼠肿瘤接种及处理。具体地，用1×PBS重悬H929细胞，制备成细胞浓度为5×10 6个/mL细胞悬液。NOG小鼠(年龄4-6周，体重15-17g，雌性)右侧背部剃毛，皮下注射H929细胞悬液，注射体积0.2mL/只，即接种量为1×10 6个细胞/只小鼠。肿瘤细胞接种后7天，将小鼠肿瘤体积在50.82～104.36mm 3的小鼠分组，分别为PBS载剂组、ADI-38497 PG抗体组(文中也简称为“PG Ab组”)，每组7只小鼠。分组完成后，于第7日进行抗体给药，每只小鼠给药体积10mL/kg，给药频率为每周1次，给药方式为腹腔注射。每周2次监测小鼠体重、肿瘤组织最大长轴(L)和最大宽轴(W)。
图5A显示在皮下接种人BCMA表达阳性H929肿瘤细胞的免疫缺陷荷瘤小鼠中ADI-38497 PG抗体的治疗效应。结果显示，在高表达BCMA的H929肿瘤模型中，施用PG抗体产生显著的抗肿瘤效应。
图5B显示了该实验中小鼠的体重变化。结果显示，在高表达BCMA的H929肿瘤模型中，施用PG抗体后，小鼠体重无显著性变化。
因此，ADI-38497 PG抗体具有显著的抗BCMA高表达肿瘤效应，并且无明显毒副效应。
实施例8 ADI-38497 PG抗体在体内抗系统性肿瘤效应研究
首先，制备了H929-luc细胞。具体而言，用H929细胞(购自南京科佰生物科技有限公司)包装含GFP-萤光素酶基因的慢病毒，并用获得的慢病毒感染H929细胞，后续经流式细胞术分选，得到GFP和萤光素酶双表达的H929-luc细胞系。
然后，用1×PBS重悬H929-luc细胞，制备成细胞浓度为25×10 6个/mL细胞悬液。NOG 小鼠(年龄4-6周，体重15-17g，雌性)尾静脉注射H929-luc细胞悬液，注射体积0.2mL/只。肿瘤细胞接种后14天，腹腔注射底物D-Luciferin(15mg/mL)，注射体积为10mL/kg/只小鼠，底物注射10分钟后用IVIS spectrum成像分析。将荧光信号在1.17×10 7～1.43×10 8photons/sec的小鼠分组，分别为载剂组、PG Ab，0.3mg/kg组、PG Ab，3mg/kg组，每组6-7只小鼠。分别配置浓度为0.03mg/mL和0.3mg/mL的抗体，分组完成后，于第14日开始进行抗体给药，每只小鼠给药体积10mL/kg，给药频率为每周1次，给药方式为腹腔注射。
图6A显示在尾静脉接种人H929-luc肿瘤细胞的免疫缺陷荷瘤小鼠中不同剂量PG抗体抗肿瘤药效。结果显示，在系统性肿瘤模型中，PG抗体在给药后1周左右开始产生抗肿瘤药效，并呈现剂量依赖性；药效维持2周后逐渐减弱。
图6B显示上述实验中小鼠体重变化。结果显示，治疗期间各处理组小鼠体重平稳上升，提示PG抗体处理未诱导明显毒性。
实施例9 ADI-38497 PG抗体的体内毒理研究
进行了小鼠肿瘤接种及处理。具体地，用1×PBS重悬H929细胞，制备成细胞浓度为5×10 6个/mL细胞悬液。NOG小鼠(年龄4-6周，体重15-17g，雌性)右侧背部剃毛，皮下注射5×10 6个/mL的H929细胞悬液，注射体积0.2mL/只。肿瘤细胞接种后6天，将小鼠肿瘤体积在38.49～104.77mm 3的小鼠分组，如表7所示，分别为未荷瘤的载剂组、荷瘤的载剂组、PG Ab组，每组24只小鼠。配置浓度为1mg/mL的抗体，分组完成后，抗体给药，每只小鼠给药体积10mL/kg，给药频率为每周1次，给药次数3次，给药方式为腹腔注射。每周2次监测小鼠体重、肿瘤组织最大长轴(L)和最大宽轴(W)。第1次抗体给药前、第3次抗体给药前及实验终点取外周血，每组4只小鼠，用于血液学和血生化检测。
表7各组的处理和给药剂量
组别 细胞系 处理 浓度
1 无肿瘤负荷 载剂 N/A
2 H929 载剂 N/A
3 H929 PG抗体 10mg/kg(QW×3)
图7A显示在皮下接种人H929肿瘤细胞的免疫缺陷荷瘤小鼠中PG抗体的治疗效应。结果显示，PG抗体具有抗肿瘤效应。
图7B显示本实验中小鼠体重变化。结果显示，PG抗体治疗期间体重相较对照小鼠无明显变化，提示PG抗体未诱导明显毒性反应。
图7C和图7D显示上述实验中小鼠血液学和血生化检测结果。结果显示，PG抗体治疗期间小鼠血液学和血生化指标较对照小鼠无明显变化，表明PG抗体治疗未产生毒性反应。
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。
示例性序列
Figure PCTCN2022137266-appb-000008
Figure PCTCN2022137266-appb-000009
Figure PCTCN2022137266-appb-000010
Figure PCTCN2022137266-appb-000011
Figure PCTCN2022137266-appb-000012
Figure PCTCN2022137266-appb-000013

Claims (17)

  1. 特异性结合BCMA的抗体或抗原结合片段，其包含
    (a)SEQ ID NO:27所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:36所示的轻链可变区氨基酸序列中的3个CDR；或者与所述6个CDR区具有单个CDR或者多个CDR不超过每个CDR区2个或1个氨基酸变化的变体；
    (b)SEQ ID NO:45所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:54所示的轻链可变区氨基酸序列中的3个CDR；或者与所述6个CDR区具有单个CDR或者多个CDR不超过每个CDR区2个或1个氨基酸变化的变体；
    (c)SEQ ID NO:81所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:90所示的轻链可变区氨基酸序列中的3个CDR；或者与所述6个CDR区具有单个CDR或者多个CDR不超过每个CDR区2个或1个氨基酸变化的变体；或
    (d)SEQ ID NO:99所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:108所示的轻链可变区氨基酸序列中的3个CDR；或者与所述6个CDR区具有单个CDR或者多个CDR不超过每个CDR区2个或1个氨基酸变化的变体；
    其中所述氨基酸变化是氨基酸的添加、缺失或取代。
  2. 特异性结合BCMA的抗体或抗原结合片段，其包含重链可变区和轻链可变区，其中：
    (a)所述重链可变区包含根据Kabat编号的GSIVSSSYYWT(SEQ ID NO:19)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；SISIAGSTYYNPSLKS(SEQ ID NO:20)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARDRGDTILDV(SEQ ID NO:21)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的RASQSISRYLN(SEQ ID NO:28)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；AASSLQS(SEQ ID NO:29)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQKYFDIT(SEQ ID NO:30)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    (b)所述重链可变区包含根据Kabat编号的GSIVSSSYYWT(SEQ ID NO:37)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；SISIAGSTYYNPSLKS(SEQ ID NO:38)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARDRGDQILDV(SEQ ID NO:39)所示的HCDR3、 或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的RASQSISRYLN(SEQ ID NO:46)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；AASSLQS(SEQ ID NO:47)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQKYFDIT(SEQ ID NO:48)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    (c)所述重链可变区包含根据Kabat编号的GTFSNDVIS(SEQ ID NO:73)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；VIIPIFGIANYAQKFQG(SEQ ID NO:74)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARGRGYYSSWLLDI(SEQ ID NO:75)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的QASQDITNYLN(SEQ ID NO:82)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；DASNLET(SEQ ID NO:83)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQAFDLIT(SEQ ID NO:84)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；或
    (d)所述重链可变区包含根据Kabat编号的GTFSNDVIS(SEQ ID NO:91)所示的HCDR1、或所述HCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；VIIPIFGIANYAQKFQG(SEQ ID NO:92)所示的HCDR2、或所述HCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和ARGRGYYSSWLHDI(SEQ ID NO:93)所示的HCDR3、或所述HCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；所述轻链可变区包含根据Kabat编号的QASQDITNYLN(SEQ ID NO:100)所示的LCDR1、或所述LCDR1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；DASNLET(SEQ ID NO:101)所示的LCDR2、或所述LCDR2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和QQAFDLIT(SEQ ID NO:102)所示的LCDR3、或所述LCDR3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    其中所述氨基酸变化是氨基酸的添加、缺失或取代。
  3. 根据权利要求2所述的特异性结合BCMA的抗体或抗原结合片段，其包含重链可变区和轻链可变区，其中
    (a)所述重链可变区包含GSIVSSSYYWT(SEQ ID NO:19)所示的HCDR1；SISIAGSTYYNPSLKS(SEQ ID NO:20)所示的HCDR2；和ARDRGDTILDV(SEQ ID NO:21)所示的HCDR3；所述轻链可变区包含RASQSISRYLN(SEQ ID NO:28)所示的LCDR1；AASSLQS(SEQ ID NO:29)所示的LCDR2；和QQKYFDIT(SEQ ID NO:30)所示的LCDR3；
    (b)所述重链可变区包含GSIVSSSYYWT(SEQ ID NO:37)所示的HCDR1；SISIAGSTYYNPSLKS(SEQ ID NO:38)所示的HCDR2；和ARDRGDQILDV(SEQ ID NO:39)所示的HCDR3；所述轻链可变区包含RASQSISRYLN(SEQ ID NO:46)所示的LCDR1； AASSLQS(SEQ ID NO:47)所示的LCDR2；和QQKYFDIT(SEQ ID NO:48)所示的LCDR3；
    (c)所述重链可变区包含GTFSNDVIS(SEQ ID NO:73)所示的HCDR1；VIIPIFGIANYAQKFQG(SEQ ID NO:74)所示的HCDR2；和ARGRGYYSSWLLDI(SEQ ID NO:75)所示的HCDR3；所述轻链可变区包含QASQDITNYLN(SEQ ID NO:82)所示的LCDR1；DASNLET(SEQ ID NO:83)所示的LCDR2；和QQAFDLIT(SEQ ID NO:84)所示的LCDR3；或
    (d)所述重链可变区包含GTFSNDVIS(SEQ ID NO:91)所示的HCDR1；VIIPIFGIANYAQKFQG(SEQ ID NO:92)所示的HCDR2；和ARGRGYYSSWLHDI(SEQ ID NO:93)所示的HCDR3；所述轻链可变区包含QASQDITNYLN(SEQ ID NO:100)所示的LCDR1；DASNLET(SEQ ID NO:101)所示的LCDR2；和QQAFDLIT(SEQ ID NO:102)所示的LCDR3。
  4. 根据权利要求1至3中任一项所述的特异性结合BCMA的抗体或抗原结合片段，其包含重链可变区和轻链可变区，其中
    (a)重链可变区包含SEQ ID NO:27的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:36的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    (b)重链可变区包含SEQ ID NO:45的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:54的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    (c)重链可变区包含SEQ ID NO:81的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:90的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；或
    (d)重链可变区包含SEQ ID NO:99的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:108的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
  5. 根据权利要4所述的特异性结合BCMA的抗体或抗原结合片段，其包含重链可变区和轻链可变区，其中，所述抗体或抗原结合片段包含
    (a)SEQ ID NO:27所示的重链可变区和SEQ ID NO:36所示的轻链可变区；
    (b)SEQ ID NO:45所示的重链可变区和SEQ ID NO:54所示的轻链可变区；
    (c)SEQ ID NO:81所示的重链可变区和SEQ ID NO:90所示的轻链可变区；或
    (d)SEQ ID NO:99所示的重链可变区和SEQ ID NO:108所示的轻链可变区。
  6. 根据权利要求1至5中任一项所述的特异性结合BCMA的抗体或抗原结合片段，其是IgG1、IgG2、IgG3或IgG4抗体；任选地，其是IgG1或IgG4抗体；任选地，其是IgG1抗体。
  7. 根据权利要求1至6中任一项所述的特异性结合BCMA的抗体或抗原结合片段，其中所述抗原结合片段是Fab、Fab’、F(ab’)2、Fv、单链Fv、单链Fab或双体抗体(diabody)。
  8. 根据权利要求1至6中任一项所述的特异性结合BCMA的抗体或抗原结合片段，其还包含突变Fc结构域，其中根据EU编号的P329位置处的氨基酸突变为甘氨酸(G)，与未突变的亲本抗体Fc结构域的Fcγ受体结合相比，突变Fc结构域的Fcγ受体结合降低；例如，所述突变Fc结构域是IgG1、IgG2、IgG3或IgG4抗体的突变Fc结构域，优选地，所述突变Fc结构域是IgG1或IgG4抗体的突变Fc结构域；更优选地，所述突变Fc结构域是IgG1抗体的突变Fc结构域；
    例如，所述抗体或抗原结合片段包含SEQ ID NO:111所示的重链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列且其中根据EU编号的P329位置处的氨基酸突变为G；
    例如，所述抗体或抗原结合片段包含SEQ ID NO:111所示的重链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列且其中根据EU编号的P329位置处的氨基酸突变为G；和SEQ ID NO:112所示的轻链恒定区序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    例如，所述抗体或抗原结合片段包含SEQ ID NO:111所示的重链恒定区序列和SEQ ID NO:112所示的轻链恒定区序列。
  9. 根据权利要求1至8中任一项所述的特异性结合BCMA的抗体或抗原结合片段，其具有以下一个或多个特性：
    (1)以高亲和力结合BCMA，例如人BCMA、食蟹猴BCMA和小鼠BCMA，例如，所述抗BCMA抗体或其抗原结合片段与BCMA之间结合的K D是约10 -9M至约10 -12M，如通过ForteBio动力学结合测定法所测量；
    (2)特异性地与细胞表面表达的BCMA结合；
    (3)对表达BCMA的细胞具有ADCC细胞毒杀伤效应；
    (4)对表达BCMA的细胞具有ADCP杀伤效应；
    (5)阻断、抑制表达人BCMA的细胞(尤其是多发性骨髓瘤细胞)的生长、和/或杀死所述细胞；和
    (6)对表达BCMA的肿瘤具有体内抗肿瘤效应，并且无明显毒副效应。
  10. 分离的核酸，其编码权利要求1至9中任一项的抗BCMA抗体或其抗原结合片段。
  11. 包含权利要求10的核酸的载体，优选地所述载体是表达载体。
  12. 包含权利要求10的核酸或权利要求11的载体的宿主细胞，优选地，所述宿主细胞是原核的或真核的，更优选的选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞，最优选地，所述宿主细胞是HEK293细胞或CHO细胞。
  13. 制备权利要求1至9中任一项的抗BCMA抗体或其抗原结合片段的方法，所述方法包括在适于表达编码权利要求1至9中任一项的抗BCMA抗体或其抗原结合片段的核酸的条件下培养权利要求12的宿主细胞，任选地分离所述抗BCMA抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述抗BCMA抗体或其抗原结合片段。
  14. 包含权利要求1至9中任一项的抗BCMA抗体或其抗原结合片段的缀合物、融合物或双特异性抗体。
  15. 药物组合物，其包含权利要求1至9中任一项的抗BCMA抗体或其抗原结合片段、或权利要求14的缀合物、融合物或双特异性抗体，以及任选地可药用载体。
  16. 权利要求1至9中任一项的抗BCMA抗体或其抗原结合片段、权利要求14的缀合物、融合物或双特异性抗体、或权利要求15的药物组合物的用途，用于制备在受试者中预防或治疗B细胞相关疾病的药物，例如，所述B细胞相关病症选自：B细胞恶性肿瘤、浆细胞恶性肿瘤、自身免疫疾病，优选地选自：多发性骨髓瘤、非霍奇金淋巴瘤、恶性潜能不确定的B细胞增殖、淋巴瘤样肉芽肿病、移植后淋巴增生病症、免疫调节病症、类风湿性关节炎、重症肌无力、特发性血小板减少性紫癜、抗磷脂综合征、恰加斯病、格雷夫斯病、韦格纳肉芽肿、结节性多动脉炎、舍格伦氏综合征、寻常天疱疮、硬皮病、多发性硬化症、ANCA相关血管炎、古德帕斯丘氏病、川崎病、自身免疫性溶血性贫血和急进性肾小球肾炎、重链病、原发性或免疫细胞相关的淀粉样变性、或意义未明的单克隆丙种球蛋白病，优选地，所述B细胞相关病况是B细胞恶性肿瘤，更优选地，多发性骨髓瘤(MM)或非霍奇金淋巴瘤(NHL)。
  17. 检测样品中BCMA的试剂盒，所述试剂盒包含权利要求1至9中任一项的抗BCMA抗体或其抗原结合片段，用于实施以下步骤：
    (a)将样品与权利要求1至9中任一项的抗BCMA抗体或其抗原结合片段接触；和
    (b)检测所述抗BCMA抗体或其抗原结合片段和BCMA间的复合物的形成；任选地，所述抗BCMA抗体或其抗原结合片段是被可检测地标记的。
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