WO2019149269A1 - 全人源的抗b细胞成熟抗原(bcma)单链抗体及其应用 - Google Patents

全人源的抗b细胞成熟抗原(bcma)单链抗体及其应用 Download PDF

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WO2019149269A1
WO2019149269A1 PCT/CN2019/074419 CN2019074419W WO2019149269A1 WO 2019149269 A1 WO2019149269 A1 WO 2019149269A1 CN 2019074419 W CN2019074419 W CN 2019074419W WO 2019149269 A1 WO2019149269 A1 WO 2019149269A1
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seq
amino acid
acid sequence
antibody
bcma
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PCT/CN2019/074419
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French (fr)
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李志远
翟天航
周帅祥
俞德超
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信达生物制药(苏州)有限公司
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Priority to JP2020536606A priority Critical patent/JP7438953B2/ja
Priority to AU2019214183A priority patent/AU2019214183B2/en
Priority to US16/759,956 priority patent/US11807663B2/en
Priority to CA3081125A priority patent/CA3081125A1/en
Priority to CN201980007072.XA priority patent/CN111601825B/zh
Priority to CN202211597400.6A priority patent/CN116041516A/zh
Priority to EP19747676.5A priority patent/EP3693392A4/en
Publication of WO2019149269A1 publication Critical patent/WO2019149269A1/zh
Priority to AU2022204817A priority patent/AU2022204817A1/en
Priority to JP2023126384A priority patent/JP2023153933A/ja

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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95

Definitions

  • the present invention relates to novel antibodies and antibody fragments, particularly single chain antibodies (e.g., single chain scFv), that specifically bind to B cell mature antigen (BCMA).
  • the invention also relates to nucleic acids encoding such antibodies and antibody fragments, vectors, and host cells expressing the nucleic acids.
  • the invention also relates to compositions comprising the antibodies of the invention, and their use in therapy and diagnosis.
  • BCMA B cell mature antigen
  • CD269 or TNFRSF17 is a member of the tumor necrosis factor receptor family.
  • BCMA is a type III transmembrane protein with a cysteine-rich domain (CRD) characteristic of members of the TNFR family in the extracellular domain (ECD), which forms a ligand binding motif.
  • CCD cysteine-rich domain
  • ECD extracellular domain
  • BCMA is functionally related to the TNFR family members, the transmembrane activator CMAL interacting factor (TACI) and the BAFF receptor (BAFF-R).
  • TACI transmembrane activator CMAL interacting factor
  • BAFF-R BAFF receptor
  • BCMA showed some similarity to TACI in the CRD at the N-terminus of the transmembrane domain.
  • human BCMA has approximately 65% and 85% amino acid sequence identity with the BCMA of mouse and macaque on the extracellular domain ECD, respectively.
  • BCMA can bind B cell activator receptor (BAFF) and B cell proliferation-inducing ligand (APRIL) to promote the survival of B cells at different developmental stages.
  • BAFF B cell activator receptor
  • APRIL B cell proliferation-inducing ligand
  • APRIL also known as G70, is a member of the TNF ligand family. According to reports in the literature, APRIL is associated with prostate cancer, breast cancer, Alzheimer's disease, immune disease, inflammation, and gastrointestinal disorders. See Hahne et al. (1998), T. Exp. Med. 188: 1185-90. Binding of soluble APRIL to BCMA promotes survival of bone marrow (BM) plasma cells and plasmablasts (see, BLOOD, May 2014, Vol. 123, No. 20, p3128-3138); binding to TACI can lead to T cells Independent antibody response, B cell regulation, and type-switching recombination (see Vincent et al., Nature Reviews Rheumotology, 2014 Vol. 10: 365-373).
  • BAFF is a member of another TNF ligand family. BAFF binds to BCMA and promotes the survival of plasma cells. BAFF binds to BAFF receptors (BAFF-R) expressed on the surface of B cells, plasmablasts, and plasma cells to promote survival and maturation of immature B cells. In addition, BAFF can also bind to TACI, resulting in T cell-independent antibody responses, B cell regulation, and type-switching recombination (see Vincent et al., Nature Reviews Rheumotology, 2014 Vol. 10: 365-373).
  • BAFF-R BAFF receptors
  • BCMA In non-tumor cells, BCMA is predominantly expressed in plasma cells and mature B cell subsets. In 60-70% of patients with multiple myeloma (MM), BCMA is also expressed on the surface of cancerous plasma cells. Serum BCMA levels are elevated in MM patients, and elevated levels are associated with disease status, treatment response, and overall survival. BCMA gene-deficient mice have normal B cell levels, but their plasma cell life cycle is significantly shortened. Therefore, BCMA is an ideal target for multiple myeloma immunotherapy.
  • MM multiple myeloma
  • a single-stranded scFv antibody is a small molecule, genetically engineered antibody that is linked to the heavy chain variable region (VH) and the light chain variable region (VL) of a natural antibody by genetic engineering at the DNA level (usually through a stretch of A small molecule recombinant antibody made by synthetically linked peptides (or "linkers").
  • VH heavy chain variable region
  • VL light chain variable region
  • scFv single-chain antibodies have the following advantages: contain intact antibody variable regions, retain antigen-specificity and binding activity of the original antibody; do not contain the Fc region of the antibody molecule, and thus have weak immunogenicity,
  • the human body is not easy to produce an immune reaction; it has a small molecular weight, is highly penetrating, and is easy to penetrate into tissues.
  • the antibody molecule can be used for imaging diagnosis or treatment, and can enter the inside of tissues which cannot be reached by general intact antibodies; it can form functional without glycosylation modification.
  • the antibody molecule is advantageous for mass production of the prokaryotic expression system; it is easy to operate, and is suitable for use as a genetic engineering component to prepare other antigen-specific binding molecules having novel properties, such as full-length antibody, scFv-Fc and the like.
  • the present invention satisfies this need by providing a fully human single-chain antibody that binds BCMA with high target specificity and high affinity, particularly to BCMA expressed on the surface of tumor cells, and has low side effects.
  • the fully human single-chain antibody of the present invention is not only suitable for use in the diagnosis or treatment of tumors and cancer alone, but, more advantageously, is suitable as a genetic engineering component for the preparation of other diagnostic and therapeutic molecules with high BCMA targeting. For example, various forms of antibodies, scFv-Fc, and antibody-based fusions and conjugates and the like.
  • the present invention provides a fully human anti-human BCMA antibody and its encoding gene and application.
  • the present inventors screened a human antibody against human BCMA from a human antibody library displayed on the surface of yeast, and obtained a variable region gene sequence, and constructed a complete
  • the human single-stranded scFv antibody and its fusion construct with the human Fc region are expressed and purified by mammalian cells to obtain a scFv-hFc recombinant single-chain antibody molecule.
  • the recombinant single-chain antibody molecule of the present invention binds not only to high-affinity to non-membrane-bound human BCMA, but also to cell surface-expressed BCMA with high affinity.
  • the present invention provides antibodies, particularly single-chain antibodies, and nucleic acid molecules encoding the same, which specifically bind to BCMA, and their use in therapy and diagnosis.
  • the invention provides an antibody or antigen-binding fragment thereof that specifically binds BCMA, preferably a human BCMA protein.
  • the antibody of the invention is a single chain antibody.
  • the antibody of the invention is a single chain scFv antibody.
  • the antibody of the invention is a scFv-Fc antibody.
  • an antibody of the invention binds to a human BCMA protein with a KD of from about 100 nM to 5 nM, wherein the KD value is measured according to, for example, a biofilm layer optical interference technique (eg, Fortebio assay).
  • an antibody of the invention binds to a human surface BCMA protein expressed on a cell surface with an EC50 of about 40 nM to 4 nM, wherein the EC50 value is measured, for example, by flow cytometry (eg, FACS).
  • flow cytometry eg, FACS
  • the invention provides the use of an anti-BCMA antibody or fragment thereof of the invention in the treatment of a condition associated with BCMA.
  • an antibody of the invention comprises a VH region sequence of any of the antibodies set forth in Table 1, or a variant thereof. In other embodiments, an antibody of the invention comprises a VL region sequence of any of the antibodies set forth in Table 1, or a variant thereof. In other embodiments, an antibody of the invention comprises a VH and VL sequence pair of any of the antibodies set forth in Table 1, or variants thereof. In other embodiments, an antibody of the invention comprises one, two or three CDRs (preferably three CDRs) of a VH region sequence of any of the antibodies set forth in Table 1, or variants thereof.
  • an antibody of the invention comprises one, two or three CDRs (preferably three CDRs) of the VL region sequence of any of the antibodies set forth in Table 1, or variants thereof. In some embodiments, an antibody of the invention comprises the six CDR region sequences of any of the antibodies set forth in Table 1, or variants thereof. In one embodiment, the CDR sequences of the antibodies are the CDR sequences set forth in Table 2.
  • an antibody of the invention is a single chain scFv antibody.
  • the scFv antibody comprises a VH sequence, a VL sequence and a linker.
  • the scFv antibody comprises, from the N-terminus to the C-terminus, a VL domain-linker-VH domain, or a VH domain-linker-VL domain.
  • the invention further provides a scFv-Fc antibody formed by fusion of a single chain scFv antibody of the invention and a wild type or altered Fc region.
  • the Fc region of an antibody of the invention is low or afucosylated.
  • the scFv antibody is fused to the Fc region via a hinge region.
  • the invention relates to fusions and conjugates constructed based on antibodies of the invention, in particular single chain antibodies.
  • the invention relates to methods and compositions for treating a B cell associated disorder, wherein an effective amount of an antibody of the invention or antigen binding fragment thereof, or a fusion or conjugate of the invention is administered to the subject.
  • the B cell associated disorder is selected from the group consisting of: a B cell malignancy, a plasma cell malignancy, an autoimmune disease, preferably selected from the group consisting of: multiple myeloma, non-Hodgkin's lymphoma, B with uncertain malignant potential Cell proliferation, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorders, immunomodulatory disorders, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves Disease, Wegener's granulomatosis, nodular polyarteritis, Sjogren's syndrome, pemphigus vulgaris, scleroderma
  • the B cell associated disorder is a B cell malignancy, preferably multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).
  • MM multiple myeloma
  • NHS non-Hodgkin's lymphoma
  • an antibody molecule, fusion or conjugate of the invention is used in combination with other therapeutic agents.
  • the invention relates to a method and kit for detecting BCMA in a sample, wherein the method comprises: (a) contacting the sample with an antibody of the invention or antigen-binding fragment, fusion or conjugate thereof; and b) detecting the formation of a complex between the antibody or antigen-binding fragment, fusion or conjugate thereof and BCMA protein.
  • the sample is from a patient with multiple myeloma (MM). The detection can be in vitro or in vivo.
  • Figure 1 shows the affinity of an exemplary anti-BCMA antibody of the invention obtained from yeast display library screening and multiple myeloma cell line NCI-H929 cells as determined by flow cytometry.
  • Figure 2 is a schematic representation of an expression vector cloning strategy for an exemplary scFv-hFc recombinant single chain antibody of the invention.
  • Figure 3 shows the affinity of an exemplary scFv-hFc recombinant single chain antibody of the invention to NCI-H929 cells as determined by flow cytometry.
  • Figure 4 shows exemplary CDR sequences of antibodies of the invention.
  • Figure 5 shows an exemplary VH sequence of an antibody of the invention.
  • Figure 6 shows an exemplary VL sequence of an antibody of the invention.
  • Figure 7 shows exemplary amino acid sequences of human BCMA antigen and its extracellular domain (ECD).
  • Figure 8 shows the amino acid sequence and nucleotide sequence of the linker, hinge region and Fc region used to construct the exemplary scFv-Fc constructs of the invention and the reference scFv-Fc construct construct.
  • the term "antigen binding molecule” refers to a molecule, such as a protein or polypeptide, comprising an antigen binding region or antigen binding portion capable of binding to a target antigen.
  • the target antigen is a B cell mature antigen (BCMA)
  • the antigen-binding molecule that binds to BCMA is also referred to as a BCMA-binding molecule.
  • Antigen binding molecules include, for example, antibodies and antigen binding fragments thereof, single chain scFv antibodies, various fusions and conjugates constructed based on scFv, such as scFv-Fc antibodies.
  • the antigen binding portion of an antibody typically comprises amino acid residues from a “complementarity determining region” or “CDR”.
  • CDR complementarity determining region
  • BCMA binding molecules are used interchangeably with “antibody of the invention” or “anti-BCMA antibodies” depending on the context.
  • antibody refers to a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen.
  • the term encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, single chain antibodies or multi-chain antibodies, monospecific or multispecific antibodies (eg, bispecific antibodies), fully human antibodies, or A chimeric or humanized antibody, a full length antibody, and an antibody fragment, as long as they exhibit a desired antigen binding activity.
  • “whole antibodies” (which are used interchangeably herein with “full length antibodies”, “complete antibodies” and “intact antibodies”) comprise at least two heavy (H) chains and two light chains ( L).
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain CL.
  • a variable region is a domain involved in the binding of an antibody to its antigen in the heavy or light chain of an antibody.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits multiple effector functions.
  • the light chain of an antibody can be assigned to one of two types (referred to as kappa ( ⁇ ) and lambda ( ⁇ )) based on the amino acid sequence of its constant domain.
  • the heavy chain of an antibody can be divided into five major different types depending on the amino acid sequence of its heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, and several of these types can be further subdivided into subclasses, such as , IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2.
  • the heavy chain constant regions corresponding to different antibody types are referred to as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively. See, for example, Fundamental Immunology, Ch. 7 (Paul, W. Ed., Second Edition, Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety.
  • antibody fragment refers to a molecule that is not an intact antibody and that comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds.
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab', F(ab') 2 , Fab'-SH, Fv, single chain Fv, diabody, triabody, tetra-chain antibody ( Tetrabody), minibody, single domain antibody (sdAb); and multispecific antibodies formed from antibody fragments.
  • a Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains, for example, a Fab fragment can be obtained by digestion of a complete antibody by papain.
  • the light chain (L chain) and heavy chain (H chain) of the Fab can be fused by a linker to form a single polypeptide chain, i.e., a single chain Fab (scFab) (see, for example, US20070274985A1).
  • F(ab') 2 which is a dimer of Fab', is a divalent antibody fragment by digesting a complete antibody under the disulfide bond of the hinge region by pepsin.
  • F(ab') 2 can be reduced under neutral conditions by disrupting the disulfide bond in the hinge region, converting from the F(ab') 2 dimer to the Fab' monomer.
  • the Fab' monomer is essentially a Fab fragment with a hinge region.
  • the Fv fragment consists of the VL and VH domains of one arm of the antibody.
  • a recombinant method encoding a gene encoding two domains VL and VH of an Fv fragment independently by a nucleic acid sequence encoding a linker (linker) can be recombinantly expressed to form a single-chain Fv, in which the single-chain Fv is The VH and VL regions are paired to provide an antigen binding site.
  • a diabody is an antibody fragment having two antigen-binding sites comprising VL and VH linked by a short linker in the same polypeptide chain. In the diabody, since the linker is too short, the two domains of VH and VL on the same chain cannot be paired, but are forced to pair with the complementary domain on the other chain and produce two antigen-binding sites.
  • Double-stranded antibodies can be bivalent or bispecific. A more detailed description of double-stranded antibodies can be found, for example, in EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129-134 (2003); and Hollinger et al, PNAS USA 90:6444-6448 (1993). ).
  • a single domain antibody generally refers to an antibody in which a single variable domain (eg, a heavy chain variable domain (VH) or a light chain variable domain (VL), derived from a camelid heavy chain antibody
  • VH heavy chain variable domain
  • VL light chain variable domain
  • monoclonal antibody denotes an antibody obtained from a population of substantially homogeneous antibodies, ie, in addition to a possible variant antibody that is normally present in minor amounts (eg, containing a natural mutation or in a monoclonal antibody preparation) In addition to the variant antibodies produced during the production process, the individual antibodies that make up the population are identical and/or bind to the same epitope.
  • Monoclonal antibodies can be prepared by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, yeast display methods, and methods of using transgenic animals comprising all or part of a human immunoglobulin locus.
  • human antibody or “full human antibody” are used interchangeably herein to refer to an antibody comprising a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Moreover, if the antibody contains a constant region, the constant region is also derived from the human germline immunoglobulin sequence.
  • Human antibodies of the invention can include amino acids that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or point-specific mutagenesis in vitro or in vivo somatic mutations), such as in CDRs, particularly in CDR3.
  • the term "human antibody” is not intended to include antibodies in which the CDR sequences are derived from the germline of other mammalian species (eg, a mouse) and are grafted into human framework sequences.
  • the term "recombinant human antibody” includes all human antibodies that are produced, expressed, produced or isolated by recombinant means, for example, (a) an animal (eg, a mouse) that has been transgenic or transchromosomal with a human immunoglobulin gene or An antibody isolated from a hybridoma prepared therefrom, (b) an antibody isolated from a host cell expressing a human antibody, such as a transfectoma, (c) an antibody isolated from a recombinant human antibody library, such as a yeast display library, and d) an antibody produced, expressed, produced or isolated by any other means including splicing of the human immunoglobulin gene to other DNA sequences.
  • recombinant human antibodies have a framework region and a CDR region derived from the variable region of a human germline immunoglobulin sequence.
  • the recombinant human antibody can be subjected to in vitro mutagenesis (or somatic mutagenesis in vivo when using a human Ig sequence transgenic animal), the amino acid sequence of the VH and VL regions of the recombinant antibody thus obtained, although derived from and associated with human germline VH and VL sequences, they are not naturally found in human antibody germline libraries in vivo.
  • chimeric antibody refers to an antibody from which a variable region sequence is derived from one species, the constant region sequence is derived from another species, eg, wherein the variable region sequence is derived from a mouse antibody, and the constant region sequence is derived from a human antibody. .
  • humanized antibody refers to an antibody that binds a CDR sequence derived from a germline of another mammalian species, such as a mouse, to a human framework sequence. Additional framework region modifications can be made within the human framework sequence.
  • an “isolated” antibody is one that has been separated from components in its natural environment.
  • the antibody is purified to greater than 95% or 99% purity by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse) Phase HPLC) determination.
  • electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography eg, ion exchange or reverse Phase HPLC
  • An epitope is an antigenic region to which an antibody binds.
  • Epitopes may be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed by tertiary folding of the protein.
  • BCMA and “B-cell maturation antigen” are used interchangeably and include variants, isoforms, species homologs of human BCMA and similar to at least one identical epitope to BCMA (eg, human BCMA). Things.
  • Figure 7 shows an exemplary human BCMA sequence (SEQ ID NO: 74).
  • BCMA proteins may also include fragments of BCMA, such as extracellular domains as well as fragments of extracellular domains, such as fragments that retain the ability to bind to any of the antibodies of the invention.
  • the term "specifically binds” means that the antibody binds antigen selectively or preferentially. If in biophotometric interferometry, the antibody is at about 5 x 10 -7 M or lower, about 1 x 10 -7 M or lower, about 5 x 10 -8 M or lower, about 1 x 10 -8 M or lower, about 5 x 10 -9 M or lower K D , which binds to human BCMA, is an antibody that "specifically binds to human BCMA".
  • affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of molecule X for its partner Y can generally be represented by an equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant and the binding rate constant (k dis and k on , respectively ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio Kinetic Binding Assay herein.
  • An antibody “competingly binds to a reference antibody that binds to an antigen such as BCMA” refers to an antibody that blocks the binding of the reference antibody to an antigen (eg, BCMA) by 50% or more in a competition assay, and conversely, The reference antibody also blocked the binding of the antibody to an antigen (e.g., BCMA) by 50% or more in a competition assay.
  • An exemplary competition test is described in "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).
  • a competitively bound antibody can bind to the same epitope region as the reference antibody, such as the same epitope, an adjacent epitope, or an overlapping epitope.
  • Fc region as used herein is used to define the C-terminal region of an immunoglobulin heavy chain containing 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 of the heavy chain or from Pro230 to the carboxy terminus.
  • the C-terminal lysine (Lys447) of the Fc-region may or may not be present.
  • the numbering of amino acid residues in an Fc-region or constant region is based on the EU numbering system, also known as the EU index, such as Kabat, EA, etc., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service , National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • variant refers to a deletion, deletion, which has been substituted by at least 1, for example 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acids. And/or an antibody that has an amino acid altered target antibody region (eg, a heavy chain variable region or a light chain variable region or a heavy chain CDR region or a light chain CDR region), wherein the variant substantially retains the antibody molecule prior to the alteration Biological characteristics.
  • the invention encompasses variants of any of the antibodies described herein.
  • the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the altered pre-antibody.
  • the alteration does not result in loss of binding of the antibody variant to the antigen, but may optionally impart properties such as increased antigen affinity and different effector functions.
  • the heavy chain variable region or the light chain variable region of the antibody, or each CDR region may be altered individually or in combination.
  • the amino acid change in one or more or all three heavy chain CDRs does not exceed one, two, three, four, five, six, seven, eight, nine Or 10.
  • the amino acid changes in one or more or all three of the light chain CDRs are no more than one, two, three, four, five, six, seven, eight, nine Or 10. In some embodiments, the amino acid change in one or more or all of the six CDRs does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 One. Preferably, the amino acid is changed to an amino acid substitution, preferably a conservative substitution. In some embodiments, the antibody variant and the parent antibody have at least 80%, 85%, 90% or 95% or 99% or more amino acid identity over the region of the antibody sequence of interest.
  • an antibody of the invention has at least 80%, 85%, 90%, 91%, 92%, 93% over the heavy chain variable region, as compared to any of the antibodies listed in Table 1. 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.
  • the antibody of the invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94% of the light chain variable region compared to any of the antibodies listed in Table 1. , 95%, 96%, 97%, 98%, or 99% or higher sequence identity.
  • the antibody of the invention has at least 80%, 85%, 90%, 91%, 92% of the heavy chain variable region and the light chain variable region as compared to any of the antibodies listed in Table 1. , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.
  • sequence identity refers to the same degree of nucleotide-by-nucleotide or amino acid-based sequences in a comparison window.
  • the “percent sequence identity” can be calculated by comparing the two optimally aligned sequences in a comparison window to determine the presence of the same nucleic acid base in both sequences (eg, A, T, C, G, I) Or the same amino acid residues (eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) The number of positions is obtained to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (ie, the window size), and the result is multiplied by 100 to generate a sequence identity percentage.
  • Optimal alignments for determining percent sequence identity can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN
  • Megalign DNASTAR
  • One of skill in the art can determine suitable parameters for aligning the sequences, including any algorithms needed to achieve maximum alignment within the full length sequence being compared or within the target sequence region.
  • the amino acid sequence identity percentage is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence in a preferred embodiment according to the Kabat numbering rule.
  • the antibody region of interest eg, the entire variable region of the heavy or light chain, or portions thereof, eg, one or more CDR regions
  • the percent sequence identity between the target antibody region and the reference antibody region is: the number of positions occupied by the same amino acid in both the target and reference antibody regions divided by the total number of aligned positions in the two regions (vacancies are not counted) and Multiply by 100 to get the percentage.
  • sequence identity may be distributed throughout the heavy chain variable region and/or the entire light chain variable region, or the percent sequence identity may be limited to only the framework region, while the corresponding CDR region The sequence remains 100% identical.
  • BCMA binding molecules and compositions of the invention are BCMA binding molecules and compositions of the invention.
  • One aspect of the invention provides antibodies, particularly single-chain antibodies (e.g., single-chain scFv antibodies) that bind BCMA (especially membrane-bound BCMA) with high target specificity and high affinity.
  • single-chain antibodies e.g., single-chain scFv antibodies
  • BCMA especially membrane-bound BCMA
  • BCMA for example, human BCMA
  • high affinity for example with a KD value of less than 100 nM, such as less than 50 nM, such as 5-30 nM, preferably less than 10 nM;
  • BCMA cell surface expressed BCMA
  • high affinity eg, with an EC50 value of less than 100 nM, such as less than 50 nM, such as 1-40 nM, preferably less than 20 nM, more preferably less than 10 or 5 nM;
  • the present invention is an anti-BCMA antibody molecule with high affinity, e.g., by the following equilibrium dissociation constant (K D) to human BCMA (e.g. SEQ ID NO: 74 polypeptide) binding, the K D of less than about 100nM , less than or equal to about 80 nM, 70 nM, 60 nM, or 50 nM, more preferably less than or equal to about 40 nM, 30 nM or 20 nM, more preferably less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM or 2 nM, for example, is determined by using a bio-optical interference assay (eg, Fortebio affinity measurement).
  • K D equilibrium dissociation constant
  • human BCMA e.g. SEQ ID NO: 74 polypeptide
  • the anti-BCMA antibody molecule of the invention binds to human BCMA (eg, the polypeptide of SEQ ID NO: 74) with a dissociation rate constant (K dis ) of less than 3 ⁇ 10 -2 , 1.5 ⁇ 10 -2 , 5 ⁇ 10 -3 or 3 ⁇ 10 -3 s -1 , for example, about 1.46 ⁇ 10 -3 s -1 .
  • the binding rate constant (K on ) of the anti-BCMA antibody molecule to BCMA is greater than 1 ⁇ 10 4 , 5 ⁇ 10 4 , 1 ⁇ 10 5 , 5 ⁇ 10 5 or 8 ⁇ 10 5 M ⁇ 1 s. -1 , for example, with a BCMA of about 7.29 ⁇ 10 5 M -1 s -1 .
  • an anti-BCMA antibody molecule of the invention binds cells expressing BCMA with high affinity, preferably a multiple myeloma cell line expressing human BCMA (eg, NCI-H929) on the cell surface, preferably, by flow cytometry
  • the antibody binds to the cells with an EC50 value of less than about 200 nM, 150 nM or 100 nM, preferably less than or equal to about 80 nM, 70 nM, 60 nM, or 50 nM, more preferably less than or equal to about 40 nM, 30 nM, as determined by FACS. Or 20 nM, more preferably less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM or 2 nM.
  • the antibody molecule binds to human BCMA comprising the amino acid sequence of SEQ ID NO:74. In some embodiments, the antibody molecule binds to an epitope on the BCMA, preferably on the extracellular domain of BCMA.
  • the antibody molecule is a full length antibody. In other embodiments, the antibody molecule is an antibody fragment.
  • the antibody molecule of the present invention may comprise or may be Fab, scFab, Fab', F(ab') 2 , Fab'-SH, Fv, single-chain scFv antibody, diabody, tri-chain antibody, tetra Chain antibody, minibody, or single domain antibody (sdAb).
  • the antibody molecule of the invention is a single chain scFv antibody.
  • the antibody molecule of the invention comprises an scFv and an Fc region linked thereto.
  • the antibody molecules of the invention are of all human origin.
  • a “variable region” or “variable domain” is a domain of a heavy or light chain of an antibody that is involved in the binding of an antibody to its antigen.
  • the heavy chain variable region (VH) and the light chain variable region (VL) can be further subdivided into hypervariable regions (HVRs, also known as complementarity determining regions (CDRs)) with more conserved regions interposed therebetween (ie, frameworks) District (FR)).
  • HVRs hypervariable regions
  • CDRs complementarity determining regions
  • FRs frameworks
  • Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL domain is sufficient to confer antigen-binding specificity.
  • antibodies that bind to a particular antigen can be isolated by screening a library of complementary VL or VH domains using a VH or VL domain from an antibody that binds to the antigen (see, for example, Portolano, S. et al, J. Immunol. 150 (1993). ) 880-887; Clackson, T. et al, Nature 352 (1991) 624-628).
  • VH or “VH domain” includes the heavy chain variable region VH of a full length antibody, Fv, scFv, dsFv, Fab, scFab, or other antibody fragments disclosed herein.
  • VL or “VL domain” includes the full-length antibody, Fv, scFv, dsFv, Fab, scFab, or the light chain variable region VL of other antibody fragments disclosed herein.
  • an anti-BCMA antibody molecule of the invention comprises: (i) an antigen binding region identical to an antigen binding region (eg, a heavy chain variable region and a light chain variable region pair) of any of the antibodies listed in Table 1. Or (ii) having an antigen binding region of (i) having, for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity in amino acid sequence Antigen binding domain.
  • the anti-BCMA antibody molecule of the invention comprises: (i) a heavy chain variable region identical to the heavy chain variable region of any of the antibodies listed in Table 1; or (ii) and (i) a chain variable region having, for example, a heavy chain variable region of at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or greater in amino acid sequence; or (iii a variant of the heavy chain variable region of (i), wherein said variant comprises at least one and no more than 30, 20 or 10 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions), and preferably said variants A total of no more than 10, preferably 5-0, amino acid changes (preferably amino acid substitutions) are included in the three heavy chain complementarity determining region (CDR) regions.
  • CDR complementarity determining region
  • the anti-BCMA antibody molecule of the invention comprises: (i) a light chain variable region identical to the light chain variable region of any of the antibodies listed in Table 1; or (ii) lighter than (i) a chain variable region having, for example, a light chain variable region of at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or greater identity in amino acid sequence; or (iii a variant of the light chain variable region of (i), wherein said variant comprises at least one and no more than 30, 20 or 10 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions), and preferably said variant A total of no more than 10, preferably 5-0, amino acid changes (preferably amino acid substitutions) are included in the three light chain complementarity determining regions (CDRs).
  • CDRs three light chain complementarity determining regions
  • the anti-BCMA antibody molecule of the invention comprises a heavy chain variable region and a light chain variable region
  • heavy chain variable region is selected from the group consisting of
  • a heavy chain variable region identical to the heavy chain variable region of any of the antibodies listed in Table 1; or (ii) and the heavy chain variable region of (i) having, for example, at least 80% of the amino acid sequence, a heavy chain variable region of 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity; or (iii) a variant of the heavy chain variable region of (i), wherein The variant comprises at least one and no more than 30, 20 or 10 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions), and preferably the variant comprises a total of 3 heavy chain complementarity determining region (CDR) regions No more than 10, preferably 5-0 amino acid changes (preferably amino acid substitutions);
  • CDR heavy chain complementarity determining region
  • light chain variable region is selected from the group consisting of:
  • a light chain variable region identical to the light chain variable region of any of the antibodies listed in Table 1; or (ii) and the light chain variable region of (i) having, for example, at least 80% of the amino acid sequence, a light chain variable region of 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity; or (iii) a variant of the light chain variable region of (i), wherein The variant comprises at least one and no more than 30, 20 or 10 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions), and preferably the variant comprises no total of 3 light chain complementarity determining regions (CDRs) More than 10, preferably 5-0, amino acid changes (preferably amino acid substitutions).
  • CDRs light chain complementarity determining regions
  • the invention provides an anti-BCMA antibody, or variant thereof, comprising the amino acid sequence of a heavy chain variable region and a light chain variable region pair of any of the antibodies listed in Table 1.
  • the antibody comprises an amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 4/31, 5/41, 5/42, 10/46, 16/50, 17/58, 23/64 , 27/59, 27/71, and 27/72.
  • the variant has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity on the VH and/or VL amino acid sequence. Or at least one and no more than 30, 20 or 10 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included on the VH and/or VL amino acid sequences.
  • the invention provides an anti-BCMA antibody comprising a heavy chain variable region VH and a light chain variable region VL, said VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, and 6, VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 41, 42 and 43.
  • X represents any amino acid, preferably an amino acid residue at a corresponding position of SEQ ID NO: 4 or 5, or a conservatively substituted residue thereof.
  • X represents any amino acid, preferably an amino acid residue at the corresponding position of SEQ ID NO: 41 or 42, or a conservatively substituted residue thereof.
  • the invention provides an anti-BCMA antibody comprising a heavy chain variable region VH and a light chain variable region VL, said VH comprising the amino acid sequence of SEQ ID NO: 4, and said VL comprising SEQ ID NO: Amino acid sequence of 31.
  • the invention also provides variants of the antibody, such as variants having at least 95-99% identity on VH and/or VL or containing no more than 10 amino acid changes.
  • an anti-BCMA antibody of the invention comprises a heavy chain variable region VH and a light chain variable region VL, said VH comprising the amino acid sequence of SEQ ID NO: 5, and said VL comprising selected from the group consisting of SEQ ID NO: Amino acid sequences of 41 and 42.
  • the invention also provides variants of the antibody, such as variants having at least 95-99% identity on VH and /VL or containing no more than 10 amino acid changes.
  • the invention provides an anti-BCMA antibody comprising a heavy chain variable region VH and a light chain variable region VL, said VH comprising the amino acid sequence of SEQ ID NO: 10, and said VL comprising SEQ ID NO: Amino acid sequence of 46.
  • the invention also provides variants of the antibody, such as variants having at least 95-99% identity on VH and/or VL or containing no more than 10 amino acid changes.
  • an anti-BCMA antibody of the invention comprises a heavy chain variable region VH and a light chain variable region VL, said VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 17 or 19, and said VL comprising An amino acid sequence selected from the group consisting of SEQ ID NOS: 50 and 58.
  • X represents any amino acid, preferably an amino acid residue at the corresponding position of SEQ ID NO: 16 or 17, or a conservatively substituted residue thereof.
  • the invention provides an anti-BCMA antibody comprising a heavy chain variable region VH and a light chain variable region VL, said VH comprising the amino acid sequence of SEQ ID NO:16, and said VL comprising SEQ ID NO: Amino acid sequence of 50.
  • the invention also provides variants of the antibody, such as variants having at least 95-99% identity on VH and/or VL or containing no more than 10 amino acid changes.
  • the invention provides an anti-BCMA antibody comprising a heavy chain variable region VH and a light chain variable region VL, said VH comprising the amino acid sequence of SEQ ID NO:17, and said VL comprising SEQ ID NO: Amino acid sequence of 58.
  • the invention also provides variants of the antibody, such as variants having at least 95-99% identity on VH and/or VL or containing no more than 10 amino acid changes.
  • the invention provides an anti-BCMA antibody comprising a heavy chain variable region VH and a light chain variable region VL, said VH comprising the amino acid sequence of SEQ ID NO:23, and said VL comprising SEQ ID NO: 64 amino acid sequence.
  • the invention also provides variants of the antibody, such as variants having at least 95-99% identity on VH and/or VL or containing no more than 10 amino acid changes.
  • the invention provides an anti-BCMA antibody comprising a heavy chain variable region VH and a light chain variable region VL, the VH comprising the amino acid sequence of SEQ ID NO:27, NO: amino acid sequences of 59, 71, 72 and 73.
  • X represents any amino acid, preferably an amino acid residue at the corresponding position of SEQ ID NO: 71 or 72, or a conservatively substituted residue thereof.
  • the invention provides an anti-BCMA antibody comprising a heavy chain variable region VH and a light chain variable region VL, said VH comprising the amino acid sequence of SEQ ID NO: 27, and said VL comprising SEQ ID NO: Amino acid sequence of 59.
  • the invention also provides variants of the antibody, such as variants having at least 95-99% identity on VH and/or VL or containing no more than 10 amino acid changes.
  • the invention provides an anti-BCMA antibody comprising a heavy chain variable region VH and a light chain variable region VL, said VH comprising the amino acid sequence of SEQ ID NO: 27, and said VL comprising SEQ ID NO: Amino acid sequence of 71 or 72.
  • the invention also provides variants of the antibody, such as variants having at least 95-99% identity on VH and/or VL or containing no more than 10 amino acid changes.
  • the heavy chain variable region of the antibody of the invention is on one or more CDRs (preferably all 3 CDRs) regions relative to said reference heavy chain variable region amino acid sequence Containing no more than 10, preferably no more than 5 (eg, 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the light chain variable region VL of the antibody of the invention is in one or more CDRs (preferably all 3 CDRs) regions relative to said reference light chain variable region amino acid sequence
  • a “complementarity determining region” or “CDR region” or “CDR” (used interchangeably herein with the hypervariable region “HVR”) is an amino acid region of the antibody variable region that is primarily responsible for binding to an epitope.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.
  • the CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3.
  • CDR complementarity determining region
  • the Kabat complementarity determining region is determined based on sequence variability and is most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ( 1991)). Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
  • AbM HVR is a compromise between the Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.
  • the "Contact" HVR is based on an analysis of the complex crystal structures available. The residues of each of the HVR/CDRs of these HVRs are as follows, according to different CDR determination protocols.
  • the HVR can also be an HVR sequence located at the Kabat residue position according to the Kabat numbering system:
  • Position 24-36 or 24-34 (LCDR1) in VL, position 46-56 or 50-56 (LCDR2), and position 89-97 or 89-96 position (LCDR3); and position 26-35 in VH or 27-35B (HCDR1), position 50-65 or 49-65 (HCDR2), and position 93-102, 94-102 or 95-102 (HCDR3).
  • the HVR of an antibody of the invention is an HVR sequence located at the Kabat residue position according to the Kabat numbering system:
  • the HVR can also be determined based on having the same Kabat numbering position as the reference CDR sequence (e.g., any of the exemplary CDRs of the invention).
  • CDR or “CDR sequence” or “HVR” or “HVR sequence” encompasses an HVR or CDR sequence determined in any of the above manners.
  • the HCDR1 of an antibody of the invention is a CDR sequence determined according to the AbM protocol; and the remaining CDRs are CDR sequences determined according to the Kabat protocol.
  • the CDR sequences of the invention are as shown in Table 2.
  • Antibodies with different specificities have different CDRs.
  • the CDRs differ between antibodies and antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding.
  • the minimal overlap region can be determined to provide a "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of the CDR.
  • residues of the remainder of the CDR sequences can be determined by the structure of the antibody and protein folding. Accordingly, the invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • an antibody of the invention comprises at least one, two, three, four, five or six CDRs identical to the corresponding CDRs of any of the antibodies listed in Table 1, or variants thereof. In some embodiments, an antibody of the invention comprises at least one, two, or three HCDRs, or variants thereof, identical to the corresponding heavy chain CDRs of any of the antibodies listed in Table 1. In some embodiments, an antibody of the invention comprises at least one, two, or three HCDRs, or variants thereof, identical to the corresponding light chain CDRs of any of the antibodies listed in Table 1.
  • a CDR variant is a CDR that has been modified by at least one, for example 1 or 2 or 3 amino acid substitutions, deletions and/or insertions, wherein the antigen-binding molecule comprising the CDR variant substantially retains the unmodified CDR
  • the biological properties of the antigen binding molecule for example, maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability).
  • each CDR may be modified individually or in combination.
  • the amino acid modification is an amino acid substitution, especially a conservative amino acid substitution, such as the preferred conservative amino acid substitutions listed in Table A.
  • an antibody of the invention comprises a heavy chain variable region comprising a heavy chain complementarity determining region 3 (HCDR3), said HCDR3:
  • HCDR3 heavy chain complementarity determining region 3
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, and the heavy chain complementarity determining region 3 (HCDR3) and the light chain complementarity determining region 3 of the antibody ( LCDR3):
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region, HCDR1, HCDR2, and HCDR3 of the heavy chain variable region:
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region comprising a heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising
  • the chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein said HCDR1, HCDR2, HCDR3 and LCDR3:
  • an antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein said LCDR1, LCDR2 and LCDR3:
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region comprising a heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising
  • the chain variable region includes light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, LCDR3, and HCDR3 are:
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region comprising a heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising
  • the chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the antibody:
  • an antibody of the invention or antigen-binding fragment thereof comprises:
  • the antibody or antigen-binding fragment of the invention comprises HCDR1, 2 and 3 sequences and LCDR1, 2 and 3 selected from the group consisting of the heavy chain variable region (VH) and the light chain variable region (VL) sequence:
  • the invention provides an HCDR3 selected from the group consisting of SEQ ID NOs: 3, 9, 13, 14, 15, 22, and 26, and an antibody or antigen-binding fragment comprising the HCDR3.
  • the invention provides a HCDR3 and LCDR3 sequence combination selected from the group consisting of: and an antibody or antigen-binding fragment comprising the combination: (i) HCDR3 of SEQ ID NO: 3, and selected from SEQ ID NO: 30, LCDR3 of 38, 39 and 40; or (ii) HCDR3 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 45; or (iii) HCDR3 selected from SEQ ID NOS: 13, 14 and 15, and selected from LCDR3 of SEQ ID NOS: 49 and 55; or (iv) HCDR3 of SEQ ID NO: 22, and LCDR3 of SEQ ID NO: 63; or (v) HCDR3 of SEQ ID NO: 26, and selected from SEQ ID NO: LCDR3 of 56, 68, 69, and 70.
  • the invention also provides variants of the CDR combinations, for example variants comprising at least one and no more than 20, 10 or 5 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) on the CDRs.
  • the invention also provides an anti-BCMA antibody or antigen-binding fragment comprising the variant.
  • the invention provides a combination of CDR sequences selected from the group consisting of an antibody or antigen-binding fragment comprising the combination: (i) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and SEQ ID NO: 3 HCDR3; (ii) HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 9; (iii) HCDR1 of SEQ ID NO: 11, SEQ ID NO: HCDR2 of 12 and HCDR3 of SEQ ID NO: 13 or 14 or 15; (iv) HCDR1 of SEQ ID NO: 20, HCDR2 of SEQ ID NO: 21, and HCDR3 of SEQ ID NO: 22; (v) SEQ ID NO: HCDR1 of 24, HCDR2 of SEQ ID NO: 25, and HCDR3 of SEQ ID NO: 26.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO
  • the invention provides a heavy chain CDR combination (HCDR1, HCDR2, and HCDR3, respectively, selected from the group consisting of: SEQ ID NOs: 1/2/3, 7/8/9, 11 /12/13, 11/12/14, 20/21/22, and 24/25/26.
  • the invention also provides variants of the heavy chain CDR combinations, in a preferred embodiment, the variants comprise at least one and no more than 20, 10 or 5 amino acid changes on the three CDR regions (preferably Amino acid substitutions, preferably conservative substitutions).
  • the invention also provides an anti-BCMA antibody comprising the heavy chain CDR combination or the variant.
  • the invention provides a CDR combination selected from the group consisting of an antibody or antigen-binding fragment comprising the combination: (i) LCDR1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 29, and SEQ ID NO LCDR3 of: 30; (ii) LCDR1 of SEQ ID NO: 32 or 33 or 34, LCDR2 of SEQ ID NO: 35 or 36 or 37, and LCDR3 of SEQ ID NO: 38 or 39 or 40; (iii) SEQ ID NO: 32, LCDR1, SEQ ID NO: 44, LCDR2, and SEQ ID NO: 45, LCDR3; (iv) SEQ ID NO: 47, LCDR1, SEQ ID NO: 48, LCDR2, and SEQ ID NO: 49, LCDR3 (v) LCDR1 of SEQ ID NO: 51, LCDR2 of SEQ ID NO: 54, and LCDR3 of SEQ ID NO: 55; (vi) LCDR1 of SEQ ID NO: 61, LCDR2 of SEQ ID NO: 62,
  • the invention provides a light chain CDR combination (LCDR1, LCDR2, and LCDR3, respectively, in a sequence selected from the group consisting of: SEQ ID NOs: 28/29/30, 32/35/38, 33/36/39, 32/44/45, 47/48/49, 51/54/55, 61/62/63, 52/62/56, 65/62/68, and 66/62/69.
  • the invention also provides variants of the light chain CDR combination, in a preferred embodiment, the variant comprises at least one and no more than 20, 10 or 5 amino acid changes on the three CDR regions (preferably Amino acid substitutions, preferably conservative substitutions).
  • the invention also provides an anti-BCMA antibody or antigen-binding fragment comprising the light chain CDR combination or the variant.
  • the invention provides a CDR combination selected from the group consisting of an antibody or antigen-binding fragment comprising the same: (i) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and SEQ ID NO : HCDR3 of 3, LCDR1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 29, and LCDR3 of SEQ ID NO: 30; (ii) HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 32 or 33 or 34, LCDR2 of SEQ ID NO: 35 or 36 or 37, and LCDR3 of SEQ ID NO: 38 or 39 or 40; (iii) SEQ ID NO: 7 HCDR1, HCDR2 of SEQ ID NO: 8, and HCDR3 of SEQ ID NO: 9, LCDR1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 44, and LCDR3 of SEQ
  • HCDR2 of SEQ ID NO: 25, and HCDR3 of SEQ ID NO: 26 LCDR1 of SEQ ID NO: 52, LCDR2 of SEQ ID NO: 62, and LCDR3 of SEQ ID NO: 56;
  • the invention provides a combination of heavy and light chain CDRs selected from the group consisting of HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2, and LCDR3, respectively: SEQ ID NOs: 1/2/ 3/28/29/30, 1/2/3/32/35/38, 1/2/3/33/36/39, 7/8/9/32/44/45, 11/12/13/ 47/48/49, 11/12/14/51/54/55, 20/21/22/61/62/63, 24/25/26/52/62/56, 24/25/26/65/ 62/68, and 24/25/26/66/62/69.
  • the invention also provides variants of the CDR combinations, in a preferred embodiment, the variant comprises at least one and no more than 20, 10 or 5 amino acid changes (preferably amino acid substitutions) on the six CDR regions , preferably conservative substitution).
  • the invention also provides an anti-BCMA antibody or antigen-binding fragment comprising the heavy chain and light chain CDR combinations or the variants.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, LCDR1, SEQ ID of SEQ ID NO: NO: 29 LCDR2, and SEQ ID NO: 30 LCDR3.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, LCDR1, SEQ ID of SEQ ID NO: NO: 35 LCDR2, and SEQ ID NO: 38 LCDR3.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, LCDR1, SEQ ID of SEQ ID NO: NO: 36 LCDR2, and SEQ ID NO: 39 LCDR3.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO:7, HCDR2 of SEQ ID NO:8, and HCDR3 of SEQ ID NO:9, LCDR1, SEQ ID of SEQ ID NO:32 NO: 44 LCDR2, and SEQ ID NO: 45 LCDR3.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO:11, HCDR2 of SEQ ID NO:12, and HCDR3 of SEQ ID NO:13, LCDR1, SEQ ID of SEQ ID NO:47 NO: 48 LCDR2, and SEQ ID NO: 49 LCDR3.
  • the antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO:11, HCDR2 of SEQ ID NO:12, and HCDR3 of SEQ ID NO:14, LCDR1, SEQ ID of SEQ ID NO:51 NO: 54 LCDR2, and SEQ ID NO: 55 LCDR3.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO: 20, HCDR2 of SEQ ID NO: 21, and HCDR3 of SEQ ID NO: 22, LCDR1, SEQ ID of SEQ ID NO: 61 NO: 62 LCDR2, and SEQ ID NO: 63 LCDR3.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO: 24, HCDR2 of SEQ ID NO: 25, and HCDR3 of SEQ ID NO: 26, LCDR1, SEQ ID of SEQ ID NO: NO: 62 LCDR2, and SEQ ID NO: 56 LCDR3.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO:24, HCDR2 of SEQ ID NO:25, and HCDR3 of SEQ ID NO:26, LCDR1, SEQ ID of SEQ ID NO:65 NO: 62 LCDR2, and SEQ ID NO: 68 LCDR3.
  • an antibody or antigen-binding fragment thereof of the invention comprises HCDR1 of SEQ ID NO: 24, HCDR2 of SEQ ID NO: 25, and HCDR3 of SEQ ID NO: 26, LCDR1, SEQ ID of SEQ ID NO: NO: 62 LCDR2, and SEQ ID NO: 69 LCDR3.
  • X in SEQ ID NO: 15 represents any amino acid residue, preferably an amino acid residue at a corresponding position of SEQ ID NO: 13 or 14, or a conservatively substituted residue thereof, preferably S or R or Conservative substitution of residues.
  • X in SEQ ID NO: 34 represents any amino acid residue, preferably an amino acid residue at the corresponding position of SEQ ID NO: 32 or 33, or a conservatively substituted residue thereof.
  • X in SEQ ID NO: 37 represents any amino acid residue, preferably an amino acid residue at a corresponding position of SEQ ID NO: 35 or 36, or a conservatively substituted residue thereof.
  • X in SEQ ID NO: 40 represents any amino acid residue, preferably an amino acid residue at a corresponding position of SEQ ID NO: 38 or 39, or a conservatively substituted residue thereof.
  • X in SEQ ID NO: 67 represents any amino acid residue, preferably an amino acid residue at the corresponding position of SEQ ID NO: 65 or 66, or a conservatively substituted residue thereof.
  • X in SEQ ID NO: 70 represents any amino acid residue, preferably an amino acid residue at the corresponding position of SEQ ID NO: 68 or 69, or a conservatively substituted residue thereof.
  • conservative substitution refers to an amino acid change that results in the replacement of a certain amino acid with a chemically similar amino acid.
  • Conservative substitution tables that provide functionally similar amino acids are well known in the art.
  • the conservatively substituted residue is derived from the conservative substitution table A below, preferably the preferred substitution residue shown in Table A.
  • the present invention provides fully human antibodies that specifically bind to BCMA (e.g., human BCMA) isolated and characterized as in the Examples.
  • BCMA e.g., human BCMA
  • the variable region sequences of these exemplary antibodies of the invention are set forth in Table 1 below.
  • Exemplary CDR sequences for these antibodies are given in Table 2 below (see also Figure 4).
  • the invention also provides variants of the above antibodies.
  • the amino acid sequence of the antibody or the nucleic acid encoding the amino acid sequence has been mutated, but still has at least 60%, 65%, 70%, 75%, 80%, 85%, 90 with the sequence described in Table 1. % or 95% or higher identity.
  • the antibody comprises a mutated variable region amino acid sequence, wherein the variable region has been mutated no more than 1, 2, 3, 4, 5 or 10 when compared to the variable region sequence shown in Table 1. Amino acids, but retaining substantially the same antigen binding activity.
  • VH and VL amino acid sequences and nucleotide sequences encoding the amino acid sequences
  • binding assays known in the art (e.g., ELISA, and other assays described in the Examples section).
  • VH sequences from a particular VH/VL pair with a structurally similar VH sequence.
  • VL sequences from a particular VH/VL pairing are preferably replaced with structurally similar VL sequences.
  • the invention also provides variants of the above antibodies.
  • the antibody has been mutated in the amino acid sequence of one or more or all of the six CDR regions or the nucleic acid encoding the amino acid sequence.
  • the amino acid sequence of the mutated CDR region has been mutated by no more than 1, 2, 3, 4 or 5 amino acids when compared to the corresponding CDR regions of Table 1, but retaining substantially the same antigen binding active.
  • each of the antibodies of Table 1 can bind to BCMA and the antigen binding specificity is mainly provided by the CDR1, 2 and 3 regions
  • the VH CDR1, 2 and 3 sequences and the VL CDR1, 2 and 3 sequences can be "mixed” And match” (ie, the CDRs from different antibodies can be mixed and matched, but each antibody preferably contains VH CDRs 1, 2 and 3 and VL CDRs 1, 2 and 3) to produce additional molecules of the invention that bind to BCMA.
  • Binding of such "mixed and matched" antibodies to BCMA can be tested using binding assays known in the art (e.g., ELISA, SET, Biacore) and those described in the Examples.
  • an antigen-binding fragment of an antibody described herein can comprise VH CDRs 1, 2 and 3, or VL CDRs 1, 2 and 3, wherein the fragment binds to BCMA in a single domain.
  • the antibody of the invention is a single chain scFv antibody.
  • single-chain scFv antibody or “scFv” or “single-stranded scFv” refers to a single polypeptide comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an immunoglobulin or antibody.
  • VH heavy chain variable region
  • VL light chain variable region
  • the VH and VL regions of a single chain scFv antibody of the invention are covalently linked together by a linker peptide, such as a flexible linker peptide.
  • linker peptide such as a flexible linker peptide.
  • the term "flexible linker peptide” is a peptide linker composed of amino acids.
  • Each of the variable domains in the antibody, such as the VH and VL regions, can be joined by such a peptide linker.
  • Peptide linkers are typically enriched in glycine, which exhibits flexibility, and serine or threonine, which exhibits solubility. For example, glycine and/or serine residues can be used alone or in combination.
  • Non-limiting examples of flexible linker peptides or peptide linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008), WO 2012/138475, and WO 2014/087010, the entire contents of each of which are hereby incorporated by reference.
  • the linker in the construction of scFv, preferably, the linker will facilitate pairing of VH and VL and does not interfere with the formation of a functionally effective antigen binding site for the VH and VL pairs.
  • a scFv single chain antibody of the invention comprises a flexible linker peptide or peptide linker consisting of a peptide bond-linked amino acid residue.
  • the amino acid is selected from the group consisting of twenty natural amino acids.
  • the one or more amino acids are selected from the group consisting of glycine, serine, threonine, alanine, valine, asparagine, glutamine, and lysine.
  • the one or more amino acids are selected from the group consisting of Gly, Ser, Thr, Lys, Pro, and Glu.
  • the linker is from about 1 to 30 amino acids in length, or from about 10 to about 25 amino acids, from about 15 to about 20 amino acids, or from about 10 to about 20 amino acids, or any intervening Amino acid length. In a preferred embodiment, the linker has a length of 15-25 amino acid residues, and in a more preferred embodiment, a length of 15-18 amino acid residues. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.
  • Examples of peptide linkers that can be used in the present invention include: glycine polymer (G) n; glycine-serine polymer (G 1-5 S 1-5 )n, where n is at least 1, 2, 3, 4 or 5 An integer; a glycine-alanine polymer; an alanine-serine polymer; and other flexible linkers known in the art.
  • G glycine polymer
  • n is at least 1, 2, 3, 4 or 5 An integer
  • a glycine-alanine polymer an alanine-serine polymer
  • other flexible linkers known in the art.
  • the linker between VH and VL may consist entirely of a flexible linker peptide, or the linker may consist of a flexible linker peptide moiety and one or more portions that impart a less flexible structure.
  • the peptide linker is GSTSGSGKPGSGEGSTKG (SEQ ID NO: 93).
  • the nucleotide sequence encoding amino acid sequence SEQ ID NO: 93 is given in SEQ ID NO:94.
  • the nucleotide sequence encoding amino acid sequence SEQ ID NO: 93 is given in SEQ ID NO:97.
  • the peptide linker is a Gly/Ser linker peptide.
  • a linker can comprise an amino acid sequence (G 4 S)n, wherein n is an integer equal to or greater than 1, eg, n is an integer from 1-7.
  • the linker is (G4S)3.
  • the linker is (G4S)4.
  • the linker is (G4S)6G2.
  • KESGSVSSEQLAQFRSLD (Bird et al, 1988, Science 242: 423-426), GGRRGGGS; LRQRDGERP; LRQKDGGGSERP; LRQKD (GGGS) 2 ERP.
  • a computer program capable of modeling the DNA-binding site and the peptide itself can be used ( Desjarlais & Berg, PNAS 90: 2256-2260 (1993), PNAS 91: 11099-11103 (1994)), or by rational design of flexible joints by phage or yeast display methods.
  • VH and VL may be taken in either direction.
  • the scFv comprises from the N-terminus to the C-terminus: VH-linker-VL; or VL-linker-VH.
  • the single chain scFv antibodies of the invention comprise from the N-terminus to the C-terminus: VL-linker-VH.
  • VL is covalently linked to the N-terminus of VH via its linker at its C-terminus.
  • polypeptide fragments having a particular function can be inserted between the VL and VH domains, such as a polypeptide fragment having a function of modulating an immune response, or a polypeptide fragment having a cell solvent or cell killing.
  • a single chain antibody can be stabilized by introducing a disulfide bond in the scFv.
  • the framework regions of the VH and VL of the scFv can be joined by introducing an intrachain or interchain disulfide bond.
  • one amino acid residue of each of the antibodies VH and VL can be mutated to a cysteine, for example according to the Kabat numbering system, position 44 of VH and position 100 of VL, or 105 and VL of VH 43.
  • the single-chain scFv polypeptide antibodies of the invention can be expressed by nucleic acids comprising VH- and VL-encoding sequences as described by Huston et al. (Proc. Nat. Acad. Sci. USA, 85: 5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.
  • a single-chain scFv antibody of the invention is expressed in a eukaryotic cell, such as a yeast cell, a mammalian cell, such as a H293 cell, or a CHO cell.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising an antigen binding region of amino acid sequence SEQ ID NO: 99 or a variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:99.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:100.
  • an anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 4 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 31 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO:4. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:31. In certain embodiments, the anti-BCMA scFv comprises a VH having the sequence set forth in SEQ ID NO: 4 and a VL comprising the sequence set forth in SEQ ID NO:31. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence of SEQ ID NO: 4 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 31.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO:3 and LCDR3 of SEQ ID NO:30.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, and VH CDR3 of SEQ ID NO: 3.
  • the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO:28, VL CDR2 of SEQ ID NO:29, and VL CDR3 of SEQ ID NO:30.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, and VH CDR3 of SEQ ID NO: 3, and VL CDR1 of SEQ ID NO: VL CDR2 of SEQ ID NO:29 and VL CDR3 of SEQ ID NO:30.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising the antigen binding region of amino acid sequence SEQ ID NO: 102 or a variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:102.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:103.
  • an anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 5 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 41 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO:5. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:41. In certain embodiments, the anti-BCMA scFv comprises VH of an amino acid having the sequence set forth in SEQ ID NO: 5 and VL comprising an amino acid having the sequence set forth in SEQ ID NO:41. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence set forth in SEQ ID NO: 5 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 41.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO:3 and LCDR3 of SEQ ID NO:38.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, and VH CDR3 of SEQ ID NO: 3.
  • the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO: 32, VL CDR2 of SEQ ID NO: 35, and VL CDR3 of SEQ ID NO: 38.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, and VH CDR3 of SEQ ID NO: 3, and VL CDR1 of SEQ ID NO: VL CDR2 of SEQ ID NO: 35 and VL CDR3 of SEQ ID NO: 38.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising an antigen binding region of amino acid sequence SEQ ID NO: 105 or a variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:105.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:106.
  • an anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 5 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 42 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO:5. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:42. In certain embodiments, the anti-BCMA scFv comprises a VH having the amino acid sequence set forth in SEQ ID NO: 5 and a VL comprising the amino acid sequence set forth in SEQ ID NO:42. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence of SEQ ID NO: 5 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 42.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO:3 and LCDR3 of SEQ ID NO:39.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, and VH CDR3 of SEQ ID NO: 3.
  • the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO:33, VL CDR2 of SEQ ID NO:36, and VL CDR3 of SEQ ID NO:39.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, and VH CDR3 of SEQ ID NO: 3, and VL CDR1 of SEQ ID NO: VL CDR2 of SEQ ID NO: 36 and VL CDR3 of SEQ ID NO: 39.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising the antigen binding region of amino acid sequence SEQ ID NO: 108 or variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:108.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:109.
  • an anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 10 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 46 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:46. In certain embodiments, the anti-BCMA scFv comprises VH of an amino acid having the sequence set forth in SEQ ID NO: 10 and VL comprising an amino acid having the sequence set forth in SEQ ID NO:46. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence of SEQ ID NO: 10 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 46.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO:9 and LCDR3 of SEQ ID NO:45.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:7, VH CDR2 of SEQ ID NO:8, and VH CDR3 of SEQ ID NO:9.
  • the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO: 32, VL CDR2 of SEQ ID NO: 44, and VL CDR3 of SEQ ID NO: 45.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:7, VH CDR2 of SEQ ID NO:8, and VH CDR3 of SEQ ID NO:9, and VL CDR1 of SEQ ID NO:32 VL CDR2 of SEQ ID NO: 44 and VL CDR3 of SEQ ID NO: 45.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising the antigen binding region of amino acid sequence SEQ ID NO: 111 or a variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:111.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:112.
  • an anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 16 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 50 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO:16. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:50. In certain embodiments, the anti-BCMA scFv comprises VH of an amino acid having the sequence set forth in SEQ ID NO:16 and VL comprising an amino acid having the sequence set forth in SEQ ID NO:50. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence of SEQ ID NO: 16 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 50.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO: 13 and LCDR3 of SEQ ID NO: 49.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:11, VH CDR2 of SEQ ID NO:12, and VH CDR3 of SEQ ID NO:13.
  • the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO: 47, VL CDR2 of SEQ ID NO: 48, and VL CDR3 of SEQ ID NO: 49.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:11, VH CDR2 of SEQ ID NO:12, and VH CDR3 of SEQ ID NO:13, and VL CDR1 of SEQ ID NO:47 VL CDR2 of SEQ ID NO:48 and VL CDR3 of SEQ ID NO:49.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising an antigen binding region of amino acid sequence SEQ ID NO: 114 or a variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:114.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:115.
  • an anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 17 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 58 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO:17. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:58. In certain embodiments, the anti-BCMA scFv comprises a VH comprising an amino acid of the sequence set forth in SEQ ID NO: 17 and a VL comprising an amino acid having the sequence set forth in SEQ ID NO:58. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence of SEQ ID NO: 17 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 58.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO:14 and LCDR3 of SEQ ID NO:55. In certain embodiments, the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:11, VH CDR2 of SEQ ID NO:12, and VH CDR3 of SEQ ID NO:14. In certain embodiments, the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO: 51, VL CDR2 of SEQ ID NO: 54, and VL CDR3 of SEQ ID NO: 55.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:11, VH CDR2 of SEQ ID NO:12, and VH CDR3 of SEQ ID NO:14, and VL CDR1 of SEQ ID NO:51 VL CDR2 of SEQ ID NO: 54 and VL CDR3 of SEQ ID NO: 55.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising an antigen binding region of amino acid sequence SEQ ID NO: 120 or a variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:120.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:121.
  • an anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 64 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO:23. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:64. In certain embodiments, the anti-BCMA scFv comprises VH of an amino acid having the sequence set forth in SEQ ID NO:23 and VL comprising an amino acid having the sequence set forth in SEQ ID NO:64. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence of SEQ ID NO: 23 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 64.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO:22 and LCDR3 of SEQ ID NO:63.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 20, VH CDR2 of SEQ ID NO: 21, and VH CDR3 of SEQ ID NO: 22.
  • the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO: 61, VL CDR2 of SEQ ID NO: 62, and VL CDR3 of SEQ ID NO: 63.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 20, VH CDR2 of SEQ ID NO: 21, and VH CDR3 of SEQ ID NO: 22, and VL CDR1 of SEQ ID NO: 61 VL CDR2 of SEQ ID NO: 62 and VL CDR3 of SEQ ID NO: 63.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising the antigen binding region of amino acid sequence SEQ ID NO: 117 or variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:117.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:118.
  • the anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 27 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 59 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO:27. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:59. In certain embodiments, the anti-BCMA scFv comprises VH of an amino acid having the sequence set forth in SEQ ID NO:27 and VL comprising an amino acid having the sequence set forth in SEQ ID NO:59. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence of SEQ ID NO: 27 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 59.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO:26 and LCDR3 of SEQ ID NO:56. In certain embodiments, the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:24, VH CDR2 of SEQ ID NO:25, and VH CDR3 of SEQ ID NO:26. In certain embodiments, the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO: 52, VL CDR2 of SEQ ID NO: 62, and VL CDR3 of SEQ ID NO: 56.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 24, VH CDR2 of SEQ ID NO: 25, and VH CDR3 of SEQ ID NO: 26, and VL CDR1 of SEQ ID NO: VL CDR2 of SEQ ID NO: 62 and VL CDR3 of SEQ ID NO: 56.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising the antigen binding region of amino acid sequence SEQ ID NO: 123 or variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:123.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:124.
  • the anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 27 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 71 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO:27. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:71. In certain embodiments, the anti-BCMA scFv comprises VH of an amino acid having the sequence set forth in SEQ ID NO:27 and VL comprising an amino acid having the sequence set forth in SEQ ID NO:71. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence set forth in SEQ ID NO: 27 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 71.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO:26 and LCDR3 of SEQ ID NO:68. In certain embodiments, the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:24, VH CDR2 of SEQ ID NO:25, and VH CDR3 of SEQ ID NO:26. In certain embodiments, the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO: 65, VL CDR2 of SEQ ID NO: 62, and VL CDR3 of SEQ ID NO: 68.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:24, VH CDR2 of SEQ ID NO:25, and VH CDR3 of SEQ ID NO:26, and VL CDR1 of SEQ ID NO:65 VL CDR2 of SEQ ID NO: 62 and VL CDR3 of SEQ ID NO: 68.
  • the antibody of the invention is an anti-BCMA scFv or antigen-binding fragment thereof comprising the antigen binding region of amino acid sequence SEQ ID NO: 126 or variant thereof, and specifically binding to a BCMA polypeptide (eg, having an amino acid The BCMA polypeptide of SEQ ID NO: 74 or a fragment thereof).
  • the variant has at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity to SEQ ID NO:126.
  • the anti-BCMA scFv is encoded by the nucleotide of SEQ ID NO:127.
  • the anti-BCMA scFv antibody comprises: an amino acid comprising the amino acid sequence of SEQ ID NO: 27 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto
  • the heavy chain variable region of the sequence, and the light chain comprising the amino acid sequence of SEQ ID NO: 72 or having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto A region, and optionally a linker between the heavy chain variable region and the light chain variable region, such as a linker peptide.
  • the linker comprises the amino acid sequence of SEQ ID NO:93.
  • the anti-BCMA scFv comprises a VH having the amino acid sequence of SEQ ID NO:27. In certain embodiments, the anti-BCMA scFv comprises VL having the amino acid sequence of SEQ ID NO:72. In certain embodiments, the anti-BCMA scFv comprises VH of an amino acid having the sequence set forth in SEQ ID NO:27 and VL comprising an amino acid having the sequence set forth in SEQ ID NO:72. In certain embodiments, the anti-BCMA scFv comprises 3 HCDR sequences of VH of the sequence set forth in SEQ ID NO: 27 and/or 3 LCDR sequences of VL of the sequence of SEQ ID NO: 72.
  • the anti-BCMA scFv comprises HCDR3 of SEQ ID NO:26 and LCDR3 of SEQ ID NO:69. In certain embodiments, the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO:24, VH CDR2 of SEQ ID NO:25, and VH CDR3 of SEQ ID NO:26. In certain embodiments, the anti-BCMA scFv comprises: VL CDR1 of SEQ ID NO: 66, VL CDR2 of SEQ ID NO: 62, and VL CDR3 of SEQ ID NO: 69.
  • the anti-BCMA scFv comprises: VH CDR1 of SEQ ID NO: 24, VH CDR2 of SEQ ID NO: 25, and VH CDR3 of SEQ ID NO: 26, and VL CDR1 of SEQ ID NO: VL CDR2 of SEQ ID NO: 62 and VL CDR3 of SEQ ID NO: 69.
  • Antibodies with an Fc region have several advantages, including, but not limited to, anomeric functions mediated by Fc regions, such as CDC and ADCC immunological activities; formation of bivalent antibodies by dimerization of the Fc region, which can provide strong Antigen binding affinity, and/or altering plasma half-life and renal clearance; bivalent antibodies can be internalized at a rate different from monovalent Fab or scFv antibodies, altering immune function or vector function. For example, alpha emitters do not require internalization to kill target cells, but many drugs and toxins will benefit from internalization of immune complexes.
  • a scFv-Fc antibody formed by fusion of a single chain scFv antibody of the invention with an antibody Fc region is provided.
  • the antibody comprises a single chain scFv antibody of the invention and a wild type or altered Fc region.
  • the antibody comprises, from the N-terminus to the C-terminus: Fc-VH-linker-VL or Fc-VL-linker-VH; or preferably VH-linker-VL-Fc or VL-linker-VH -Fc.
  • the Fc is linked to the variable region (VH or VL) by a hinge region.
  • the Fc is an Fc region from a human immunoglobulin, preferably a human IgGl or IgG4 Fc region.
  • the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 132, or comprises at least one, two or three, but no more than 20, and 10 amino acid sequences relative to SEQ ID NO: 132 Or a 5 amino acid altered amino acid sequence, or a sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO: 132.
  • a single chain scFv antibody of the invention is joined to the Fc region by a hinge region.
  • the hinge region is a C8 hinge region, eg, comprising at least one, two or three, but not more than the amino acid sequence set forth in SEQ ID NO: 95, or relative to the amino acid sequence of SEQ ID NO: 95 Amino acid sequence of 5 amino acid changes.
  • the invention provides an antibody that specifically binds to a BCMA polypeptide (eg, a BCMA polypeptide having the amino acid sequence of SEQ ID NO: 74 or a fragment thereof), and comprises a mutation selected from the group consisting of SEQ ID NO:
  • a BCMA polypeptide eg, a BCMA polypeptide having the amino acid sequence of SEQ ID NO: 74 or a fragment thereof
  • amino acid sequences of some exemplary ScFv-Fc antibodies of the invention and the amino acid and nucleotide sequences of the single-stranded scFvs used to construct them are set forth in Table 3 below.
  • Table 3 also shows the amino acid sequence and nucleotide sequence of the reference scFv-Fc recombinant single chain antibody constructed based on the description of US20170226216A1.
  • the amino acid sequence and nucleotide sequence of the linker and hinge used in these scFv-Fc antibodies are shown in Fig. 8.
  • Antibody name Amino acid sequence of the scFv used DNA sequence of scFv used Amino acid sequence of scFv-hFc ADI-34848 scFv-hFc SEQ ID NO:99 SEQ ID NO: 100 SEQ ID NO: 101 ADI-34849 scFv-hFc SEQ ID NO: 102 SEQ ID NO: 103 SEQ ID NO: 104 ADI-34850 scFv-hFc SEQ ID NO:105 SEQ ID NO:106 SEQ ID NO:107 ADI-34854 scFv-hFc SEQ ID NO:108 SEQ ID NO:109 SEQ ID NO: 110 ADI-34846 scFv-hFc SEQ ID NO: 111 SEQ ID NO: 112 SEQ ID NO: 113 ADI-34857 scFv-hFc SEQ ID NO: 114 SEQ ID NO:115 SEQ ID NO:116 ADI-34859 scFv-hFc S
  • the scFv-Fc antibodies of the invention have effector functions.
  • effector function refers to those biological activities attributable to the Fc-region of an antibody, which vary with the class of antibody.
  • the heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • Effector functions of antibodies include, for example but not limited to, C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors ( For example, down-regulation of B cell receptors; and B-cell activation.
  • the scFv-Fc antibody of the invention blocks, inhibits growth, and/or kills of BCMA-expressing cells (especially MM cells) by effector cell-mediated cytotoxicity (ADCC) activity cell.
  • an Fc region can comprise an Fc-region having one or more amino acid substitutions that increase ADCC activity, eg, substitutions at positions 298, 333, and/or 334 of the Fc-region (EU numbering of residues) .
  • the Fc-region can also be altered to result in altered (ie, increased or decreased) C1q binding and/or complement dependent cytotoxicity (CDC) (see, eg, US 6,194,551, WO 99/51642 and Idusogie, EE et al, J. Immunol. 164 (2000) 4178-4184).
  • CDC complement dependent cytotoxicity
  • the Fc can be altered to increase or decrease its degree of glycosylation and/or alter its glycosylation pattern.
  • Addition or deletion of a glycosylation site to an Fc can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.
  • one or more amino acid substitutions can be performed to eliminate one or more glycosylation sites, thereby eliminating glycosylation at that site.
  • Antibodies with altered types of glycosylation can be prepared, such as low or no fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased aliquots of GlcNac structure. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies.
  • the invention provides antibodies wherein the Fc region is low or afucosylated, thereby significantly increasing the Fc domain of the antibody and the Fc gamma receptor expressed on the effector cells (eg, The binding affinity of Fc ⁇ RIIIa), thereby resulting in antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.
  • the amount of fucose in the antibody can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%.
  • the average of fucose in the sugar chain at Asn297 can be calculated by MALDI-TOF mass spectrometry, relative to the sum of all sugar structures (eg, complex, heterozygous, and high mannose structures) attached to Asn 297.
  • the amount of fucose is thus determined, for example as described in WO 2008/077546.
  • Asn297 refers to an asparagine residue located at approximately position 297 (the EU numbering of the Fc region residues) in the Fc region; however, Asn297 may also be located approximately ⁇ 3 upstream or downstream of position 297 due to minor sequence changes in the antibody Amino acid position, ie between positions 294 and 300. See, for example, US 2003/0157108; US 2004/0093621.
  • Such antibody variants can be produced in cell lines capable of producing defucosylated or hypofucosylated antibodies.
  • Examples of such cells include Lec13 CHO cells deficient in protein fucosylation (Ripka, J. et al, Arch. Biochem. Biophys. 249 (1986): 533-545; US 2003/0157108; and WO 2004/056312, especially Is Example 11); and a knockout cell line, such as the ⁇ -1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87).
  • the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 ( ⁇ (1,6)-fucosyltransferase), thereby expressing the lack of fucoids in the Ms704, Ms705, and Ms709 cell lines.
  • FUT8 ⁇ (1,6)-fucosyltransferase
  • EP 1,176,195 also describes cell lines with a functionally disrupted FUT8 gene in which antibodies expressed in the cell line exhibit low fucosylation.
  • fucosylase residues can be used to excise the fucose residue of the antibody; for example, fucosidase alpha-L-fucosidase removes fucosyl residues from the antibody (Tarentino et al. (1975) Biochem. 14: 5516-23).
  • the present invention also contemplates antibody variants having a bisected oligosaccharide, for example, an antibody in which the biantennary oligosaccharide linked to the Fc region is bisected by GlcNAc.
  • These antibody variants may have reduced fucosylation and/or increased ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; US 6,602,684; and US 2005/0123546.
  • Antibody variants having at least one galactose residue in an oligosaccharide linked to an Fc region are also contemplated by the present invention. These antibody variants can have enhanced CDC function. These antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.
  • Non-limiting examples of in vitro assays for assessing ADCC activity of a target molecule are described in US 5,500,362 (see, for example, Hellstrom, I. et al, Proc. Nat'l Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom , I. et al., Proc. Nat'l Acad. Sci. USA 82 (1985) 1499-1502); US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361).
  • non-radioactive assays methods may be employed (see, for example, ACTI TM non-radioactive cytotoxicity assay for flow cytometry cells (CellTechnology, Inc.Mountain View, CA), and Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells suitable for these assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells.
  • the ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model as disclosed in Clynes, R. et al, Proc. Nat'l Acad. Sci. USA 95 (1998) 652-656. Evaluation.
  • C1q binding assays can also be performed to determine C1q binding and CDC activity of antibodies. See, for example, the C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402.
  • the invention also contemplates antibody variants having some but not all effector functions, which makes them ideal candidates for certain applications in which the in vivo half-life of the antibody is important, but Certain effector functions such as complement and ADCC are unnecessary or harmful.
  • In vitro and/or in vivo assays as described above can be performed to confirm reduction/depletion of CDC and/or ADCC activity.
  • an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcyR binding (and therefore is likely to lack ADCC activity), but retains FcRn binding ability.
  • the Fc region may comprise a mutation that abolishes or attenuates effector function, such as a human IgGl Fc region with mutations P329G and/or L234A and L235A, or a human IgG4 Fc region with mutations P329G and/or S228P and L235E.
  • a mutation that abolishes or attenuates effector function such as a human IgGl Fc region with mutations P329G and/or L234A and L235A, or a human IgG4 Fc region with mutations P329G and/or S228P and L235E.
  • an scFv-Fc region antibody of the invention can form a bivalent antibody by dimerization of the Fc region, thereby further increasing the total affinity and stability of the antibody, or forming a multispecificity such as bispecific Sex.
  • the Fc region may comprise i) a homodimeric Fc-region of the human IgGl subclass, or ii) a homodimeric Fc-region of the human IgG4 subclass, or iii) a heterodimeric Fc-region, wherein a) an Fc- The region polypeptide comprises the mutation T366W, while the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the mutations T366W and Y349C, and the other Fc-region polypeptide comprises the mutation T366S, L368A, Y407V and S354C, or c) one Fc-region polypeptide comprises the mutations T366W and S354C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.
  • a scFv-Fc recombinant antibody of the invention can be directly fused or conjugated to other molecules by means of a portion of the Fc region, including, but not limited to, fluorescent dyes, cytotoxins, radioisotopes, and the like, for example, for antigen quantification Studies, immobilization of antibodies for affinity measurement, targeted delivery for therapeutic agents, Fc-mediated cytotoxicity testing using immune effector cells, and many other uses.
  • the invention provides a substantially purified nucleic acid molecule encoding a polypeptide comprising a segment or domain of an antibody chain that binds BCMA as described above.
  • a nucleic acid molecule of the invention encodes an antibody chain that binds to BCMA (eg, any of the antibodies of the invention, including single-stranded scFv antibodies and scFv-Fc antibodies, and fragments thereof).
  • nucleic acids of the invention comprise a nucleotide sequence encoding a heavy chain variable region of any of the antibodies set forth in Table 1, or variants thereof, and/or a light chain variable region of a corresponding antibody shown in Table 1, or variants thereof Nucleotide sequence.
  • the nucleic acid molecule is the DNA VH sequence and/or the DNA VL sequence set forth in Table 1.
  • nucleic acid molecules of the invention comprise a nucleotide sequence that is substantially identical (eg, at least 65%, 80%, 95%, or 99% identical) to the nucleotide sequence of the nucleic acid molecule shown in Table 1. When expressed from a suitable expression vector, the polypeptide encoded by these polynucleotides is capable of exhibiting BCMA antigen binding ability.
  • polynucleotides encoding at least one CDR region and generally all three CDR regions of a heavy chain VH or light chain VL sequence from an antibody that binds BCMA as described above.
  • the polynucleotide encodes a complete or substantially complete variable region sequence of the heavy and/or light chain of the BCMA-binding antibody described above.
  • each antibody or polypeptide amino acid sequence can be encoded by a plurality of nucleic acid sequences because of codon degeneracy.
  • nucleic acid sequences of the invention comprise a nucleotide sequence encoding a heavy chain VH comprising: (i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 75-82 or having, for example, at least 80%, 90% or 99 % identity nucleotide sequence.
  • nucleotide sequence encoding a light chain VL comprising the nucleotide sequence of SEQ ID NOs: 83-92 or a nucleotide sequence having, for example, at least 80%, 90% or 99% identity thereto .
  • nucleic acid sequences of the invention encode any of the above single-stranded scFv antibodies of the invention.
  • the nucleic acid sequences of the invention encoding an scFv antibody comprise a nucleotide sequence encoding a heavy chain VH sequence and a nucleotide sequence encoding a light chain VL sequence selected from the group consisting of:
  • the nucleic acid of the invention encoding an scFv antibody further comprises a nucleotide sequence encoding a linker, such as the sequence set forth in SEQ ID NO: 94 or a sequence substantially identical thereto.
  • the nucleic acid of the invention encoding an scFv antibody comprises, or is substantially identical to, a sequence selected from the group consisting of SEQ ID NOs: 100, 103, 106, 109, 112, 115, 118, 121, 124, and 127 the sequence of.
  • a "substantially identical" nucleotide sequence means having at least 80%, 85%, 90%, 90%, 92% in sequence with a reference nucleotide sequence, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity sequence.
  • the identity of the nucleotide sequences can be determined using various sequence alignment methods well known in the art. For example, the BLAST sequence alignment search tool can be obtained from the website of NCBI (National Center for Biotechnology Information, Bethesda, MD). Typically, percent identity is performed using the default parameters of NCBI Blast.
  • polynucleotide sequences can be produced by de novo solid phase DNA synthesis or by PCR mutagenesis of existing sequences encoding BCAM-binding antibodies or binding fragments thereof (e.g., the sequences set forth in Tables 1-3).
  • Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; phosphoric acid of Brown et al., Meth. Enzymol. 68:109, 1979.
  • Patent No. 4,458,066 Introduction of mutations into a polynucleotide sequence by PCR can be as for example PCR Technology: Principles and Applications for DNA Amplification, HAErlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al, Nucleic Acids Res. 19: 967, 1991; and Eckert et al., PCR Methods and Applications, 1:17, 1991.
  • Antibodies can be produced using recombinant methods and compositions, such as described in U.S. Patent 4,816,567.
  • a vector comprising an isolated nucleic acid encoding an antibody that binds BCMA as described herein is provided.
  • This nucleic acid may encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody.
  • the vector is an expression vector.
  • the invention provides a host cell comprising the nucleic acid.
  • the host cell comprises (eg, has been transformed with a vector): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH, or (2) comprising a coding comprising the antibody VL A first vector of the nucleic acid of the amino acid sequence and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH.
  • the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells, HEK293 cells, or lymphoid cells (eg, Y0, NSO, Sp20 cells).
  • a method of making an anti-BCMA antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above, optionally under conditions suitable for expression of the antibody, and optionally from a host cell (or host) The antibody is recovered in the cell culture medium.
  • a nucleic acid encoding the antibody such as a nucleic acid as described above, can be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • This nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding antibody heavy and light chain variable regions).
  • a variety of expression vectors can be used to express polynucleotides encoding antibody chains that bind to BCMA, such as any of the antibodies of the invention, including scFv antibodies and full length antibodies.
  • Both viral-based expression vectors and non-viral expression vectors can be used to produce antibodies in mammalian host cells.
  • Non-viral vectors and systems comprise plasmids, episomal vectors, and artificial chromosomes, typically containing an expression cassette for expression of a protein or RNA (see, for example, Harrington et al, Nat Genet 15:345, 1997).
  • Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papillomavirus, HBP EB virus, vaccinia virus vectors, and Semliki Forest virus (SFV). See, Smith, Annu. Rev. Microbiol. 49: 807, 1995; and Rosenfeld et al, Cell 68: 143, 1992.
  • the expression vector contains a promoter operably linked to a polynucleotide encoding an antibody chain or polypeptide that binds BCMA.
  • promoter operably linked to a polynucleotide encoding an antibody chain or polypeptide that binds BCMA.
  • other regulatory elements may be required or required for efficient expression of antibody chains or fragments that bind to BCMA. These elements typically include an ATG start codon and an adjacent ribosome binding site or other sequence.
  • the efficiency of expression can be enhanced by the inclusion of enhancers suitable for the cell system used (see, for example, Scharf et al, Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al, Meth. Enzymol., 153: 516, 1987).
  • an SV40 enhancer or CMV enhancer can be used to increase expression in a mammalian host cell.
  • the expression vector can also provide a secretion signal sequence to form a fusion protein comprising a BCMA binding polypeptide.
  • the BCMA binding antibody/polypeptide sequence can also be ligated to the signal sequence prior to insertion into the vector.
  • the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:133.
  • Vectors for receiving sequences encoding an antibody light chain variable domain and a heavy chain variable domain that bind to BCMA can sometimes also encode a constant region or a portion thereof. Such vectors allow expression of the variable region as a fusion protein with the constant region, thereby resulting in the production of intact antibodies or fragments thereof.
  • such constant regions are human constant regions such as the human IgGl Fc region.
  • the Fc region fused to the variable region comprises the amino acid sequence set forth in SEQ ID NO:132.
  • Suitable host cells for cloning or expression of the vector include prokaryotic or eukaryotic cells.
  • antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required.
  • For the expression of antibody fragments and polypeptides in bacteria see, for example, US 5,648,237, US 5,789,199 and US 5,840,523. (See also Charlton, KA, see: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes antibody fragments in E. coli. expression).
  • the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized", which results in partial or Production of antibodies in a fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al, Nat. Biotech (2006) 24: 210-215.
  • Suitable host cells for expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells.
  • baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures can also be used as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describes techniques for PLANTIBODIES TM producing antibodies in transgenic plants).
  • Vertebrate cells that can be used as hosts include, for example, suspension growth-adapted mammalian cell lines can be useful.
  • mammalian host cell lines are the SV40 transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or as described, for example, in Graham, FL et al, J. Gen Virol. 36 (1997) 59. 293 cells); baby hamster kidney cells (BHK); mouse Sertoli cells (eg, TM4 cells described in Mather, JP, Biol. Reprod.
  • 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 (Hep G2) Mouse breast tumor (MMT 060562); TRI cells, such as those described in Mather, JP et al, Annals NY Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub, G. et al, Proc. Natl. Acad. Sci.
  • a mammalian host cell is used to express and produce an antibody polypeptide of the invention that binds to BCMA.
  • anti-BCMA antibodies provided herein can be screened, identified, or characterized for their physical/chemical properties and/or biological activity by various assays known in the art.
  • a yeast that binds to a target antigen of interest with high affinity can be selected from a yeast display library expressing a human antibody.
  • a variety of methods for presenting or displaying antibodies or antibody fragments on a yeast surface and screening libraries are described, for example, see US20110076752A1, US9845464B2, Boder and Wittrup, 1997, Nat. Biotechnol., 15, 553-557; Blasie et al 2004, Gene, 342, 211-218 ; Sazinsky et al. 2008, Proc. Natl. Acad. Sci. USA, 105, 20167-20172; Tasumi et al. 2009, Proc. Natl. Acad. Sci.
  • Yeast display library screening can be performed, for example, by the following non-limiting means: screening can first be performed by magnetic bead sorting (MACS).
  • MCS magnetic bead sorting
  • a yeast population presenting an IgG or antibody fragment can be contacted with a biotinylated target antigen for a period of time, followed by washing, incubation with streptavidin magnetic beads (available from Miltenyi; Biotec), by LS magnetic column (available Yeast cells captured from Miltenyi; Biotec) and enriched for binding to the target antigen. After that, multiple rounds of enrichment can be performed by FACS technology.
  • the yeast population can be exposed to a reduced concentration of biotinylated target antigen (to screen for high affinity antibodies) or antigen homologs from different species (to screen for antibodies with different species cross-reactivity) Wash the cells, resuspend in ice in a standard solution (for example, a mixture containing streptavidin-PE and anti-human LC-FITC), wash the cells and resuspend in a buffer such as FACS wash buffer.
  • a negative selection reagent such as the multispecific reagent (PSR) described in Xu et al. (Protein Engineering, Design & Selection, 2013, Vol 26, No. 10, pp663-670) can also be used instead of the target antigen and
  • PSR multispecific reagent
  • the IgG-presenting yeast was incubated and sorted by the same two-label and FACS as described above to attenuate the non-specific binding of the antibody and subsequent drug-forming problems.
  • the antibodies of the invention may be identified or characterized for their antigen binding activity, for example by known methods, such as ELISA, aLISA, Western blot, antibodies or reverse phase arrays, and the methods described in the Examples.
  • antibodies can be spotted on a glass or nitrocellulose chip.
  • the slides were blocked and incubated with a solution containing BCMA, washed to remove unbound antibody, and bound antibodies were detected with fluorescently labeled corresponding secondary antibodies. Fluorescence signals were measured by a fluorescent slide scanner.
  • recombinant BCMA cell supernatant, cell or tissue lysates, body fluids, and the like, are spotted on a glass or nitrocellulose chip.
  • the slides were blocked and the array was incubated with antibodies against specific epitopes on BCMA. Unbound antibody is washed away and the bound antibody is detected with a fluorescently labeled corresponding secondary antibody. Fluorescent signals were measured by a fluorescent slide scanner (Dernick, G. et al, J. Lipid Res., 52 (2011) 2323-2331).
  • Antibodies can also be detected using the ForteBio assay.
  • the ForteBio affinity assay can be performed according to the existing method (Estep, P et al, High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8).
  • the AHQ sensor can be lined down in the assay buffer for 30 minutes and then detected on line for 60 seconds to establish a baseline. Thereafter, the AHQ sensor loaded with the purified antibody was exposed to 100 nM antigen for 5 minutes, and the sensor was transferred to assay buffer for 5 minute offline measurement. Kinetic analysis was performed using a 1:1 binding model.
  • Binding of antibodies to cells expressing BCMA on the surface can also be detected by flow cytometry.
  • H929 cells expressing BCMA can be incubated with serially diluted antibodies in PBS 1% BSA for a period of time (eg, 30 minutes) on ice.
  • the secondary antibody eg, phycobiliprotein-labeled secondary antibody
  • the secondary antibody is then incubated with light in PBS 1% BSA on ice for a period of time (eg, 30 minutes).
  • Cells were analyzed by flow cytometry after washing the cells.
  • Flow cytometry can be performed on an Accuri C6 system (BD Biosciences) and the EC50 values were calculated using Graphpad software.
  • the invention provides a fusion or conjugate comprising an antibody of the invention.
  • a fusion or conjugate can be produced by fusing or conjugating an antibody of the invention to a heterologous molecule.
  • an antibody polypeptide of the invention can be fused or conjugated to one or more heterologous molecules, including but not limited to proteins/polypeptides/peptides, labels, drugs, and cytotoxic agents.
  • Methods of fusing or conjugating a protein, polypeptide or peptide or chemical molecule to an antibody are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929 and EP 367,166.
  • 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.
  • an antibody of the invention can be fused or conjugated to a heterologous molecule in the form of a full length antibody or antibody fragment.
  • the single chain scFv antibodies of the invention are used for fusion or conjugation.
  • a fusion protein comprising a single chain scFv of the invention is provided. Such fusion proteins can be readily prepared by recombinant methods known in the art.
  • a conjugate comprising a single chain scFv of the invention is provided, for example, a conjugate comprising a scFv of the invention and a non-proteinaceous drug molecule.
  • Linkers can be used to covalently link different entities in the fusions and/or conjugates of the invention.
  • Linkers include chemical linkers or single-stranded peptide linkers.
  • a single chain antibody of the invention such as an scFv antibody, is fused to other peptides or proteins via a peptide linker.
  • a single chain antibody of the invention eg, an scFv antibody, is conjugated to other molecules, such as a label or drug molecule, via a chemical linker.
  • Peptide linkers that can be used to form the invention include peptides consisting of amino acid residues. Such linker peptides are generally flexible, allowing the antigen binding portion to which they are attached, such as scFv, to move independently.
  • the length of the linker peptide can be readily determined by one skilled in the art based on actual conditions, for example, at least 4-15 amino acids in length, or longer, such as about 20-25 amino acids.
  • coupling agents are N-succinimide-3-(2-pyridyldithio)propionate (SPDP), succinimide-4-(N-maleimidomethyl)cyclohexane a difunctional derivative of an alkyl-1-carboxylate (SMCC), an iminosulfane (IT), an imidate (such as dimethyl dimethyl imidate), an active ester (such as suberic acid II) Succinimide ester), aldehyde (such as glutaraldehyde), diazide compound (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivative (such as double-(paired to double) Nitrobenzoyl)-ethylenediamine), diisocyanate (such as toluene 2,6-diisocyanate), and bis-active fluorine compound (e.g., 1,5-succinimide-3-(2-pyridyldithio)propionate (SPDP
  • the linker can be a "cleavable linker" that facilitates release of the polypeptide upon delivery to the target site.
  • a "cleavable linker” that facilitates release of the polypeptide upon delivery to the target site.
  • an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimethyl linker or a disulfide containing linker can be used (Chari et al, Cancer Research 52 (1992) 127-131; US 5,208,020). .
  • the invention provides the use of an anti-BCMA antibody, fusion or conjugate of the invention in the 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.
  • the term "detecting" as used herein includes quantitative or qualitative detection. Exemplary detection methods include, but are not limited to, immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic hybridization of antibody molecules, ELISA assays, PCR-technology (e.g., RT-PCR).
  • the biological sample comprises a bodily fluid, a cell, or a tissue.
  • the biological sample is a blood, serum or other liquid sample of biological origin.
  • an anti-BCMA antibody, fusion or conjugate is provided for use in a diagnostic or detection method.
  • a method of detecting the presence of BCMA in a biological sample comprises contacting a biological sample with an anti-BCMA antibody, fusion or conjugate described herein under conditions that allow binding of the anti-BCMA antibody, fusion or conjugate to BCMA, and detecting the anti-antibody Whether a complex is formed between the BCMA antibody, fusion or conjugate and BCMA.
  • Such methods can be in vitro or in vivo methods.
  • an anti-BCMA antibody, fusion or conjugate is used to select a subject that is suitably treated with an anti-BCMA antibody, for example, when BCMA is a biomarker for patient selection.
  • Exemplary conditions 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 patient with multiple myeloma (MM) with an antibody, fusion or conjugate of the invention comprising determining B cells, preferably malignant B, of the patient Whether the cell expresses a BCMA protein on the surface of the B cell, wherein the B cell expresses a BCMA protein on its surface, the patient will likely respond and use a therapeutic agent targeting BCMA (eg, an anti-BCMA antibody) ) for treatment.
  • an anti-BCMA antibody can be conjugated to a diagnostic or detectable agent.
  • the invention provides a kit for use in diagnosis or detection comprising any of the anti-BCMA antibodies, fusions or conjugates of the invention.
  • the invention relates to a method of treating a B cell associated disorder comprising administering to the subject an effective amount of an antibody of the invention or antigen binding fragment thereof, or a fusion or conjugate of the invention.
  • mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse).
  • domesticated animals eg, cows, sheep, cats, dogs, and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits eg, mice and large mouse.
  • rodents eg, mice and large mouse.
  • the subject is a human.
  • treatment refers to the clinical intervention intended to alter the natural course of the disease in an individual being treated. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or mitigating the disease state, and alleviating or improving the prognosis.
  • B cell-associated disorders are disorders associated with abnormal B cell activity, including, but not limited to, B cell malignancies, plasma cell malignancies, autoimmune diseases.
  • Exemplary conditions that can be treated with BCMA antibodies include, for example, multiple myeloma, non-Hodgkin's lymphoma, B cell proliferation with uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorders, immunomodulatory disorders, Rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves' disease, Wegener's granulomatosis, nodular polyarteritis, Sjogren's Syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, and progressive glomerulonephritis, heavy chain disease , primary or
  • an antibody of the invention is a fully human antibody comprising a VH region of a fully human origin and a full human VL region amino acid sequence, such as the antibodies shown in Table 1, and Table 3 A single-stranded scFv and an scFv-Fc antibody comprising the human hFc fragment constructed therefrom.
  • the conjugates and fusions of the invention are conjugates and fusions comprising a fully human antibody, such as a fully human single chain scFv.
  • the antibodies, fusions and conjugates of the invention are particularly useful for therapeutic applications in humans.
  • the antibodies, fusions and conjugates of the invention are used to treat a human B cell related disorder, such as a B cell malignancy, preferably multiple myeloma (MM) or non-Hodgkin's lymph Tumor (NHL).
  • a human B cell related disorder such as a B cell malignancy, preferably multiple myeloma (MM) or non-Hodgkin's lymph Tumor (NHL).
  • MM myeloma
  • NHS non-Hodgkin's lymph Tumor
  • the anti-tumor effects of the anti-BCMA antibodies, fusions, and conjugates of the invention include, but are not limited to, for example, reducing tumor volume, reducing the number of tumor cells, reducing tumor cell proliferation, or reducing tumor cell survival.
  • Multiple myeloma is a B cell malignancy of mature plasma cells.
  • the clonal plasma cells in the bone marrow are abnormally proliferating and can invade adjacent bones and sometimes invade the blood.
  • Variants of multiple myeloma include: dominant multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory multiple myeloma, IgD multiple myeloma, osteosclerosing myeloma, Bone solitary plasmacytoma and extramedullary plasmacytoma. (See, for example, Braunwald et al. (eds.), Harrison's Principles of Internal Medicine, 15th Ed. (McGraw-Hill 2001).
  • Non-Hodgkin's lymphoma can be divided into invasive (rapidly growing) and inert (slowly growing).
  • Non-Hodgkin's lymphoma includes: Burkitt's lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblasts Severe cell lymphoma, precursor B lymphoblastic lymphoma, and mantle cell lymphoma. Lymphomas that occur after bone marrow or stem cell transplantation are usually B-cell non-Hodgkin's lymphoma.
  • the BCMA antibodies, fusions and conjugates of the invention can be administered in combination with other therapeutic modalities for the treatment of such diseases as tumors.
  • Such other forms of treatment 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 anti-neoplastic agents.
  • compositions comprising any one or more of the BCMA-binding antibody molecules, fusions and conjugates, polynucleotides, vectors, or host cells herein are also contemplated by the present invention.
  • Compositions include, but are not limited to, pharmaceutical compositions.
  • the pharmaceutical compositions can be used for administration to cells or animals alone or in combination with one or more other therapeutic modalities.
  • compositions of the antibodies, fusions and conjugates of the invention can be prepared, for example, by bringing antibodies, fusions and conjugates of the desired purity, together with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical) Science, 16th Edition, Osol, A. (ed.) (1980)) is mixed and prepared in the form of a lyophilized formulation or an aqueous solution.
  • pharmaceutically acceptable carriers Remington's Pharmaceutical Science, 16th Edition, Osol, A. (ed.) (1980)
  • Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methyl sulfide Preservatives (such as octadecyl dimethyl benzyl ammonium chloride; chlorhexidine ammonium; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as Methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) polypeptide; protein, such as serum white Protein, gelatin or immunoglobulin; hydrophilic polymer such as poly(vinylpyrrolidone); amino acid such as glycine, glutamine, aspara
  • Exemplary lyophilized antibody formulations are described in US 6,267,958.
  • Aqueous antibody preparations include those described in US 6,171,586 and WO 2006/044908, the latter including a histidine-acetate buffer.
  • the formulations herein may also contain more than one active ingredient as needed for the particular indication being treated, preferably those having complementary activities and not adversely affecting each other.
  • active ingredients are suitably present in combination in amounts effective for the intended purpose.
  • Yeast display technology fully human antibodies that specifically bind screening BCMA from greater than 6 total diversity of synthetic antibody library of 1 ⁇ 10 8 (the design and construction of the library can be found in WO 2009036379, WO2010105256, WO2012009568).
  • the screening process is as follows: First, the first round of screening is performed by magnetic bead sorting (MACS) and using biotin-labeled, Fc-fused recombinant human BCMA to screen six different synthetic antibody libraries; The round of screening was performed according to the method of Chao et al.
  • MCS magnetic bead sorting
  • Batch optimization is a regular part of the initial screening step. Briefly, the heavy chain region of the antibody population enriched by the initial screening (the diversity of this stage is between 10 3 and 10 4 ) is separated and recombined with the natural human light chain sequence library in the yeast. carry out. This process is called light chain batch shuffle (LCBS), and finally the antibody library with heavy chain/light chain pairing diversity between 10 7 and 10 8 with human BCMA binding tendency is finally prepared. .
  • the antibody library was further enriched with human, monkey and murine BCMA-specific antibodies via a round of magnetic bead screening and four-round flow screening as described in the paragraph above. As described above, human, monkey, and murine fusion proteins of BCMA and Fc, and His-tagged human BCMA monomer protein were used for positive screening, and PSR was negatively screened.
  • the enriched yeast population containing the specific antibody sequence is spread on an agar plate to obtain a yeast monoclonal colony containing a specific antibody gene. Clones were picked and their variable regions were sequenced using the sanger method. Approximately 460 unique H3:L3 antibodies (i.e., antibodies with unique heavy chain CDR3 and light chain CDR3 domain pairs) were identified. Some antibodies were then obtained by yeast expression and purification using protein A affinity chromatography.
  • NCI-H929 ATCC, CRL-9068 cells
  • adjust the cell density to 2x10 6 /ml
  • the anti-BCMA antibody was started from a concentration of 400 nM, and serially diluted in a 3-fold gradient in PBS containing 0.1% bovine serum albumin (BSA) for 12 points, 100 ⁇ l of diluted antibody was added to each well, and incubated at 4 ° C for 30 min;
  • BSA bovine serum albumin
  • test results are shown in Figure 1 and Table 4 below. It can be confirmed from the experimental results that 10 antibodies have a strong affinity with NCI-H929 cells at a concentration of 400 nM, and their affinity with NCI-H929 is gradually decreased as the antibody concentration is diluted.
  • a recombinant protein expression vector of the single antibody variant variable region (scFv) of the above 10 antibodies and a human Fc fragment was constructed, respectively.
  • a recombinant single-chain antibody expression vector (as a negative control) of the ADI-34819 antibody, and a recombinant single-chain antibody expression vector (as a standard control) based on the huBCMA-10 sequence disclosed in US20170226216A1 were also constructed.
  • the amino acid sequences of these scFv-Fc recombinant proteins constructed and their corresponding coding nucleotide sequences are shown in Table 3 above.
  • mice ⁇ light chain signal peptide (METDTLLLWVLLLWVPGSTG, SEQ ID NO: 133; coding sequence ATGGAGACCGACACCCTGCTGCTCTGGGTCCTGCTGC TGTGGGT GCCCGGATCCACAGGA, SEQ ID NO: 134) and human IgG1 Fc coding sequence (SEQ ID) were digested by restriction endonuclease.
  • pDD1-hFc vector was constructed based on the pTT5 vector, by insertion of the signal peptide and hFc coding gene), and the synthetic scFv sequence was cloned into a light chain signal by homologous recombination. A fusion expression is formed between the peptide and the gene encoding the hFc.
  • Figure 2 A schematic of the construction of the vector is shown in Figure 2.
  • the volume required transfected HEK293 cells were passaged, the day before transfection the cells were adjusted to a density of 1.2x10 6 / ml.
  • OptiMEM medium (Gibco, 31985-070) was used as a transfection buffer, and 30 ⁇ g of the plasmid carrying the scFv-hFc recombinant single-chain antibody-encoding gene was separately mixed, and the mixture was allowed to stand for 5 min.
  • PEI polyethyleneimine
  • the cell stock solution was centrifuged at 8000 r/min for 40 min, then filtered using a 0.45 ⁇ m filter, and stored at 4 ° C until use.
  • the packing was equilibrated with 10 ml of binding/washing buffer (20 mM Tris + 150 mM NaCl (pH 7.2)) prior to purification.
  • the kinetic constant of the antibody molecule is determined based on the biofilm optical interference technique (BLI) of the fiber biosensor.
  • the basic principle of BLI is: when a biomolecule is bound to the surface of the sensor, a biofilm is formed.
  • the biofilm interferes with the waveform of the light transmitted through the sensor, and the interference phenomenon is detected by phase shifting, so that the binding can be detected.
  • the change in the number of sensor molecules; the kinetic curve is fitted according to the change of the real-time response value, and the binding constant (Kon), the dissociation constant (Kdis), and the affinity (KD) are calculated.
  • the Fortebio device model selected for the experiment was Octet Red 96, and the ForteBio affinity assay was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8) proceed.
  • the specific process is:
  • Reference scFv-Fc is the level of affinity of the scFv-hFc recombinant protein (see Example 3, SEQ ID NO: 131) constructed according to scFv in the BCMA-10 sequence of US20170226216 A1.
  • the affinity (KD value) of 10 single-chain antibodies to the monovalent human BCMA (BCMA-His) was comparable to that of the reference single-chain antibody and BCMA-His.
  • ADI-34857 and ADI-34860 two single-chain antibodies have the closest affinity to BCMA-His and the reference single-chain antibody.
  • NCI-H929 ATCC, CRL-9068 cells
  • adjust the cell density to 2x10 6 /ml
  • centrifuge 100 ⁇ l per well in a 96-well microplate centrifuge at 400 G for 5 min, and remove the supernatant.
  • test results are shown in Figure 3 and Table 6 below.
  • EC50 value the affinity of other single-chain antibodies to NCI-H929 cells

Abstract

提供一种特异性结合B细胞成熟抗原(BCMA)的新型抗体和抗体片段，尤其是全人源的单链抗体scFv。还提供编码这些抗体的核酸、载体和表达所述核酸的宿主细胞，以及包含前述抗体的组合物、及其在治疗和诊断中的用途。

Description

全人源的抗B细胞成熟抗原(BCMA)单链抗体及其应用 技术领域
本发明涉及特异性结合B细胞成熟抗原(BCMA)的新型抗体和抗体片段，尤其是单链抗体(如单链scFv)。本发明还涉及编码这些抗体和抗体片段的核酸、载体和表达所述核酸的宿主细胞。此外，本发明也涉及包含本发明抗体的组合物、及其在治疗和诊断中的用途。
背景技术
B细胞成熟抗原(BCMA)，又名CD269或TNFRSF17，是肿瘤坏死因子受体家族成员。BCMA是III型跨膜蛋白，在胞外结构域(ECD)中具有TNFR家族成员所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与TNFR家族成员，跨膜激活物CMAL相互作用因子(TACI)和BAFF受体(BAFF-R)在功能上相关。BCMA在跨膜结构域N端的CRD中表现出与TACI具有一定的相似性。此外，人BCMA与小鼠和猕猴的BCMA在胞外结构域ECD上分别具有大约65％和85％氨基酸序列同一性。
研究表明，BCMA可以结合B细胞激活因子受体(BAFF)和B细胞增殖诱导配体(APRIL)，促进B细胞在不同发育阶段的存活。而异常的信号传导可促使B细胞异常增殖，导致自身免疫疾病和肿瘤形成。参见Rickert等人,Immunological Reviews2011,第244卷：115-133。
APRIL，又称作G70，是TNF配体家族的成员。根据文献报道，APRIL与***癌、乳腺癌、阿尔茨海默氏病、免疫疾病、炎症、和胃肠道紊乱相关。参见Hahne等(1998),T.Exp.Med.188:1185-90。可溶性APRIL与BCMA的结合可以促进骨髓(BM)浆细胞和浆母细胞的存活(参见，BLOOD,2014年5月，123卷,20期，p3128-3138)；而与TACI的结合可以导致T细胞非依赖性的抗体反应、B细胞调节、和类型转换重组(参见Vincent等人在Nature Reviews Rheumotology,2014第10卷：365-373)。
BAFF是另一TNF配体家族成员。BAFF与BCMA结合，可以促进浆细胞的存活。BAFF与B细胞、浆母细胞和浆细胞表面上表达的BAFF受体(BAFF-R)结合，可以促进未成熟B细胞的存活和成熟。此外，BAFF也可以与TACI结合，导致T细胞非依赖性的抗体反应、B细胞调节、和类型转换重组(参见Vincent等人在Nature Reviews Rheumotology,2014第10卷：365-373)。
在非肿瘤细胞中，BCMA主要表达在浆细胞和成熟B细胞亚群中。而在60-70％的多发性骨髓瘤(MM)患者中，BCMA也在癌化的浆细胞表面表达。在MM患者中血清BCMA水平升高，并且升高的水平与疾病状况、治疗反应、和整体存活相关。BCMA基因缺陷小鼠有着正常的B细胞水平，但是其浆细胞的生存周期会显著缩短。因此，BCMA是多发性骨髓瘤免疫治疗的理想靶点。
单链scFv抗体是一种小分子的基因工程抗体，它是在DNA水平上利用基因工程方法将天然抗体的重链可变区(VH)和轻链可变区(VL)连接(通常通过一段人工合成的连接肽(或“接头”)连接)而成的小分子重组抗体。与完整抗体分子相比，scFv单链抗体具有以下优点：含有完整的抗体可变区，保留原抗体的抗原特异性和结合活性；不含有抗体分子的Fc区，因而免疫原性弱，用于人体不易产生免疫反应；分子量小，穿透性强、易于渗入组织，用于显像 诊断或治疗时可以进入一般完整抗体不能达到的组织内部；不需要进行糖基化修饰即可形成有功能的抗体分子，利于原核表达***大量生产；易于操作，适用于作为基因工程构件，制备具有新性质的其它抗原特异性结合分子，例如全长抗体、scFv-Fc等。
鉴于BCMA在B细胞恶性肿瘤，尤其是多发性骨髓瘤中作为治疗靶点的有效性，本领域仍然需要新的BCMA特异性结合分子。本发明通过提供以高靶特异性和高亲合性结合BCMA，尤其是与肿瘤细胞表面上表达的BCMA结合，并具有低副作用的全人单链抗体，满足了这方面的需求。本发明的全人单链抗体不仅适于单独用于肿瘤和癌症的诊断或治疗中，而且，更有利地的是，适于作为基因工程构件制备具有高BCMA靶向性的其它诊断和治疗分子，例如各种形式的抗体、scFv-Fc以及基于抗体的融合物和缀合物等。
发明概述
本发明提供了全人源抗人BCMA抗体及其编码基因与应用。通过基因工程手段和酵母表面展示技术，本发明人从展示在酵母表面的人抗体库中筛选出抗人BCMA的全人源抗体，并获得其可变区基因序列，在此基础上构建了全人单链scFv抗体及其与人Fc区的融合构建体，经哺乳动物细胞表达、纯化获得scFv-hFc重组单链抗体分子。本发明的重组单链抗体分子不仅以高亲和力与非膜结合型人BCMA结合，也以高亲和力以细胞表面表达的BCMA结合。
因此，本发明提供特异性结合BCMA的抗体，尤其是单链抗体、及编码其的核酸分子、及它们在治疗和诊断中的用途。
在一个方面，本发明提供特异性地结合BCMA(优选人BCMA蛋白质)的抗体或其抗原结合片段。在一个优选实施方案中，本发明抗体是单链抗体。在一个优选实施方案中，本发明抗体是单链scFv抗体。在另一优选实施方案中，本发明抗体是scFv-Fc抗体。在一些实施方案中，本发明的抗体以大约100nM至5nM的KD结合人BCMA蛋白，其中KD值按照例如生物膜层光学干涉技术(例如Fortebio检测法)测量。在一些实施方案中，本发明的抗体以大约40nM至4nM的EC50结合细胞表面表达的人BCMA蛋白，其中EC50值按照例如流式细胞术(例如FACS)测量。在本发明的一些实施方案中，本发明提供本发明的抗BCMA抗体或其片段在治疗与BCMA相关病症中的用途。
在一些实施方案中，本发明抗体包含表1所示任一抗体的VH区序列或其变体。在另一些实施方案中，本发明抗体包含表1所示任一抗体的VL区序列或其变体。在另一些实施方案中，本发明抗体包含表1所示任一抗体的VH和VL序列对、或其变体。在另一些实施方案中，本发明抗体包含表1所示任一抗体的VH区序列的一个、两个或三个CDR(优选三个CDR)、或其变体。在另一些实施方案中，本发明抗体包含表1所示任一抗体的VL区序列的一个、两个或三个CDR(优选三个CDR)、或其变体。在一些实施方案中，本发明抗体包含表1所示任一抗体的6个CDR区序列、或其变体。在一个实施方案中，抗体的CDR序列是表2所示的CDR序列。
在一些实施方案中，本发明抗体是单链scFv抗体。优选地，scFv抗体包含VH序列、VL序列和接头。优选地scFv抗体从N端到C端包含：VL结构域-接头-VH结构域，或VH结构域-接头-VL结构域。
在一些实施方案中，本发明还提供由本发明单链scFv抗体和野生型或改变的Fc区融合形成的scFv-Fc抗体。在一些实施方案中，本发明的抗体的Fc区是低或无岩藻糖基化的。在一些实施方案中，scFv抗体通过铰链区与Fc区融合。
再一方面，本发明涉及基于本发明抗体，尤其是单链抗体构建的融合物和缀合物。
再一方面，本发明涉及治疗B细胞相关病症的方法和组合物，其中向所述受试者施用有效量的本发明抗体或其抗原结合片段、或本发明的融合物或缀合物。在一些实施方案中，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。在一些优选的实施方案中，B细胞相关病症是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，本发明抗体分子、融合物或缀合物与其它治疗剂联用。
再一方面，本发明涉及检测样品中BCMA的方法和试剂盒，其中所述方法包括：(a)将所述样品与本发明抗体或其抗原结合片段、融合物或缀合物接触；和(b)检测所述抗体或其抗原结合片段、融合物或缀合物和BCMA蛋白之间复合物的形成。在一些实施方案中，样品来自多发性骨髓瘤(MM)患者。所述检测可以是体外的或体内的。
附图简述
图1显示，通过流式细胞术测定的，自酵母展示文库筛选获得的本发明示例性抗BCMA抗体与多发性骨髓瘤细胞系NCI-H929细胞的亲和力。
图2示意性显示本发明的示例性scFv-hFc重组单链抗体的表达载体克隆策略。
图3显示，通过流式细胞术测定的，本发明示例性scFv-hFc重组单链抗体与NCI-H929细胞的亲和力。
图4显示本发明抗体的示例性CDR序列。
图5显示本发明抗体的示例性VH序列。
图6显示本发明抗体的示例性VL序列。
图7显示人BCMA抗原及其胞外结构域(ECD)的示例性氨基酸序列。
图8显示用于构建本发明示例性scFv-Fc构建体和参照scFv-Fc构建体结构的接头、铰链区和Fc区的氨基酸序列和核苷酸序列。
发明详述
除非明确指明相反，否则本发明的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。这些方法的描述可以参见，例如，Sambrook等人，Molecular Cloning:A Laboratory Manual(第3版,2001)；Sambrook等人，Molecular Cloning:A Laboratory Manual(第2版,1989)；Maniatis等人，Molecular Cloning:A Laboratory Manual(1982)；Ausubel等人，Current Protocols in Molecular Biology(John Wiley和Sons,2008年7月更新)；Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates和Wiley-Interscience；Glover,DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford,1985)；Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992)；Transcription and Translation(B.Hames&S.Higgins,Eds.,1984)；Perbal,A Practical Guide to Molecular Cloning(1984)；Harlow和Lane,Antibodies,(Cold Spring Harbor Laboratory Press,Cold  Spring Harbor,N.Y.,1998)Current Protocols in Immunology Q.E.Coligan,A.M.Kruisbeek,D.H.Margulies,E.M.Shevach和W.Strober,eds.,1991)；Annual Review of Immunology；以及期刊专著如Advances in Immunology。
定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
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。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项的任意两项或多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。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。
在本文中，术语“抗原结合分子”是指包含能够与靶抗原结合的抗原结合区或抗原结合部分的分子，例如蛋白质或多肽。在本发明中，当靶抗原是B细胞成熟抗原(BCMA)时，结合BCMA的抗原结合分子也称作BCMA结合分子。抗原结合分子包括例如抗体及其抗原结合片段、单链scFv抗体、基于scFv构建的各种融合物和缀合物，例如scFv-Fc抗体。如本领域技术人员明了的，抗体的抗原结合部分通常包含来自“互补决定区”或“CDR”的氨基酸残基。在一些情况下，根据上下文，“BCMA结合分子”与“本发明抗体”或“抗BCMA抗体”可以互换使用。
在本文中，术语“抗体”是指至少包含轻链或重链免疫球蛋白可变区的多肽，所述免疫球蛋白可变区特异性识别并结合抗原。该术语涵盖各种抗体结构，包括、但不限于单克隆抗体、多克隆抗体、单链抗体或多链抗体、单特异性或多特异性抗体(例如双特异性抗体)、全人源抗体或嵌合抗体或人源化抗体、全长抗体和抗体片段，只要它们呈现期望的抗原结合活性即可。
本领域技术人员明了，“全抗体”(在本文中可与“全长抗体”、“完全抗体”和“完整抗体”互换使用)包含至少两条重链(H)和两条轻链(L)。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。可变区是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。抗体的轻链可以基于其恒定结构域的氨基酸序列归入两种类型(称为kappa(κ)和lambda(λ))中的一种。抗体的重链可以取决于其重链恒定区的氨基酸序列而划分为主要5种不同的类型：IgA、IgD、IgE、IgG和IgM，并且这些类型中的几种可以进一步划分成亚类，如，IgG1、IgG2、IgG3和IgG4、IgA1以及IgA2。对应于不同抗体类型的重链恒定区分别称作α、δ、ε、γ和μ。参见例如Fundamental Immunology，Ch.7(Paul，W.编辑，第二版，Raven Press，N.Y.(1989))(其为所有目的以其整体在此引作参考)。
术语“抗体片段”是指并非完整抗体的分子，其包含完整抗体中用于结合该完整抗体所结合的抗原的部分。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、scFab、Fab’、F(ab’) 2、Fab’-SH、Fv、单链Fv、双链 抗体(diabody)、三链抗体(triabody)、四链抗体(tetrabody)、微抗体(minibody)、单结构域抗体(sdAb)；以及从抗体片段形成的多特异性抗体。Fab片段是一种由VL、VH、CL和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。可以借接头将Fab的轻链(L链)和重链(H链)融合构建成单一多肽链，即单链Fab(scFab)(参见例如US20070274985A1)。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体可以产生F(ab') 2，其为Fab’的二聚体，是二价的抗体片段。F(ab') 2可以在中性条件下通过破坏铰链区中的二硫键而被还原，从F(ab') 2二聚体转化为Fab'单体。Fab'单体基本上是具有铰链区的Fab片段。Fv片段由抗体单臂的VL和VH结构域组成。另外，可以使用重组方法，将独立编码Fv片段的两个结构域VL和VH的基因，通过编码连接肽(接头)的核酸序列连接在一起，重组表达形成单链Fv，在该单条蛋白链中VH区和VL区配对提供抗原结合位点。双链抗体是具有两个抗原结合位点的抗体片段，该片段在同一多肽链中包含通过短接头连接的VL和VH。在双链抗体中，由于接头过短，同一链上的VH和VL两个结构域之间无法配对，而被迫与另一链上的互补结构域配对并且产生两个抗原结合位点。双链抗体可以是二价的或双特异性的。双链抗体的更详细描述可以参见例如，EP 404,097；WO 1993/01161；Hudson等人,Nat.Med.9:129-134(2003)；以及Hollinger等人，PNAS USA 90:6444-6448(1993)。三链抗体和四链抗体和微抗体也描述于Hudson等人，Nat.Med.9:129-134(2003)，和邵荣光等人(编辑)，抗体药物研究与应用，人民卫生出版社(2013)。单结构域抗体(sdAb)通常指这样的抗体，其中单个可变结构域(例如，重链可变结构域(VH)或轻链可变结构域(VL)、衍生自骆驼科重链抗体的重链可变结构域、衍生自鱼类IgNAR的VH样单结构域(v-NAR))即可赋予抗原结合，而不需要与另一可变结构域相互作用以识别靶抗原。(关于抗体片段的更详细的描述，也可以参见：基础免疫学(Fundamental Immunology),W.E.Paul编辑,Raven Press,N.Y.(1993)。
本文中使用的术语“单克隆抗体”表示，得自基本上同质的抗体群体的抗体，即，除了通常以很少量存在的可能变体抗体(例如，含有天然突变或在单克隆抗体制品的生产过程中产生的变体抗体)以外，构成所述群体的各个抗体是相同的和/或结合相同表位。单克隆抗体可以通过多种技术来制备，所述技术包括、但不限于杂交瘤方法、重组DNA方法、酵母展示方法，和使用包含人免疫球蛋白基因座的全部或部分的转基因动物的方法。
术语“人抗体”或“全人源抗体”在本文中可以互换使用，指包括其中构架区和CDR区二者均源自人种系免疫球蛋白序列的可变区的抗体。而且，如果抗体含有恒定区，恒定区也源自人种系免疫球蛋白序列。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸(例如，通过体外随机或点特异诱变或体内体细胞突变引入的突变)，例如在CDR——尤其在CDR3中。然而，如本文所使用的，术语“人抗体”不意欲包括其中的CDR序列衍生自其他哺乳动物物种(如，小鼠)的种系而移植入人构架序列的抗体。
如本文所用，术语“重组人抗体”包括所有通过重组方式制备、表达、产生或分离的人抗体，例如，(a)自用人免疫球蛋白基因进行转基因或转染色体的动物(例如小鼠)或由其制备的杂交瘤分离的抗体，(b)自转化成表达人抗体的宿主细胞例如转染瘤分离的抗体，(c)自重组、组合人抗体文库例如酵母展示文库分离的抗体，和(d)通过包括剪接人免疫球蛋白基因至其他DNA序列的任意其他方式制备、表达、产生或分离的抗体。这些重组人抗体具有构架区和CDR区源自人种系免疫球蛋白序列的可变区。然而，在某些实施方案中，可以对重组人抗体进行体外诱变(或使用人Ig序列转基因动物时为体内体细胞诱变)，由此得到的重组抗体的VH和VL区的氨基酸序列，尽管源自人种系VH和VL序列并与之相关、但是并不天然存在于体 内的人抗体种系库中。
术语“嵌合抗体”是指可变区序列源自一物种、恒定区序列源自另一物种的抗体，例如，其中可变区序列源自小鼠抗体、恒定区序列源自人抗体的抗体。
术语“人源化抗体”是指将源自其他哺乳动物物种例如小鼠种系的CDR序列接到人构架序列上的抗体。可以在人构架序列内进行额外的构架区修饰。
“分离的”抗体是已经与它的天然环境中的组分分离的抗体。在一些实施方案中，将抗体纯化至大于95％或99％纯度，所述纯度通过例如电泳(例如，SDS-PAGE、等电聚焦(IEF)、毛细管电泳)或色谱(例如，离子交换或反相HPLC)确定。关于评价抗体纯度的方法的综述，参见，例如，Flatman，S.等，J.Chrom.B 848(2007)79-87。
表位是抗体所结合的抗原区域。表位可以由连续的氨基酸形成或者通过蛋白的三级折叠而并置的非连续氨基酸形成。
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。图7显示了一个示例性的人BCMA序列(SEQ ID NO:74)。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结合能力的片段。
术语“特异性结合”表示抗体选择性地或优先地结合抗原。如果在生物光干涉测量中，抗体以大约5x10 -7M或更低、大约1x10 -7M或更低、大约5x10 -8M或更低、大约1x10 -8M或更低、大约5x10 -9M或更低的K D，与人BCMA结合，则该抗体是“与人BCMA特异性结合”的抗体。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(K D)代表，平衡解离常数是解离速率常数和结合速率常数(分别是k dis和k on)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
与结合例如BCMA的抗原的参考抗体“竞争结合的抗体”是指这样的抗体，该抗体在竞争检验中阻断参考抗体与抗原(例如BCMA)的结合达到50％或更多，并且反过来，在竞争检验中参考抗体也阻断该抗体与抗原(例如BCMA)的结合达50％或更多。示例性竞争检验描述于：“Antibodies”,Harlow and Lane(Cold Spring Harbor Press,Cold Spring Harbor,NY)。竞争结合的抗体可以与参考抗体结合相同的表位区，例如相同表位、相邻表位或重叠表位。
本文中的术语“Fc区”用于定义含有至少一部分的恒定区的免疫球蛋白重链的C-末端区域。该术语包括天然序列Fc-区和变体Fc-区。在一个实施方案中，人IgG重链Fc-区从重链的Cys226或从Pro230延伸至羧基端。然而，Fc-区的C-端赖氨酸(Lys447)可以存在或可以不存在。除非本文中另外指出，Fc-区或恒定区中的氨基酸残基的编号根据EU编号***，也称为EU索引，如Kabat，E.A.等，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health，Bethesda，MD(1991)，NIH Publication 91-3242中所述。
与抗体相关的术语“变体”在本文中指，包含已经通过至少1个，例如1-30，或1-20或1-10个，例如1或2或3或4或5个氨基酸取代、缺失和/或***而具有氨基酸改变的目标抗体区域(例如重链可变区或轻链可变区或重链CDR区或轻链CDR区)的抗体，其中变体基本上保持改变之前的抗体分子的生物学特性。在一方面，本发明涵盖在本文中所述及的任何抗体的变体。在一个实施方案中，抗体变体保持改变前抗体的至少60％,70％,80％,90％,或100％的生物学活性(例如抗原结合能力)。在一些实施方案中，所述改变不导致抗体变体丧失 对抗原的结合，但任选地可以赋予诸如提高的抗原亲和力和不同的效应子功能等性质。可以理解的，抗体的重链可变区或轻链可变区、或各CDR区可以单独改变或组合改变。在一些实施方案中，在一个或多个或全部三个重链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。在一些实施方案中，在一个或多个或全部三个轻链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。在一些实施方案中，在一个或多个或全部6个CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。优选地，所述氨基酸改变为氨基酸取代，优选保守取代。在一些实施方案中，抗体变体与亲本抗体在目的抗体序列区域上具有至少80％、85％、90％或95％或99％或更高的氨基酸同一性。例如，在一个实施方案中，本发明抗体，与表1所列任一抗体相比，在重链可变区上具有至少80％、85％、90％,91％,92％,93％,94％,95％,96％,97％,98％,或99％或更高的序列同一性。在再一实施方案中，本发明抗体与表1所列任一抗体相比，在轻链可变区上具有至少80％、85％、90％,91％,92％,93％,94％,95％,96％,97％,98％,或99％或更高的序列同一性。在再一实施方案中，本发明抗体与表1所列任一抗体相比，在重链可变区和轻链可变区上具有至少80％、85％、90％,91％,92％,93％,94％,95％,96％,97％,98％,或99％或更高的序列同一性。
在本文中，“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
在本发明中，就抗体序列而言，氨基酸序列同一性百分数通过将候选抗体序列与参考抗体序列最佳比对后，在一个优选方案中按照Kabat编号规则进行最佳比对后，予以确定。比对后，将目标抗体区域(例如，重链或轻链的整个可变区、或其部分例如一个或多个CDR区)与参考抗体的相同区域进行比较。目标抗体区域和参考抗体区域之间的序列同一性百分数为：在目标和参考抗体区域两者中被相同氨基酸占据的位置的数目除以两个区域的比对位置总数(空位不计入)并乘以100得到的百分数。在本文中，在不指定目标抗体区域的情况下，将适用于在参考抗体序列的全长上进行比对。在一些实施方案中，就抗体而言，序列同一性可以分布在整个重链可变区和/或整个轻链可变区上，或序列百分数同一性可以仅限定于构架区，而对应CDR区的序列保持100％相同。
A.本发明的BCMA结合分子和组合物
I.本发明的抗BCMA抗体
本发明一方面提供以高靶特异性和高亲合性结合BCMA(尤其是膜结合性BCMA)的抗体，尤其是单链抗体(例如单链scFv抗体)。
本发明的抗体具有以下一个或多个特性：
(i)以高亲和力，例如以小于100nM，例如小于50nM，例如5-30nM，优选小于10nM的 KD值，与BCMA(例如，人BCMA)结合；
(ii)以高亲和力，例如以小于100nM，例如小于50nM，例如1-40nM，优选小于20nM，更优选小于10或5nM的EC50值，与细胞表面表达的BCMA(例如人BCMA)结合；
(iii)与BCMA上，尤其是BCMA的胞外域ECD上的表位特异性结合(例如，与表1所列任一抗体识别相同或相似的表位)；
(iv)显示与表1所列任一抗体相同或相似的结合亲和力和/或特异性；
(v)抑制(例如，竞争性抑制)本文所述的抗体分子，例如表1所示的任一抗体分子，与BCMA的结合；
(vi)与表1所示的任一抗体结合相同或重叠的表位；
(vii)与表1所示的任一抗体竞争结合BCMA和/或结合BCMA上的相同表位；
(viii)结合人BCMA并且与猴BCMA交叉反应；
(ix)具有本文所述的抗体分子,例如表1所列任一抗体分子的一个或多个生物学特性；
(x)具有本文所述的抗体分子，例如表1所示任一抗体的一种或多种药代动力学特性；
(xi)抑制BCMA的一种或多种活性，从而导致例如以下一者或多者：减少表达BCMA的B细胞或浆细胞的数量、抑制所述细胞的存活或增殖；
(xii)基本上不与BAFF-R或TACI结合；
(xiii)抑制或减少BCMA与其配体，例如与BAFF或APRIL或与两者的结合；
在一些实施方案中，本发明抗BCMA抗体分子以高亲和力，例如，以下述解离平衡常数(K D)与人BCMA(例如SEQ ID NO:74的多肽)结合，所述K D小于约100nM，小于或等于大约80nM、70nM、60nM、或50nM，更优选地小于或等于大约40nM、30nM或20nM，更优选地小于或等于大约10nM、9nM、8nM、7nM、6nM、5nM、4nM、3nM或2nM，例如，通过使用生物光干涉测定法(例如Fortebio亲和测量法)测定。
在一些实施方案中，本发明抗BCMA抗体分子与人BCMA(例如SEQ ID NO:74的多肽)结合的解离速率常数(K dis)小于3×10 -2、1.5×10 -2、5×10 -3或3×10 -3s -1，例如约1.46×10 -3s -1。在一些实施方案中，抗BCMA抗体分子与BCMA结合的结合速率常数(K on)大于1×10 4、5×10 4、1×10 5、5×10 5或8×10 5M -1s -1，例如以约7.29×10 5M -1s -1的K a与BCMA结合。
在一些实施方案中，本发明抗BCMA抗体分子以高亲和力结合表达BCMA的细胞，优选地在细胞表面表达人BCMA的多发性骨髓瘤细胞系(例如NCI-H929)，优选地，以流式细胞术(例如FACS)测定，所述抗体与细胞结合的EC50值小于大约200nM、150nM或100nM，优选地，小于或等于大约80nM、70nM、60nM、或50nM，更优选地小于或等于大约40nM、30nM或20nM，更优选地小于或等于大约10nM、9nM、8nM、7nM、6nM、5nM、4nM、3nM或2nM。
在一个实施方案中，抗体分子与包含氨基酸序列SEQ ID NO:74的人BCMA结合。在一些实施方案中，抗体分子结合BCMA上，优选BCMA的胞外域上的表位。
在一些实施方案中，抗体分子是全长抗体。在另一些实施方案中，抗体分子是抗体片段。例如，本发明的抗体分子可以包含或可以是Fab、scFab、Fab’、F(ab’) 2、Fab’-SH、Fv、单链scFv抗体、双链抗体(diabody)、三链抗体、四链抗体、微抗体、或单结构域抗体(sdAb)。在一个优选的实施方案，本发明抗体分子是单链scFv抗体。在一个优选的实施方案，本发明抗体分子包含scFv及与其连接的Fc区。在一个优选的实施方案，本发明抗体分子是全人源的。
抗体可变区
“可变区”或“可变结构域”是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。重链可变区(VH)和轻链可变区(VL)可以进一步再划分为高变区(HVR，又称作互补决定区(CDR))，其间插有较保守的区域(即，构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。在一些情况下，单个VH或VL结构域足以赋予抗原-结合特异性。此外，结合特定抗原的抗体可以使用来自结合所述抗原的抗体的VH或VL结构域筛选互补VL或VH结构域文库而分离(参见，例如，Portolano，S.等，J.Immunol.150(1993)880-887；Clackson，T.等，Nature 352(1991)624-628)。在本文中，“VH”或“VH结构域”包括全长抗体、Fv、scFv、dsFv、Fab、scFab或本文公开的其它抗体片段的重链可变区VH。在本文中，“VL”或“VL结构域”包括全长抗体、Fv、scFv、dsFv、Fab、scFab或本文公开的其它抗体片段的轻链可变区VL。
在一个实施方案中，本发明抗BCMA抗体分子包含：(i)与表1所列任一抗体的抗原结合区(例如，重链可变区和轻链可变区对)相同的抗原结合区；或(ii)与(i)的抗原结合区在氨基酸序列上具有例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的抗原结合区。
在再一个实施方案中，本发明抗BCMA抗体分子包含：(i)与表1所列任一抗体的重链可变区相同的重链可变区；或(ii)与(i)的重链可变区在氨基酸序列上具有例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的重链可变区；或(iii)(i)的重链可变区的变体，其中所述变体包含至少一个且不超过30、20或10个氨基酸改变(优选氨基酸取代，优选保守取代)，且优选地所述变体在3个重链互补决定区(CDR)区中包含总共不超过10个、优选5-0个氨基酸改变(优选氨基酸取代)。
在再一个实施方案中，本发明抗BCMA抗体分子包含：(i)与表1所列任一抗体的轻链可变区相同的轻链可变区；或(ii)与(i)的轻链可变区在氨基酸序列上具有例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的轻链可变区；或(iii)(i)的轻链可变区的变体，其中所述变体包含至少一个且不超过30、20或10个氨基酸改变(优选氨基酸取代，优选保守取代)，且优选地所述变体在3个轻链互补决定区(CDR)中包含总共不超过10个、优选5-0个氨基酸改变(优选氨基酸取代)。
在再一个实施方案中，本发明抗BCMA抗体分子包含重链可变区和轻链可变区，
其中所述重链可变区选自：
(i)与表1所列任一抗体的重链可变区相同的重链可变区；或(ii)与(i)的重链可变区在氨基酸序列上具有例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的重链可变区；或(iii)(i)的重链可变区的变体，其中所述变体包含至少一个且不超过30、20或10个氨基酸改变(优选氨基酸取代，优选保守取代)，且优选地所述变体在3个重链互补决定区(CDR)区中包含总共不超过10个、优选5-0个氨基酸改变(优选氨基酸取代)；
且其中所述轻链可变区选自：
(i)与表1所列任一抗体的轻链可变区相同的轻链可变区；或(ii)与(i)的轻链可变区在氨基酸序列上具有例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的轻链可变区；或(iii)(i)的轻链可变区的变体，其中所述变体包含至少一个且不超过30、20或10个氨基酸改变(优选氨基酸取代，优选保守取代)，且优选地所述变体在3个轻链互补决定区(CDR)中包含总共不超过10个、优选5-0个氨基酸改变(优选氨基酸取代)。
在一些实施方案中，本发明提供包含表1所列任一抗体的重链可变区和轻链可变区对的氨基酸序列的抗BCMA抗体、或其变体。在一个优选实施方案中，所述抗体包含选自以下的 氨基酸序列对：SEQ ID NOs：4/31,5/41,5/42,10/46,16/50,17/58,23/64,27/59,27/71,和27/72。在一个优选实施方案中，所述变体在VH和/或VL氨基酸序列上具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性、或在VH和/或VL氨基酸序列上包含至少一个且不超过30、20或10个氨基酸改变(优选氨基酸取代，优选保守取代)。
在一些实施方案中，本发明提供包含重链可变区VH和轻链可变区VL的抗BCMA抗体，所述VH包含选自SEQ ID NO:4、5和6的氨基酸序列，且所述VL包含选自SEQ ID NO:31、41、42和43的氨基酸序列。在一些实施方案中，在SEQ ID NO:6的氨基酸序列中，X代表任何氨基酸，优选地代表在SEQ ID NO:4或5的相应位置处的氨基酸残基或其保守取代残基。在一些实施方案中，在SEQ ID NO:43的氨基酸序列中，X代表任何氨基酸，优选地代表在SEQ ID NO:41或42的相应位置处的氨基酸残基或其保守取代残基。
在一些实施方案中，本发明提供包含重链可变区VH和轻链可变区VL的抗BCMA抗体，所述VH包含SEQ ID NO:4的氨基酸序列，且所述VL包含SEQ ID NO:31的氨基酸序列。本发明也提供该抗体的变体，例如在VH和/或VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在一些实施方案中，本发明抗BCMA抗体包含重链可变区VH和轻链可变区VL，所述VH包含SEQ ID NO:5的氨基酸序列，且所述VL包含选自SEQ ID NO:41和42的氨基酸序列。本发明也提供该抗体的变体，例如在VH和/VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在一些实施方案中，本发明提供包含重链可变区VH和轻链可变区VL的抗BCMA抗体，所述VH包含SEQ ID NO:10的氨基酸序列，且所述VL包含SEQ ID NO:46的氨基酸序列。本发明也提供该抗体的变体，例如在VH和/或VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在一些实施方案中，本发明抗BCMA抗体包含重链可变区VH和轻链可变区VL，所述VH包含选自SEQ ID NO:16、17或19的氨基酸序列，且所述VL包含选自SEQ ID NO:50和58的氨基酸序列。在一些实施方案中，在SEQ ID NO:19的氨基酸序列中，X代表任何氨基酸，优选地代表在SEQ ID NO:16或17的相应位置处的氨基酸残基或其保守取代残基。
在一些实施方案中，本发明提供包含重链可变区VH和轻链可变区VL的抗BCMA抗体，所述VH包含SEQ ID NO:16的氨基酸序列，且所述VL包含SEQ ID NO:50的氨基酸序列。本发明也提供该抗体的变体，例如在VH和/或VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在一些实施方案中，本发明提供包含重链可变区VH和轻链可变区VL的抗BCMA抗体，所述VH包含SEQ ID NO:17的氨基酸序列，且所述VL包含SEQ ID NO:58的氨基酸序列。本发明也提供该抗体的变体，例如在VH和/或VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在一些实施方案中，本发明提供包含重链可变区VH和轻链可变区VL的抗BCMA抗体，所述VH包含SEQ ID NO:23的氨基酸序列，且所述VL包含SEQ ID NO:64的氨基酸序列。本发明也提供该抗体的变体，例如在VH和/或VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在一些实施方案中，本发明提供包含重链可变区VH和轻链可变区VL的抗BCMA抗体，所述VH包含SEQ ID NO:27的氨基酸序列，且所述VL包含选自SEQ ID NO:59、71、72和73的氨基酸序列。在一些实施方案中，在SEQ ID NO:73的氨基酸序列中，X代表任何氨基 酸，优选地代表在SEQ ID NO:71或72的相应位置处的氨基酸残基或其保守取代残基。
在一些实施方案中，本发明提供包含重链可变区VH和轻链可变区VL的抗BCMA抗体，所述VH包含SEQ ID NO:27的氨基酸序列，且所述VL包含SEQ ID NO:59的氨基酸序列。本发明也提供该抗体的变体，例如在VH和/或VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在一些实施方案中，本发明提供包含重链可变区VH和轻链可变区VL的抗BCMA抗体，所述VH包含SEQ ID NO:27的氨基酸序列，且所述VL包含SEQ ID NO:71或72的氨基酸序列。本发明也提供该抗体的变体，例如在VH和/或VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在上述任一实施方案中，优选地，相对于所述的参考重链可变区氨基酸序列，本发明抗体的重链可变区在1个或多个CDR(优选全部3个CDR)区域上包含不超过10个，优选不超过5个(例如，3、2、1或0个)氨基酸改变(优选氨基酸取代，优选保守取代)。
在上述任一实施方案中，优选地，相对于所述的参考轻链可变区氨基酸序列，本发明抗体的轻链可变区VL在1个或多个CDR(优选全部3个CDR)区域上包含不超过10个，优选不超过5个(例如，3、2、1或0个)氨基酸改变(优选氨基酸取代，优选保守取代)。
抗体CDR区
“互补决定区”或“CDR区”或“CDR”(在本文中与超变区“HVR”可以互换使用)，是抗体可变区中主要负责与抗原表位结合的氨基酸区域。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。
本领域公知多种用于在一个给定的VH或VL氨基酸序列中确定其CDR序列的方案。例如，Kabat互补决定区(CDR)是基于序列变异性确定的并且是最常用的(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。而Chothia指的是结构环的位置(Chothia和Lesk，J.Mol.Biol.196:901-917(1987))。AbM HVR是Kabat HVR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用。“接触性”(Contact)HVR基于对可获得的复杂晶体结构的分析。根据不同的CDR确定方案，这些HVR中的每一个HVR/CDR的残基如下所述。
Figure PCTCN2019074419-appb-000001
HVR也可以是根据Kabat编号***位于如下Kabat残基位置的HVR序列：
VL中的位置24-36或24-34(LCDR1)，位置46-56或50-56(LCDR2)，和位置89-97或89-96位置(LCDR3)；和VH中的位置26-35或27-35B(HCDR1)，位置50-65或49-65(HCDR2)，和位置93-102、94-102或95-102(HCDR3)。
在一个实施方案中，本发明抗体的HVR是根据Kabat编号***位于如下Kabat残基位置的HVR序列：
VL中的位置24-34(LCDR1)、位置50-56(LCDR2)、和位置89-97(LCDR3)，以及VH中的位置27-35B(HCDR1)、位置50-65(HCDR2)、和位置93-102(HCDR3)。
HVR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”或“HVR”或“HVR序列”涵盖以上述任一种方式确定的HVR或CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号***(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
在一个优选的实施方案中，本发明抗体的HCDR1为根据AbM方案确定的CDR序列；而其余CDR为根据Kabat方案确定的CDR序列。在另一优选的实施方案中，本发明CDR序列如表2中所示。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，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定义的其余CDR残基可以被保守氨基酸残基替代。
在一些实施方案中，本发明的抗体包含与表1所列任一抗体的对应CDR相同的至少一个、两个、三个、四个、五个或六个CDR，或其变体。在一些实施方案中，本发明的抗体包含与表1所列任一抗体的对应重链CDR相同的至少一个、两个、或三个HCDR，或其变体。在一些实施方案中，本发明的抗体包含与表1所列任一抗体的对应轻链CDR相同的至少一个、两个、或三个HCDR，或其变体。在本文中，CDR变体是已经通过至少一个，例如1或2或3个氨基酸取代、缺失和/或***而修饰的CDR，其中包含CDR变体的抗原结合分子基本上保持包含未修饰CDR的抗原结合分子的生物学特性，例如，保持至少60％,70％,80％,90％,或100％的生物学活性(例如抗原结合能力)。可以理解，各CDR可以单独修饰或组合修饰。优选地，氨基酸修饰为氨基酸取代，尤其是保守氨基酸取代，例如表A中列出的优选保守氨基酸置换。
在一些实施方案中，本发明的抗体包含重链可变区，所述重链可变区包含重链互补决定 区3(HCDR3)，所述HCDR3：
(i)与表1列出的任一抗体的重链可变区的HCDR3相同；或
(ii)相对于(i)的HCDR3，包含至少1个且不超过5个(优选1-3个或更优选1-2个)氨基酸改变(优选取代、更优选保守取代)。
在一些实施方案中，本发明的抗体或其抗原结合片段包含重链可变区和轻链可变区，且所述抗体的重链互补决定区3(HCDR3)和轻链互补决定区3(LCDR3)：
(i)与表1列出的任一抗体的重链和轻链可变区序列的HCDR3和LCDR3相同；或
(ii)相对于(i)的HCDR3和LCDR3，共包含至少1个且不超过5个(优选1-3个或更优选1-2个)氨基酸改变(优选取代、更优选保守取代)。
在一些实施方案中，本发明抗体或其抗原结合片段包含重链可变区，所述重链可变区的HCDR1、HCDR2、和HCDR3：
(i)与表1列出的任一抗体的重链可变区的HCDR1、HCDR2、和HCDR3分别相同；或
(ii)相对于(i)的HCDR1、HCDR2和HCDR3，共包含至少1个且不超过10，优选不超过5个(优选1、2或3个)氨基酸改变(优选取代、更优选保守取代)，且优选地在HCDR3区上的氨基酸改变不超过3个(例如，2、1或0个)。
在一些实施方案中，本发明抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1、HCDR2和HCDR3，且所述轻链可变区包含轻链互补决定区LCDR1、LCDR2和LCDR3，其中所述HCDR1、HCDR2、HCDR3和LCDR3：
(i)与表1列出的任一抗体的重链和轻链可变区的HCDR1、HCDR2、HCDR3和LCDR3分别相同；或
(ii)相对于(i)的HCDR1、HCDR2、HCDR3和LCDR3，共包含至少1个且不超过10个(优选1-5个，优选1、2或3个)氨基酸改变(优选取代、更优选保守取代)。
在一些实施方案中，本发明抗体或其抗原结合片段包含轻链可变区，所述轻链可变区包含轻链互补决定区LCDR1、LCDR2和LCDR3，其中所述LCDR1、LCDR2和LCDR3：
(i)与表1列出的任一抗体的轻链可变区的LCDR1、LCDR2和LCDR3分别相同；或
(ii)相对于(i)的LCDR1、LCDR2和LCDR3，共包含至少1个且不超过10个(优选1-5个，优选1、2或3个)氨基酸改变(优选取代、更优选保守取代)。
在一些实施方案中，本发明抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1、HCDR2和HCDR3，且所述轻链可变区包含轻链互补决定区LCDR1、LCDR2和LCDR3，其中所述LCDR1、LCDR2、LCDR3和HCDR3：
(i)与表1列出的任一抗体的轻链和重链可变区的LCDR1、LCDR2、LCDR3和HCDR3分别相同；或
(ii)相对于(i)的LCDR1、LCDR2、LCDR3和HCDR3，共包含至少1个且不超过10个(优选1-5个，优选1、2或3个)氨基酸改变(优选取代、更优选保守取代)。
在一些实施方案中，本发明抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1、HCDR2和HCDR3，且所述轻链可变区包含轻链互补决定区LCDR1、LCDR2和LCDR3，其中所述抗体：
(i)包含表1所列任一抗体的重链和轻链可变区的全部6个CDR区的序列；或
(ii)相对于表1所列任一抗体，在全部6个CDR区上包含不超过10个，优选不超过5个(例如，3、2、1或0个)氨基酸改变(优选氨基酸取代，优选保守取代)。
在一个实施方案中，本发明抗体或其抗原结合片段包含：
(i)如SEQ ID NO:4或5所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:31或41或42所示的轻链可变区的LCDR1、2和3序列，或者
(ii)如SEQ ID NO:10所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:46所示的轻链可变区的LCDR1、2和3序列，或者
(iii)如SEQ ID NO:16或17所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:50或58所示的轻链可变区的LCDR1、2和3序列，或者
(iv)如SEQ ID NO:23所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:64所示的轻链可变区的LCDR1、2和3序列，或者
(v)如SEQ ID NO:27所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:59或71或72所示的轻链可变区的LCDR1、2和3序列。
在一个优选的实施方案中，本发明抗体或抗原结合片段包含选自以下的重链可变区(VH)和轻链可变区(VL)的HCDR1、2和3序列和LCDR1、2和3序列：
(i)SEQ ID NO:4的VH和SEQ ID NO:31的VL；
(i)SEQ ID NO:5的VH和SEQ ID NO:41的VL；
(iii)SEQ ID NO:5的VH和SEQ ID NO:42的VL；
(iv)SEQ ID NO:10的VH和SEQ ID NO:46的VL；
(v)SEQ ID NO:16的VH和SEQ ID NO:50的VL；
(vi)SEQ ID NO:17的VH和SEQ ID NO:58的VL；
(vii)SEQ ID NO:23的VH和SEQ ID NO:64的VL；
(viii)SEQ ID NO:27的VH和SEQ ID NO:59的VL；
(ix)SEQ ID NO:27的VH和SEQ ID NO:71的VL；
(x)SEQ ID NO:27的VH和SEQ ID NO:72的VL。
在一些实施方案中，本发明提供选自SEQ ID NO:3、9、13、14、15、22、和26的HCDR3、和包含该HCDR3的抗体或抗原结合片段。
在一些实施方案中，本发明提供选自以下的HCDR3和LCDR3序列组合、和包含该组合的抗体或抗原结合片段：(i)SEQ ID NO:3的HCDR3，和选自SEQ ID NO:30、38、39和40的LCDR3；或(ii)SEQ ID NO:9的HCDR3，和SEQ ID NO:45的LCDR3；或(iii)选自SEQ ID NO:13、14和15的HCDR3，和选自SEQ ID NO:49和55的LCDR3；或(iv)SEQ ID NO:22的HCDR3，和SEQ ID NO:63的LCDR3；或(v)SEQ ID NO:26的HCDR3，和选自SEQ ID NO:56、68、69、和70的LCDR3。本发明也提供所述CDR组合的变体，例如在所述CDR上共包含至少一个且不超过20、10或5个氨基酸改变(优选氨基酸取代，优选保守取代)的变体。本发明也提供包含所述变体的抗BCMA抗体或抗原结合片段。
在另一些实施方案中，本发明提供选自以下的CDR序列组合、以及包含该组合的抗体或抗原结合片段：(i)SEQ ID NO:1的HCDR1、SEQ ID NO:2的HCDR2、和SEQ ID NO:3的HCDR3；(ii)SEQ ID NO:7的HCDR1、SEQ ID NO:8的HCDR2、和SEQ ID NO:9的HCDR3；(iii)SEQ ID NO:11的HCDR1、SEQ ID NO:12的HCDR2、和SEQ ID NO:13或14或15的HCDR3；(iv)SEQ ID NO:20的HCDR1、SEQ ID NO:21的HCDR2、和SEQ ID NO:22的HCDR3；(v)SEQ ID NO:24的HCDR1、SEQ ID NO:25的HCDR2、和SEQ ID NO:26的HCDR3。在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:11的HCDR1、SEQ ID NO: 12的HCDR2、和SEQ ID NO:13或14的HCDR3。
在再一实施方案中，本发明提供选自以下氨基酸序列组合的重链CDR组合(按顺序分别为HCDR1、HCDR2和HCDR3)：SEQ ID NOs：1/2/3,7/8/9,11/12/13,11/12/14,20/21/22,和24/25/26。本发明也提供所述重链CDR组合的变体，在一个优选实施方案中，所述变体在所述三个CDR区上共包含至少一个且不超过20、10或5个氨基酸改变(优选氨基酸取代，优选保守取代)。本发明也提供包含所述重链CDR组合或所述变体的抗BCMA抗体。
在一些实施方案中，本发明提供选自以下的CDR组合、以及包含该组合的抗体或抗原结合片段：(i)SEQ ID NO:28的LCDR1、SEQ ID NO:29的LCDR2、和SEQ ID NO:30的LCDR3；(ii)SEQ ID NO:32或33或34的LCDR1、SEQ ID NO:35或36或37的LCDR2、和SEQ ID NO:38或39或40的LCDR3；(iii)SEQ ID NO:32的LCDR1、SEQ ID NO:44的LCDR2、和SEQ ID NO:45的LCDR3；(iv)SEQ ID NO:47的LCDR1、SEQ ID NO:48的LCDR2、和SEQ ID NO:49的LCDR3；(v)SEQ ID NO:51的LCDR1、SEQ ID NO:54的LCDR2、和SEQ ID NO:55的LCDR3；(vi)SEQ ID NO:61的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:63的LCDR3；(vii)SEQ ID NO:52的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:56的LCDR3；(viii)SEQ ID NO:65或66或67的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:68或69或70的LCDR3。
在再一实施方案中，本发明提供具有选自以下氨基酸序列组合的轻链CDR组合(按顺序分别为LCDR1、LCDR2和LCDR3)：SEQ ID NOs：28/29/30,32/35/38,33/36/39,32/44/45,47/48/49,51/54/55,61/62/63,52/62/56,65/62/68,和66/62/69。本发明也提供所述轻链CDR组合的变体，在一个优选实施方案中，所述变体在所述三个CDR区上共包含至少一个且不超过20、10或5个氨基酸改变(优选氨基酸取代，优选保守取代)。本发明也提供包含所述轻链CDR组合或所述变体的抗BCMA抗体或抗原结合片段。
在一些实施方案中，本发明提供选自以下的CDR组合、以及包含该组合的抗体或抗原结合片段：(i)SEQ ID NO:1的HCDR1、SEQ ID NO:2的HCDR2、和SEQ ID NO:3的HCDR3、SEQ ID NO:28的LCDR1、SEQ ID NO:29的LCDR2、和SEQ ID NO:30的LCDR3；(ii)SEQ ID NO:1的HCDR1、SEQ ID NO:2的HCDR2、和SEQ ID NO:3的HCDR3、SEQ ID NO:32或33或34的LCDR1、SEQ ID NO:35或36或37的LCDR2、和SEQ ID NO:38或39或40的LCDR3；(iii)SEQ ID NO:7的HCDR1、SEQ ID NO:8的HCDR2、和SEQ ID NO:9的HCDR3、SEQ ID NO:32的LCDR1、SEQ ID NO:44的LCDR2、和SEQ ID NO:45的LCDR3；(iv)SEQ ID NO:11的HCDR1、SEQ ID NO:12的HCDR2、和SEQ ID NO:13或14或15的HCDR3、SEQ ID NO:47的LCDR1、SEQ ID NO:48的LCDR2、和SEQ ID NO:49的LCDR3；(v)SEQ ID NO:11的HCDR1、SEQ ID NO:12的HCDR2、和SEQ ID NO:13或14或15的HCDR3、SEQ ID NO:51的LCDR1、SEQ ID NO:54的LCDR2、和SEQ ID NO:55的LCDR3；(vi)SEQ ID NO:20的HCDR1、SEQ ID NO:21的HCDR2、和SEQ ID NO:22的HCDR3、SEQ ID NO:61的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:63的LCDR3；(vii)SEQ ID NO:24的HCDR1、SEQ ID NO:25的HCDR2、和SEQ ID NO:26的HCDR3、SEQ ID NO:52的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:56的LCDR3；(viii)SEQ ID NO:24的HCDR1、SEQ ID NO:25的HCDR2、和SEQ ID NO:26的HCDR3、SEQ ID NO:65或66或67的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:68或69或70的LCDR3。
在再一实施方案中，本发明提供选自以下氨基酸序列组合的重链和轻链CDR组合(按顺序分别为HCDR1、HCDR2和HCDR3、LCDR1、LCDR2和LCDR3)：SEQ ID NOs：1/2/3/28/29/30, 1/2/3/32/35/38,1/2/3/33/36/39,7/8/9/32/44/45,11/12/13/47/48/49,11/12/14/51/54/55,20/21/22/61/62/63,24/25/26/52/62/56,24/25/26/65/62/68,和24/25/26/66/62/69。本发明也提供所述CDR组合的变体，在一个优选实施方案中，所述变体在所述六个CDR区上共包含至少一个且不超过20、10或5个氨基酸改变(优选氨基酸取代，优选保守取代)。本发明也提供包含所述重链和轻链CDR组合或所述变体的抗BCMA抗体或抗原结合片段。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:1的HCDR1、SEQ ID NO:2的HCDR2、和SEQ ID NO:3的HCDR3、SEQ ID NO:28的LCDR1、SEQ ID NO:29的LCDR2、和SEQ ID NO:30的LCDR3。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:1的HCDR1、SEQ ID NO:2的HCDR2、和SEQ ID NO:3的HCDR3、SEQ ID NO:32的LCDR1、SEQ ID NO:35的LCDR2、和SEQ ID NO:38的LCDR3。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:1的HCDR1、SEQ ID NO:2的HCDR2、和SEQ ID NO:3的HCDR3、SEQ ID NO:33的LCDR1、SEQ ID NO:36的LCDR2、和SEQ ID NO:39的LCDR3。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:7的HCDR1、SEQ ID NO:8的HCDR2、和SEQ ID NO:9的HCDR3、SEQ ID NO:32的LCDR1、SEQ ID NO:44的LCDR2、和SEQ ID NO:45的LCDR3。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:11的HCDR1、SEQ ID NO:12的HCDR2、和SEQ ID NO:13的HCDR3、SEQ ID NO:47的LCDR1、SEQ ID NO:48的LCDR2、和SEQ ID NO:49的LCDR3。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:11的HCDR1、SEQ ID NO:12的HCDR2、和SEQ ID NO:14的HCDR3、SEQ ID NO:51的LCDR1、SEQ ID NO:54的LCDR2、和SEQ ID NO:55的LCDR3。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:20的HCDR1、SEQ ID NO:21的HCDR2、和SEQ ID NO:22的HCDR3、SEQ ID NO:61的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:63的LCDR3。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:24的HCDR1、SEQ ID NO:25的HCDR2、和SEQ ID NO:26的HCDR3、SEQ ID NO:52的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:56的LCDR3。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:24的HCDR1、SEQ ID NO:25的HCDR2、和SEQ ID NO:26的HCDR3、SEQ ID NO:65的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:68的LCDR3。
在一些实施方案中，本发明抗体或其抗原结合片段包含SEQ ID NO:24的HCDR1、SEQ ID NO:25的HCDR2、和SEQ ID NO:26的HCDR3、SEQ ID NO:66的LCDR1、SEQ ID NO:62的LCDR2、和SEQ ID NO:69的LCDR3。
在一些实施方案中，SEQ ID NO:15中的X代表任何氨基酸残基，优选在SEQ ID NO:13或14的相应位置处的氨基酸残基或其保守取代残基，优选S或R或其保守取代残基。在一些实施方案中，SEQ ID NO:34中的X代表任何氨基酸残基，优选地在SEQ ID NO:32或33的相应位置处的氨基酸残基或其保守取代残基。在一些实施方案中，SEQ ID NO:37中的X代表任何氨基酸残基，优选地在SEQ ID NO:35或36的相应位置处的氨基酸残基或其保守取代残基。在一些实施方案中，SEQ ID NO:40中的X代表任何氨基酸残基，优选地在SEQ ID  NO:38或39的相应位置处的氨基酸残基或其保守取代残基。在一些实施方案中，SEQ ID NO:67中的X代表任何氨基酸残基，优选地在SEQ ID NO:65或66的相应位置处的氨基酸残基或其保守取代残基。在一些实施方案中，SEQ ID NO:70中的X代表任何氨基酸残基，优选地在SEQ ID NO:68或69的相应位置处的氨基酸残基或其保守取代残基。
在本发明抗体的上述实施方案中，“保守性取代”是指导致某个氨基酸置换为化学上相似的氨基酸的氨基酸改变。提供功能上相似氨基酸的保守性置换表是本领域熟知的。在本发明任一实施方案中，在一个优选的方面，保守取代残基来自以下的保守替代表A，优选地为表A中所示优选置换残基。
表A
原始残基 示例性取代 优选的保守氨基酸取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp；Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
示例性抗体序列
本发明提供了如实施例中分离并表征的特异性结合BCMA(例如人BCMA)的全人源抗体。下表1中列出了本发明这些示例性抗体的可变区序列。下表2中给出这些抗体的示例性CDR序列(也见图4)。
表1.本发明示例性全人源抗体分子的重链可变区和轻链可变区的氨基酸序列和核苷酸序列。
抗体名称 VH VH DNA VL VL DNA
ADI-34848 SEQ ID NO:4 SEQ ID NO:75 SEQ ID NO:31 SEQ ID NO:83
ADI-34849 SEQ ID NO:5 SEQ ID NO:76 SEQ ID NO:41 SEQ ID NO:84
ADI-34850 SEQ ID NO:5 SEQ ID NO:76 SEQ ID NO:42 SEQ ID NO:85
ADI-34854 SEQ ID NO:10 SEQ ID NO:77 SEQ ID NO:46 SEQ ID NO:86
ADI-34846 SEQ ID NO:16 SEQ ID NO:78 SEQ ID NO:50 SEQ ID NO:87
ADI-34857 SEQ ID NO:17 SEQ ID NO:79 SEQ ID NO:58 SEQ ID NO:88
ADI-34832 SEQ ID NO:23 SEQ ID NO:81 SEQ ID NO:64 SEQ ID NO:90
ADI-34859 SEQ ID NO:27 SEQ ID NO:80 SEQ ID NO:59 SEQ ID NO:89
ADI-34860 SEQ ID NO:27 SEQ ID NO:82 SEQ ID NO:71 SEQ ID NO:91
ADI-34861 SEQ ID NO:27 SEQ ID NO:82 SEQ ID NO:72 SEQ ID NO:92
表2.示例性重链CDR和轻链CDR的氨基酸序列
抗体名称 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3
ADI-34848 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30
ADI-34849 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:32 SEQ ID NO:35 SEQ ID NO:38
ADI-34850 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:33 SEQ ID NO:36 SEQ ID NO:39
ADI-34854 SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:32 SEQ ID NO:44 SEQ ID NO:45
ADI-34846 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:13 SEQ ID NO:47 SEQ ID NO:48 SEQ ID NO:49
ADI-34857 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:14 SEQ ID NO:51 SEQ ID NO:54 SEQ ID NO:55
ADI-34832 SEQ ID NO:20 SEQ ID NO:21 SEQ ID NO:22 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63
ADI-34859 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:52 SEQ ID NO:62 SEQ ID NO:56
ADI-34860 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:65 SEQ ID NO:62 SEQ ID NO:68
ADI-34861 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:66 SEQ ID NO:62 SEQ ID NO:69
本发明也提供上述抗体的变体。在一个实施方案中，抗体的氨基酸序列或编码氨基酸序列的核酸已经被突变，但仍与表1中描述的序列具有至少60％、65％、70％、75％、80％、85％、90％或95％或更高同一性。一些实施方案，抗体包括突变的可变区氨基酸序列，其中与表1所示的可变区序列相比时，可变区中已突变了不多于1、2、3、4、5或10个氨基酸，但保留基本相同的抗原结合活性。
此外，由于上述抗体每一个都可以与BCMA结合，故可以“混合并匹配”VH和VL(氨基酸序列和编码所述氨基酸序列的核苷酸序列)以产生结合BCMA的本发明其他抗体。可以使用本领域已知的结合测定法(例如，ELISA，和实施例部分中描述的其他测定法)测试这类“混合和匹配的”抗体与BCMA的结合。在混合和匹配这些链时，优选地，将来自具体VH/VL配对的VH序列替换为结构相似的VH序列。同样，来自特定VH/VL配对的VL序列优选地替换为结构上相似的VL序列。
在另一方面，本发明也提供上述抗体的变体。在一个实施方案中，抗体在一个或多个或全部6个CDR区的氨基酸序列或编码该氨基酸序列的核酸上已经被突变。在一些实施方案中，突变的CDR区的氨基酸序列，与表1的对应CDR区相比时，已突变了不多于1、2、3、4或5个氨基酸，但保留基本相同的抗原结合活性。
此外，鉴于表1抗体的每一者均可以与BCMA结合且抗原结合特异性主要由CDR1、2和3区提供，故可以将VH CDR1、2和3序列和VL CDR1、2和3序列“混合并匹配”(即，可以混合并匹配来自不同抗体的CDR，不过每种抗体优选地含有VH CDR1、2和3和VL CDR1、2和3)，以产生结合BCMA的本发明其他分子。可以使用本领域已知的结合测定法(例如，ELISA、SET、Biacore)和实施例中描述的那些测定法，测试这类“混合和匹配的”抗体与BCMA的结合。当混合并匹配VH CDR序列时，来自特定VH序列的CDR1、CDR2和/或CDR3序列优选地替换为结构上相似的CDR序列。同样，当混合并匹配VL CDR序列时，来自特定VL序列的CDR1、CDR2和/或CDR3序列优选地替换为结构上相似的CDR序列。本领域技术人员明了，也可以通过将一个或多个VH和/或VL CDR区序列置换为来自本文中 所示抗体的结构上相似的CDR序列，以产生本发明的其它抗体。除前述之外，在一个实施方案中，本文所述抗体的抗原结合片段可以包含VH CDR1、2和3，或VL CDR1、2和3，其中该片段以单域形式与BCMA结合。
II.单链scFv抗体
在一个优选方面，本发明抗体是单链scFv抗体。
在本文中，“单链scFv抗体”或“scFv”或“单链scFv”是指，包含免疫球蛋白或抗体的重链可变区(VH)和轻链可变区(VL)的单个多肽链，在该单条蛋白链中VH区和VL区配对提供抗原结合位点。
在优选的实施方案中，本发明单链scFv抗体的VH区和VL区通过连接肽例如柔性连接肽共价连接在一起。术语“柔性连接肽”是由氨基酸组成的肽接头。通过这样的肽接头可以连接抗体中的各个可变结构域，例如VH和VL区。肽接头通常富含表现柔性的甘氨酸以及表现溶解性的丝氨酸或苏氨酸。例如可以单独或组合使用甘氨酸和/或丝氨酸残基。柔性连接肽或肽接头的非限定性例子公开于Shen等人，Anal.Chem.80(6):1910-1917(2008)、WO2012/138475和WO2014/087010，将其内容全文并入作为参考。如本领域已知的，在scFv的构建中，优选，接头将利于促使VH和VL配对，且不干扰VH和VL对形成功能有效的抗原结合位点。
在一些实施方案中，本发明scFv单链抗体包含由肽键连接的氨基酸残基组成的柔性连接肽或肽接头。在某些实施方案中，所述氨基酸选自二十种天然氨基酸。在某些其他实施方案中，一个或多个氨基酸选自甘氨酸、丝氨酸、苏氨酸、丙氨酸、脯氨酸、天冬酰胺、谷氨酰胺和赖氨酸。在一个优选实施方案中，一个或多个氨基酸选自Gly,Ser,Thr,Lys,Pro,和Glu。
在一些实施方案中，接头的长度是约1-30个氨基酸、或约10个至约25个氨基酸、约15个至约20个氨基酸或约10个至约20个氨基酸或者任意介于中间的氨基酸长度。在优选实施方案中，接头具有15-25个氨基酸残基长度，在更优选实施方案中，具有15-18个氨基酸残基的长度。在一些实施方案中，接头的长度是10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25个或者更多个氨基酸。
可以用于本发明的肽接头的实例包括：甘氨酸聚合物(G)n；甘氨酸-丝氨酸聚合物(G 1-5S 1-5)n，其中n是至少1、2、3、4或5的整数；甘氨酸-丙氨酸聚合物；丙氨酸-丝氨酸聚合物；以及本领域已知的其它柔性接头。本领域技术人员可以理解，在一些实施方案中，VH和VL之间的接头可以完全由柔性连接肽组成，或者接头可以由柔性连接肽部分以及赋予较小柔性结构的一个或多个部分组成。
在一个优选实施方案中，肽接头是GSTSGSGKPGSGEGSTKG(SEQ ID NO:93)。在一个实施方案中，编码氨基酸序列SEQ ID NO:93的核苷酸序列在SEQ ID NO:94中给出。在一个实施方案中，编码氨基酸序列SEQ ID NO:93的核苷酸序列在SEQ ID NO:97中给出。
在一个实施方案中，肽接头是Gly/Ser连接肽。在一个实施方案中，肽接头是(GxS)n接头，其中G＝甘氨酸、S＝丝氨酸，(x＝3，n＝8、9或10)或(x＝4和n＝6、7或8)，在一个实施方案中，x＝4，n＝6或7。在一些实施方案中，接头可以包括氨基酸序列(G 4S)n，其中n是等于或大于1的整数，例如，n是1－7的整数。在一个优选实施方案中，x＝4，n＝7。在一个实施方案中，接头是(G4S)3。在一个实施方案中，接头是(G4S)4。在一个实施方案中，接头是(G4S)6G2。
其它示例性接头包括但不限于下述氨基酸序列：GGG；DGGGS；TGEKP(参见，例如， Liu等人,PNAS5525-5530(1997))；GGRR(Pomerantz等人.1995,同上)；(GGGGS)n，其中n＝1、2、3、4或5(Kim等人,PNAS 93,1156-1160(1996)；EGKSSGSGSESKVD(Chaudhary等人,1990,Proc.Natl.Acad.Sci.U.S.A.87:1066-1070)；KESGSVSSEQLAQFRSLD(Bird等人,1988,Science242:423-426)，GGRRGGGS；LRQRDGERP；LRQKDGGGSERP；LRQKD(GGGS)2ERP。可选地，可以使用能够模建DNA-结合位点和肽自身的计算机程序(Desjarlais&Berg,PNAS90:2256-2260(1993),PNAS91:11099-11103(1994))，或者通过噬菌体或酵母展示方法，合理地设计柔性接头。
本发明单链scFv抗体中VH和VL可以取任一方向。在一些实施方案中，scFv从N端到C端包含：VH-接头-VL；或VL-接头-VH。在一个优选实施方案中，本发明单链scFv抗体从N端到C端包含：VL-接头-VH。在一个优选实施方案中，VL以其C末端经由接头共价连接VH的N末端。
在一些实施方案中，除了接头，VL和VH结构域之间也可以***具有特定功能的其它多肽片段，例如具有调节免疫反应功能的多肽片段、或具有引起细胞溶剂或细胞杀伤的多肽片段。
在一些实施方案中，可以通过在scFv中引入二硫键以稳定单链抗体。例如，可以通过引入链内或链间二硫键而连接scFv的VH和VL的构架区。在一个实施方案中，可以通过将抗体VH和VL的各1个氨基酸残基突变为半胱氨酸，例如根据Kabat编号***，VH的44位和VL的100位，或者VH的105位和VL的43位。
本发明的单链scFv多肽抗体可以由包括VH-和VL-编码序列的核酸表达，如Huston等人所述(Proc.Nat.Acad.Sci.USA，85：5879～5883，1988)。还可参见美国专利号5,091,513、5,132,405和4,956,778；以及美国专利公开号20050196754和20050196754。在一些实施方案中，本发明的单链scFv抗体在真核细胞中表达，例如酵母细胞、哺乳动物细胞如H293细胞或CHO细胞。在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:99的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:99具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:100的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:4的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:31的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:4的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:31的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:4所示序列的VH和包括具有SEQ ID NO:31所示序列的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:4所示序列的VH的3个HCDR序列和/或SEQ ID NO:31所示序列的VL的3个LCDR序列。在一些实施方案中，抗BCMA scFv包含SEQ ID NO:3的HCDR3和SEQ ID NO:30的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:1的VH CDR1、SEQ ID NO:2的VH CDR2、和SEQ ID NO:3的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:28的VL CDR1、SEQ ID NO:29的VL CDR2、和SEQ ID NO:30的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:1 的VH CDR1、SEQ ID NO:2的VH CDR2、和SEQ ID NO:3的VH CDR3，以及SEQ ID NO:28的VL CDR1、SEQ ID NO:29的VL CDR2、和SEQ ID NO:30的VL CDR3。
在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:102的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:102具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:103的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:5的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:41的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:5的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:41的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:5所示序列的氨基酸的VH和包括具有SEQ ID NO:41所示序列的氨基酸的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:5所示序列的VH的3个HCDR序列和/或SEQ ID NO:41所示序列的VL的3个LCDR序列。在一些实施方案中，抗BCMA scFv包含SEQ ID NO:3的HCDR3和SEQ ID NO:38的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:1的VH CDR1、SEQ ID NO:2的VH CDR2、和SEQ ID NO:3的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:32的VL CDR1、SEQ ID NO:35的VL CDR2、和SEQ ID NO:38的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:1的VH CDR1、SEQ ID NO:2的VH CDR2、和SEQ ID NO:3的VH CDR3，以及SEQ ID NO:32的VL CDR1、SEQ ID NO:35的VL CDR2、和SEQ ID NO:38的VL CDR3。
在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:105的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:105具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:106的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:5的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:42的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:5的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:42的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:5所示的氨基酸序列的VH和包括具有SEQ ID NO:42所示的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:5所示序列的VH的3个HCDR序列和/或SEQ ID NO:42所示序列的VL的3个LCDR序列。在一些实施 方案中，抗BCMA scFv包含SEQ ID NO:3的HCDR3和SEQ ID NO:39的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:1的VH CDR1、SEQ ID NO:2的VH CDR2、和SEQ ID NO:3的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:33的VL CDR1、SEQ ID NO:36的VL CDR2、和SEQ ID NO:39的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:1的VH CDR1、SEQ ID NO:2的VH CDR2、和SEQ ID NO:3的VH CDR3、以及SEQ ID NO:33的VL CDR1、SEQ ID NO:36的VL CDR2、和SEQ ID NO:39的VL CDR3。
在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:108的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:108具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:109的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:10的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:46的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:10的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:46的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:10所示序列的氨基酸的VH和包括具有SEQ ID NO:46所示序列的氨基酸的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:10所示序列的VH的3个HCDR序列和/或SEQ ID NO:46所示序列的VL的3个LCDR序列。在一些实施方案中，抗BCMA scFv包含SEQ ID NO:9的HCDR3和SEQ ID NO:45的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:7的VH CDR1、SEQ ID NO:8的VH CDR2、和SEQ ID NO:9的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:32的VL CDR1、SEQ ID NO:44的VL CDR2、和SEQ ID NO:45的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:7的VH CDR1、SEQ ID NO:8的VH CDR2、和SEQ ID NO:9的VH CDR3、以及SEQ ID NO:32的VL CDR1、SEQ ID NO:44的VL CDR2、和SEQ ID NO:45的VL CDR3。
在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:111的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:111具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:112的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:16的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:50的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:16的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:50的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:16所示序列的氨基酸的VH和包括具有SEQ ID NO:50所示序列的氨基酸的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:16所示序列的VH的3个HCDR序列和/或SEQ ID NO:50所示序列的VL的3个LCDR序列。在一些实施方案中，抗BCMA scFv包含SEQ ID NO:13的HCDR3和SEQ ID NO:49的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:11的VH CDR1、SEQ ID NO:12的VH CDR2、和SEQ ID NO:13的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:47的VL CDR1、SEQ ID NO:48的VL CDR2、和SEQ ID NO:49的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:11的VH CDR1、SEQ ID NO:12的VH CDR2、和SEQ ID NO:13的VH CDR3、以及SEQ ID NO:47的VL CDR1、SEQ ID NO:48的VL CDR2、和SEQ ID NO:49的VL CDR3。
在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:114的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:114具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:115的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:17的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:58的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:17的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:58的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:17所示序列的氨基酸的VH和包括具有SEQ ID NO:58所示序列的氨基酸的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:17所示序列的VH的3个HCDR序列和/或SEQ ID NO:58所示序列的VL的3个LCDR序列。在一些实施方案中，抗BCMA scFv包含SEQ ID NO:14的HCDR3和SEQ ID NO:55的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:11的VH CDR1、SEQ ID NO:12的VH CDR2、和SEQ ID NO:14的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:51的VL CDR1、SEQ ID NO:54的VL CDR2、和SEQ ID NO:55的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:11的VH CDR1、SEQ ID NO:12的VH CDR2、和SEQ ID NO:14的VH CDR3、以及SEQ ID NO:51的VL CDR1、SEQ ID NO:54的VL CDR2、和SEQ ID NO:55的VL CDR3。
在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:120的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:120具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:121的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:23的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:64的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:23的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:64的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:23所示序列的氨基酸的VH和包括具有SEQ ID NO:64所示序列的氨基酸的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:23所示序列的VH的3个HCDR序列和/或SEQ ID NO:64所示序列的VL的3个LCDR序列。在一些实施方案中，抗BCMA scFv包含SEQ ID NO:22的HCDR3和SEQ ID NO:63的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:20的VH CDR1、SEQ ID NO:21的VH CDR2、和SEQ ID NO:22的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:61的VL CDR1、SEQ ID NO:62的VL CDR2、和SEQ ID NO:63的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:20的VH CDR1、SEQ ID NO:21的VH CDR2、和SEQ ID NO:22的VH CDR3、以及SEQ ID NO:61的VL CDR1、SEQ ID NO:62的VL CDR2、和SEQ ID NO:63的VL CDR3。
在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:117的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:117具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:118的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:27的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:59的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:27的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:59的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:27所示序列的氨基酸的VH和包括具有SEQ ID NO:59所示序列的氨基酸的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:27所示序列的VH的3个HCDR序列和/或SEQ ID NO:59所示序列的VL的3个LCDR序列。在一些实施方案中，抗BCMA scFv包含SEQ ID NO:26的HCDR3和SEQ ID NO:56的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:24的VH CDR1、SEQ ID NO:25的VH CDR2、和SEQ ID NO:26的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:52的VL CDR1、SEQ ID NO:62的VL CDR2、和SEQ ID NO:56的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:24的VH CDR1、SEQ ID NO:25的VH CDR2、和SEQ ID NO:26的VH CDR3、以及SEQ ID NO:52的VL CDR1、SEQ ID NO:62的VL CDR2、和SEQ ID NO:56的VL CDR3。
在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:123的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:123具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:124的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:27的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:71的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:27的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:71的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:27所示序列的氨基酸的VH和包括具有SEQ ID NO:71所示序列的氨基酸的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:27所示序列的VH的3个HCDR序列和/或SEQ ID NO:71所示序列的VL的3个LCDR序列。在一些实施方案中，抗BCMA scFv包含SEQ ID NO:26的HCDR3和SEQ ID NO:68的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:24的VH CDR1、SEQ ID NO:25的VH CDR2、和SEQ ID NO:26的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:65的VL CDR1、SEQ ID NO:62的VL CDR2、和SEQ ID NO:68的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:24的VH CDR1、SEQ ID NO:25的VH CDR2、和SEQ ID NO:26的VH CDR3、以及SEQ ID NO:65的VL CDR1、SEQ ID NO:62的VL CDR2、和SEQ ID NO:68的VL CDR3。
在某些实施方式中，本发明的抗体是抗BCMA scFv或其抗原结合片段，其包含氨基酸序列SEQ ID NO:126的抗原结合区或其变体，并特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)。在一些实施方案中，变体与SEQ ID NO:126具有至少90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，抗BCMA scFv由SEQ ID NO:127的核苷酸编码。
在某些实施方式中，抗BCMA scFv抗体包括：包含SEQ ID NO:27的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的重链可变区，和包含SEQ ID NO:72的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的的轻链可变区，以及任选地在重链可变区和轻链可变区之间的接头，例如接头肽。在某些实施方式中，接头包含SEQ ID NO:93的氨基酸序列。
在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:27的氨基酸序列的VH。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:72的氨基酸序列的VL。在某些实施方式中，抗BCMA scFv包括具有SEQ ID NO:27所示序列的氨基酸的VH和包括具有SEQ ID NO:72所示序列的氨基酸的VL。在某些实施方式中，抗BCMA scFv包括SEQ ID NO:27所示序列的VH的3个HCDR序列和/或SEQ ID NO:72所示序列的VL的3个LCDR序列。在一些实施方案中，抗BCMA scFv包含SEQ ID NO:26的HCDR3和SEQ ID NO:69的LCDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:24的VH CDR1、SEQ ID NO:25的VH CDR2、和SEQ ID NO:26的VH CDR3。在某些实施方式中，抗BCMA scFv包括：SEQ ID NO:66 的VL CDR1、SEQ ID NO:62的VL CDR2、和SEQ ID NO:69的VL CDR3。在某些实施方式中，抗BCMA scFv包含：SEQ ID NO:24的VH CDR1、SEQ ID NO:25的VH CDR2、和SEQ ID NO:26的VH CDR3、以及SEQ ID NO:66的VL CDR1、SEQ ID NO:62的VL CDR2、和SEQ ID NO:69的VL CDR3。
III.scFv-Fc抗体
具有Fc区的抗体具有若干优势，包括，但不限于：可以通过Fc区介导效应子功能，例如CDC和ADCC免疫学活性；通过Fc区的二聚化功能形成二价抗体，可以提供强的抗原结合亲和力，和/或改变血浆半衰期和肾脏清除率；二价抗体可以以不同于单价Fab或scFv抗体的速率内化，改变免疫功能或载体功能。例如，α发射体不需要内化来杀伤靶细胞，但许多药物和毒素将受益于免疫复合物的内化。
因此，在一个优选的实施方案中，提供本发明单链scFv抗体与抗体Fc区融合形成的scFv-Fc抗体。在一些实施方案中，所述抗体包含本发明的单链scFv抗体和野生型或改变的Fc区。在一个优选实施方案中，所述抗体从N端到C端包含：Fc-VH-接头-VL或Fc-VL-接头-VH；或优选地VH-接头-VL-Fc或VL-接头-VH-Fc。在一个优选实施方案中，Fc通过铰链区连接到可变区(VH或VL)上。在一些实施方案中，Fc为来自人免疫球蛋白的Fc区，优选人IgG1或IgG4Fc区。在一个优选实施方案中，Fc区具有SEQ ID NO:132所示的氨基酸序列、或相对于SEQ ID NO:132的氨基酸序列包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与SEQ ID NO:132的氨基酸序列具有至少95-99％同一性的序列。在一些实施方案中，本发明的单链scFv抗体通过铰链区连接到Fc区上。在一个实施方案中，铰链区为C8铰链区，例如包含SEQ ID NO:95所示的氨基酸序列、或相对于SEQ ID NO:95的氨基酸序列包含至少一个，两个或三个，但不超过5个氨基酸改变的氨基酸序列。
在一些优选的实施方案中，本发明提供这样的抗体，其特异性结合BCMA多肽(例如，具有氨基酸序列SEQ ID NO:74的BCMA多肽或其片段)，并且包含选自SEQ ID NO:101、104、107、110、113、116、119、122、125、和128的氨基酸序列、或相对于其包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列。
下表3中列出了本发明一些示例性ScFv-Fc抗体的氨基酸序列及用于构建其的单链scFv的氨基酸序列和核苷酸序列。表3也显示了基于US20170226216A1的描述构建的参照scFv-Fc重组单链抗体的氨基酸序列和核苷酸序列。这些scFv-Fc抗体中所用的接头和铰链的氨基酸序列和核苷酸序列示于图8中。
表3：
抗体名称 所用scFv的氨基酸序列 所用scFv的DNA序列 scFv-hFc的氨基酸序列
ADI-34848 scFv-hFc SEQ ID NO:99 SEQ ID NO:100 SEQ ID NO:101
ADI-34849 scFv-hFc SEQ ID NO:102 SEQ ID NO:103 SEQ ID NO:104
ADI-34850 scFv-hFc SEQ ID NO:105 SEQ ID NO:106 SEQ ID NO:107
ADI-34854 scFv-hFc SEQ ID NO:108 SEQ ID NO:109 SEQ ID NO:110
ADI-34846 scFv-hFc SEQ ID NO:111 SEQ ID NO:112 SEQ ID NO:113
ADI-34857 scFv-hFc SEQ ID NO:114 SEQ ID NO:115 SEQ ID NO:116
ADI-34859 scFv-hFc SEQ ID NO:117 SEQ ID NO:118 SEQ ID NO:119
ADI-34832 scFv-hFc SEQ ID NO:120 SEQ ID NO:121 SEQ ID NO:122
ADI-34860 scFv-hFc SEQ ID NO:123 SEQ ID NO:124 SEQ ID NO:125
ADI-34861 scFv-hFc SEQ ID NO:126 SEQ ID NO:127 SEQ ID NO:128
参照scFv-hFc SEQ ID NO:129 SEQ ID NO:130 SEQ ID NO:131
在一些实施方案中，本发明的scFv-Fc抗体具有效应子功能。术语“效应子功能”是指，可归因于抗体的Fc-区的那些生物活性，其随抗体类别而改变。存在五种主要的抗体类别：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可以进一步分为亚类(同种型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同类别的免疫球蛋白的重链恒定结构域分别被称为α、δ、ε、γ和μ。抗体的效应子功能包括例如但不限于：C1q结合和补体依赖性细胞毒性(CDC)；Fc受体结合；抗体依赖性的细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体)的下调；和B-细胞激活。在一些实施方案中，本发明的scFv-Fc抗体通过效应细胞介导的细胞毒性(ADCC)活性而阻断、抑制表达BCMA的细胞(尤其是MM细胞)的生长、和/或杀死所述细胞。
在某些实施方案中，Fc区可以包含具有一个或多个提高ADCC活性的氨基酸置换的Fc-区，例如，Fc-区的位置298、333和/或334的置换(残基的EU编号)。在一些实施方案中，也可以对Fc-区进行改变，以导致改变的(即，提高的或降低的)C1q结合和/或补体依赖性细胞毒性(CDC)(参见，例如，US6,194,551、WO99/51642和Idusogie，E.E.等，J.Immunol.164(2000)4178-4184)。
在另一些实施方案中，可以对Fc进行改变以增加或降低其糖基化程度和/或改变其糖基化模式。对Fc的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。举例而言，可实施一或多种氨基酸取代以消除一或多个糖基化位点，由此消除该位点处的糖基化。可制备具有改变类型的糖基化的抗体，例如具有减小量的岩藻糖基残基的低或无岩藻糖化抗体或具有增加的等分GlcNac结构的抗体。这类改变的糖基化模式已显示可增加抗体的ADCC能力。
因此，在一些优选实施方案中，本发明提供这样的抗体，其Fc区是低或无岩藻糖基化的，从而可以显著地增加抗体Fc结构域与效应细胞上表达的Fcγ受体(如FcγRIIIa)的结合亲和力，由此导致抗体具有增强的抗体依赖性细胞介导的细胞毒性(ADCC)活性。例如，抗体中岩藻糖的量可以是1％至80％、1％至65％、5％至65％或20％至40％。可以通过MALDI-TOF质谱法测量，相对于与Asn 297连接的所有糖结构(例如复合型、杂合型及高甘露糖型结构)的总和，计算在Asn297处糖链内的岩藻糖的平均量，由此确定岩藻糖的量，例如WO2008/077546中所述。Asn297是指位于Fc区中大约位置297处(Fc区残基的EU编号)的天冬酰胺残基；然而，由于抗体中微小的序列变化，Asn297也可能位于位置297上游或下游约±3个氨基酸位置，即位置294与300之间。参见例如US2003/0157108；US2004/0093621。与“去岩藻糖基化”或“低岩藻糖基化”抗体变体有关的公布实例还包括：US2003/0157108；WO 2000/61739；WO2001/29246；US2003/0115614；US2002/0164328；US 2004/0093621；US2004/0132140；US2004/0110704；US2004/0110282；US2004/0109865；WO2003/085119；WO2003/084570；WO2005/035586；WO2005/035778；WO2005/053742；WO2002/031140；Okazaki，A.等，J.Mol.Biol.336(2004)1239-1249；Yamane-Ohnuki，N.等，Biotech.Bioeng.87：614(2004)614-622。可以在能够产生去岩藻糖基化或低岩藻糖基化抗体的细胞系中产生此类抗体变体。此类细胞的实例包括蛋白质岩藻糖基化缺陷的Lec13CHO细胞(Ripka，J.等，Arch.Biochem.Biophys.249(1986)：533-545；US 2003/0157108；和WO 2004/056312，尤其是实施例11)；及基因敲除细胞系，例如α-1,6-岩藻糖基转移酶基因FUT8敲除的CHO 细胞(参见，例如Yamane-Ohnuki，N.等，Biotech.Bioeng.87：614(2004)614-622；Kanda，Y.等，Biotechnol.Bioeng.94(2006)680-688；和WO 2003/085107)。再例如，细胞系Ms704、Ms705及Ms709缺乏岩藻糖基转移酶基因FUT8(α(1,6)-岩藻糖基转移酶)，从而可以在Ms704、Ms705及Ms709细胞系中表达缺乏岩藻糖的抗体。此外，EP 1,176,195也描述了具有功能受破坏的FUT8基因的细胞系，在这类细胞系中表达的抗体展现低岩藻糖化。备选地，还可使用岩藻糖苷酶切除抗体的岩藻糖残基；举例而言，岩藻糖苷酶α-L-岩藻糖苷酶自抗体去除岩藻糖基残基(Tarentino等人(1975)Biochem.14:5516-23)。
此外，本发明也考虑具有平分型(bisected)寡糖的抗体变体，例如，其中与Fc区连接的双触角寡糖被GlcNAc平分的抗体。这些抗体变体可具有降低的岩藻糖基化和/或提高的ADCC功能。这些抗体变体的实例描述于例如WO 2003/011878；US6,602,684；和US2005/0123546。本发明也考虑在与Fc区连接的寡糖中具有至少一个半乳糖残基的抗体变体。这些抗体变体可具有提高的CDC功能。这些抗体变体描述于例如WO 1997/30087；WO 1998/58964；和WO 1999/22764。
评价目标分子的ADCC活性的体外测定试验的非限制性实例描述于US5,500,362(参见，例如Hellstrom，I.等，Proc.Nat’l Acad.Sci.USA 83(1986)7059-7063；和Hellstrom，I.等，Proc.Nat’l Acad.Sci.USA 82(1985)1499-1502)；US 5,821,337(参见Bruggemann，M.等，J.Exp.Med.166(1987)1351-1361)。或者，可采用非放射性测定方法(参见例如用于流式细胞术的ACTI TM非放射性细胞毒性测定(CellTechnology，Inc.Mountain View，CA)和
Figure PCTCN2019074419-appb-000002
非放射性细胞毒性测定(Promega，Madison，WI))。适用于这些测定的效应细胞包括外周血单核细胞(PBMC)和自然杀伤(NK)细胞。备选地或另外地，可以在体内评价目标分子的ADCC活性，例如，在如Clynes，R.等，Proc.Nat’l Acad.Sci.USA 95(1998)652-656中公开的动物模型中评价。为评价补体活化，可进行CDC测定(参见，例如Gazzano-Santoro，H.等，J.Immunol.Methods202(1996)163-171；Cragg，M.S.等，Blood101(2003)1045-1052；和Cragg，M.S.和M.J.Glennie，Blood 103(2004)2738-2743)。也可进行C1q结合测定，以确定抗体的C1q结合和CDC活性。参见例如WO 2006/029879和WO 2005/100402中的C1q和C3c结合ELISA。
在某些实施方案中，本发明也考虑具有一些但非所有效应子功能的抗体变体，这使其成为某些应用的理想候选物，在所述应用中抗体的体内半衰期是重要的，但某些效应子功能(如补体和ADCC)是不必要或有害的。可进行如上所述的体外和/或体内测定试验，以确认CDC和/或ADCC活性的降低/耗竭。例如，可进行Fc受体(FcR)结合测定以确保抗体缺乏FcγR结合(因此很可能缺乏ADCC活性)，但保持FcRn结合能力。例如，Fc区可以包含消除或减弱效应子功能的突变，例如具有突变P329G和/或L234A和L235A的人IgG1 Fc区，或具有突变P329G和/或S228P和L235E的人IgG4Fc区。
在一些实施方案中，本发明的scFv-Fc区抗体可以通过Fc区的二聚化作用而形成二价抗体，从而可以进一步具有增加的抗体总亲和力和稳定性、或形成多特异性如双特异性。例如Fc区可以包含i)人IgG1亚类的同二聚Fc-区，或ii)人IgG4亚类的同二聚Fc-区，或iii)异二聚Fc-区，其中a)一个Fc-区多肽包含突变T366W，而另一个Fc-区多肽包含突变T366S、L368A和Y407V，或b)一个Fc-区多肽包含突变T366W和Y349C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和S354C，或c)一个Fc-区多肽包含突变T366W和S354C，而另一个Fc-区多肽包含突变T366S、L368A、Y407V和Y349C。
在一些实施方案中，本发明scFv-Fc重组抗体可以借组于Fc区部分而直接融合或缀合其 它分子，包括但不限于，荧光染料、细胞毒素、放射性同位素等，例如，用于抗原定量研究、将抗体固定化用于亲和力测量、用于治疗剂的靶向输送、使用免疫效应细胞的Fc-介导的细胞毒性测试以及许多其他用途。
B.多核苷酸和宿主
在一个方面，本发明提供基本上纯化的核酸分子，所述核酸分子编码包含上述结合BCMA的抗体链的区段或结构域的多肽。在一些实施方案中，本发明核酸分子编码结合BCMA的抗体链(例如本发明的任何抗体，包括单链scFv抗体和scFv-Fc抗体，及其片段)。
本发明的一些核酸包含编码表1所示任一抗体的重链可变区或其变体的核苷酸序列，和/或表1所示相应抗体的轻链可变区或其变体的核苷酸序列。在一个具体实施方案中，核酸分子是表1中列出的DNA VH序列和/或DNA VL序列。本发明的一些其他核酸分子包含与表1中所示的核酸分子的核苷酸序列基本上相同(例如，至少65％、80％、95％、或99％同一性)的核苷酸序列。从适宜的表达载体表达时，由这些多核苷酸编码的多肽能够显示BCMA抗原结合能力。
本发明中还提供多核苷酸，所述多核苷酸编码来自上文所述的结合BCMA的抗体的重链VH或轻链VL序列的至少一个CDR区和通常全部三个CDR区。一些进一步的实施方案中，多核苷酸编码上文所述的结合BCMA的抗体的重链和/或轻链的完整或基本上完整可变区序列。
如本领域技术人员明了的，因为密码子简并性，每一个抗体或多肽氨基酸序列可以由多种核酸序列编码。
本发明的一些核酸序列包含编码重链VH的核苷酸序列，其包含：(i)选自SEQ ID NOs:75-82的核苷酸序列或与其具有例如，至少80％、90％或99％同一性的核苷酸序列。一些其他核酸序列包含编码轻链VL的核苷酸序列，其包含SEQ ID NO:83-92的核苷酸序列或与其具有例如，至少80％、90％或99％同一性的核苷酸序列。
在一些实施方案中，本发明的核酸序列编码本发明的上述任何单链scFv抗体。在一些实施方案中，编码scFv抗体的本发明核酸序列包含选自以下的编码重链VH序列的核苷酸序列和编码轻链VL序列的核苷酸序列：
(i)SEQ ID NO:75的序列或与其基本相同的序列，以及SEQ ID NO:83的序列或或与其基本相同的序列，
(ii)SEQ ID NO:76的序列或与其基本相同的序列，以及SEQ ID NO:84或85的序列或或与其基本相同的序列，
(iii)SEQ ID NO:77的序列或与其基本相同的序列，以及SEQ ID NO:86的序列或或与其基本相同的序列，
(iv)SEQ ID NO:78的序列或与其基本相同的序列，以及SEQ ID NO:87的序列或或与其基本相同的序列，
(v)SEQ ID NO:79的序列或与其基本相同的序列，以及SEQ ID NO:88的序列或或与其基本相同的序列，
(vi)SEQ ID NO:80的序列或与其基本相同的序列，以及SEQ ID NO:89的序列或或与其基本相同的序列，
(v)SEQ ID NO:81的序列或与其基本相同的序列，以及SEQ ID NO:90的序列或或与其基本相同的序列，
(vi)SEQ ID NO:82的序列或与其基本相同的序列，以及SEQ ID NO:91或92的序列或或与其基本相同的序列。
在一个优选实施方案中，编码scFv抗体的本发明核酸还包含编码接头的核苷酸序列，例如SEQ ID NO:94中所示的序列或与其基本相同的序列。
在一个更优选的实施方案中，编码scFv抗体的本发明核酸包含选自SEQ ID NO:100、103、106、109、112、115、118、121、124、和127的序列，或与其基本相同的序列。
在上述任一实施方案中，在一个优选方面，“基本相同”的核苷酸序列是指与参考核苷酸序列在序列上具有至少80％,85％,90％,90％,92％,93％,94％,95％,96％,97％,98％,or 99％或更高同一性的序列。核苷酸序列的同一性可以使用本领域公知的各种序列比对方法确定。例如可以从NCBI(National Center for Biotechnology Information,Bethesda,MD)的网站上获得BLAST序列比对检索工具。典型地，百分比同一性采用NCBI Blast的默认参数进行。
可以通过从头固相DNA合成或通过PCR诱变编码结合BCAM的抗体或其结合片段的现有序列(例如，表1-3中所示的序列)，产生这些多核苷酸序列。核酸的直接化学合成可以通过本领域已知的方法完成，如Narang等人,1979,Meth.Enzymol.68:90的磷酸三酯法；Brown等人,Meth.Enzymol.68:109,1979的磷酸二酯法；Beaucage等人,Tetra.Lett.,22:1859,1981的二乙基磷酰亚胺法；和美国专利号4,458,066的固相支持法。通过PCR向多核苷酸序列引入突变可以如同例如PCR Technology:Principles and Applications for DNA Amplification,H.A.Erlich(编著),Freeman Press,NY,NY,1992；PCR Protocols:A Guide to Methods and Applications,Innis等人(编著),Academic Press,San Diego,CA,1990；Mattila等人,Nucleic Acids Res.19:967,1991；and Eckert等人,PCR Methods and Applications 1:17,1991中所述那样进行。
C.抗体的制备
抗体可以使用重组方法和组合物进行生产，例如US 4,816,567中所述。
在一个实施方案中，提供了包含编码本文所述结合BCMA的抗体的分离核酸的载体。此核酸可以编码包含抗体的VL的氨基酸序列和/或包含抗体的VH的氨基酸序列。在进一步的实施方案中，载体为表达载体。在进一步的实施方案中，本发明提供包含此核酸的宿主细胞。在一个实施方案中，宿主细胞包含(例如已经用以下载体转化)：(1)包含编码含有抗体VL的氨基酸序列和含有抗体VH的氨基酸序列的核酸的载体，或(2)包含编码含有抗体VL的氨基酸序列的核酸的第一载体和包含编码含有抗体VH的氨基酸序列的核酸的第二载体。在一个实施方案中，宿主细胞是真核的，例如中国仓鼠卵巢(CHO)细胞、HEK293细胞、或淋巴样细胞(例如Y0、NS0、Sp20细胞)。在一个实施方案中，提供制备抗BCMA抗体的方法，其中所述方法包括在适于抗体表达的条件下培养如上文提供的包含编码抗体的核酸的宿主细胞，且任选从宿主细胞(或宿主细胞培养基)中回收抗体。
对于抗BCMA抗体的重组生产，可以分离编码抗体的核酸，例如如上所述的核酸，并且***一个或多个载体内用于进一步克隆和/或在宿主细胞中表达。此核酸可以使用常规程序(例如通过使用能够与编码抗体重和轻链可变区的基因特异性结合的寡核苷酸探针)容易地分离和测序。
多种表达载体可以用来表达编码结合BCMA的抗体链(例如本发明的任何抗体，包括scFv抗体和全长抗体)的多核苷酸。基于病毒的表达载体和非病毒表达载体都可用于在哺乳动物宿主细胞中产生抗体。非病毒载体和***包含质粒、游离型载体和人工染色体，一般含有用于 表达蛋白质或RNA的表达盒(参见，例如，Harrington等人,Nat Genet 15:345,1997)。有用的病毒载体包括基于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒的载体，基于SV40、***瘤病毒、HBP EB病毒、痘苗病毒载体和Semliki Forest病毒(SFV)的载体。参见，Smith,Annu.Rev.Microbiol.49:807,1995；和Rosenfeld等人,Cell 68:143,1992。
表达载体的选择依赖于待在其中表达该载体的预期宿主细胞。一般，表达载体含有与编码结合BCMA的抗体链或多肽的多核苷酸有效连接的启动子。除启动子之外，也可能要求或需要其他调节元件用于高效表达结合BCMA的抗体链或片段。这些元件通常包括ATG起始密码子和邻近核糖体结合位点或其他序列。此外，可以通过纳入适用于所用细胞***的增强子增强表达的效率(参见，例如，Scharf等人,Results Probl.Cell Differ.20:125,1994；和Bittner等人,Meth.Enzymol.,153:516,1987)。例如，SV40增强子或CMV增强子可用于提高哺乳动物宿主细胞中的表达。
表达载体还可以提供分泌信号序列以形成含有BCMA结合多肽的融合蛋白。或者，也可以将BCMA结合抗体/多肽序列在***载体之前与信号序列连接。在一个优选实施方案中，信号肽包含如SEQ ID NO:133所示的氨基酸序列。用于接受编码结合BCMA的抗体轻链可变结构域和重链可变结构域的序列的载体有时还可以编码恒定区或其部分。此类载体允许将可变区表达为与恒定区的融合蛋白，由此导致完整抗体或其片段的产生。通常，此类恒定区是人的恒定区例如人IgG1 Fc区。在一个优选实施方案中，与可变区融合的Fc区包含如SEQ ID NO:132所示的氨基酸序列。
用于载体的克隆或表达的合适宿主细胞包括原核或真核细胞。例如，特别是当不需要糖基化和Fc效应子功能时，抗体可以在细菌中生产。对于抗体片段和多肽在细菌中的表达，参见例如US 5,648,237、US 5,789,199和US 5,840,523。(还参见Charlton,K.A.，见：Methods in Molecular Biology，第248卷，Lo,B.K.C.(编辑)，Humana Press，Totowa，NJ(2003)，pp.245-254，其中描述了抗体片段在大肠杆菌中的表达)。在表达后，抗体可以在可溶级分中与细菌细胞糊分离且可以进一步纯化。除了原核生物外，真核微生物例如丝状真菌或酵母是用于抗体编码载体的合适克隆或表达宿主，包括糖基化途径已被“人源化”的真菌和酵母菌株，这导致具有部分或完全人糖基化模式的抗体的生产。参见Gerngross，Nat.Biotech.22(2004)1409-1414；和Li,H.等，Nat.Biotech(2006)24:210-215。用于糖基化抗体表达的合适宿主细胞也可以源自多细胞生物(无脊椎动物和脊椎动物)。无脊椎动物细胞的实例包括植物和昆虫细胞。已经鉴定了众多杆状病毒株，其可以与昆虫细胞结合使用，特别用于草地贪夜蛾(Spodoptera frugiperda)细胞的转染。植物细胞培养物也可以用作宿主。参见，例如，US 5,959,177、US 6,040,498、US 6,420,548、US 7,125,978和US 6,417,429(描述用于在转基因植物中生产抗体的PLANTIBODIES TM技术)。可以用作宿主的脊椎动物细胞包括，例如，悬浮生长适应化的哺乳动物细胞系可以是有用的。有用的哺乳动物宿主细胞系的其他实例是SV40转化的猴肾CV1系(COS-7)；人胚肾系(293或例如Graham,F.L.等，J.Gen Virol.36(1997)59中描述的293细胞)；幼仓鼠肾细胞(BHK)；小鼠塞尔托利细胞(例如Mather,J.P.，Biol.Reprod.23(1980)243-251中描述的TM4细胞)；猴肾细胞(CV1)；非洲绿猴肾细胞(VERO-76)；人***细胞(HELA)；犬肾细胞(MDCK)；Buffalo大鼠肝细胞(BRL 3A)；人肺细胞(W138)；人肝细胞(Hep G2)；小鼠***肿瘤(MMT 060562)；TRI细胞，例如Mather,J.P.等，Annals N.Y.Acad.Sci.383(1982)44-68中描述的；MRC 5细胞；和FS4细胞。其他有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR -CHO细胞(Urlaub,G.等，Proc.Natl.Acad.Sci.USA 77(1980)4216-4220)；和骨髓瘤细 胞系，如Y0、NS0和Sp2/0。对于适合于抗体生产的一些哺乳动物宿主细胞系的综述，参见，例如，Yazaki,P.和Wu,A.M.，Methods in Molecular Biology，Vol.248，Lo.B.K.C.(编辑)，Humana Press，Totowa，NJ(2004)pp.255-268。在一些优选的实施方案中，哺乳动物宿主细胞用来表达并产生结合BCMA的本发明抗体多肽。
D.抗体的筛选、鉴定和表征
可以通过本领域已知的各种试验，针对其物理/化学特性和/或生物活性，筛选、鉴定、或表征本文中提供的抗BCMA抗体。
可以从表达人抗体的酵母展示文库中选择与所关注的目标抗原以高亲合力结合的酵母。已有多种在酵母表面呈递或展示抗体或抗体片段以及筛选文库的方法，参见例如US20110076752A1,US9845464B2,Boder and Wittrup,1997,Nat.Biotechnol.,15,553-557；Blasie等2004,Gene,342,211-218；Sazinsky等2008,Proc.Natl.Acad.Sci.USA,105,20167-20172；Tasumi等2009,Proc.Natl.Acad.Sci.USA,106,12891-12896；Kontermann和Dubel,2010,Antibody Engineering,Springer Protocols；Kuroda和Ueda 2011,Biotechnol.Lett.,33,1-9；Rakestraw等2011,PEDS,24,525-530；邵荣光等人(编辑)，抗体药物研究与应用，人民卫生出版社(2013)。可以例如通过以下非限制性方式进行酵母展示文库筛选：首先可以通过磁珠分选(MACS)进行筛选。例如可以将呈递IgG或抗体片段的酵母群接触生物素化的靶抗原一段时间、之后洗涤、与链霉亲和素磁珠(可获自Miltenyi；Biotec)孵育，通过在LS磁柱(可获自Miltenyi；Biotec)上捕获而富集与靶抗原结合的酵母细胞。之后，可以通过FACS技术进行多轮富集。在FACS分选中，可以将酵母群体接触降低浓度的生物素化的靶抗原(以筛选高亲和力抗体)或来自不同物种的抗原同源物(以筛选具有不同物种交叉反应性倾向的抗体)后，洗涤细胞，重悬在二标溶液(例如，包含链霉亲和素-PE和抗人LC-FITC的混合物)中冰上孵育，洗涤细胞后重悬在缓冲液如FACS洗涤缓冲液中，在流式细胞仪例如FACSAria(BD Bioscience)上分选LC-FITC阳性(IgG呈递)和链霉亲和素-PE阳性(靶结合)表型的酵母细胞，用于进一步增殖和选择。在FACS分选中，也可以使用负选择试剂，例如Xu等人(Protein Engineering,Design&Selection,2013,Vol 26,No.10,pp663-670)中描述的多特异性试剂(PSR)，替代靶抗原与IgG呈递酵母进行孵育，通过上述相同的二标和FACS分选，以消弱抗体的非特异性结合及后续的成药性问题。
对于抗体的鉴定，可以就其抗原结合活性，例如通过已知方法，如ELISA、αLISA、蛋白质印迹、抗体或反相阵列等、以及实施例中所述的方法，鉴定或表征本发明的抗体。
例如，可以将抗体点在玻璃或硝基纤维素芯片上。用含有BCMA的溶液阻断并孵育载玻片，洗涤以除去未结合的抗体，并用荧光标记的相应二抗检测结合的抗体。通过荧光载玻片扫描仪测量荧光信号。相似地，对于反相阵列，将重组BCMA、细胞上清液、细胞或组织裂解液、体液等，点在玻璃或硝基纤维素芯片上。封闭载玻片并用针对BCMA上特定表位的抗体孵育阵列。洗掉未结合的抗体，并用荧光标记的相应二抗检测结合的抗体。荧光信号通过荧光载玻片扫描仪来测量(Dernick,G.等，J.Lipid Res.，52(2011)2323-2331)。
也可以采用ForteBio测定法，检测抗体。ForteBio亲和力测定可以按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):p.270-8)进行。例如，可以将AHQ传感器在分析缓冲液中线下平衡30分钟，然后线上检测60秒建立基线。之后，将在线加载了经纯化的抗体的AHQ传感器，暴露于100nM抗原作用5分钟，将传感器转移至分析缓冲液进行5分钟的线下测量。使 用1:1结合模型进行动力学分析。
也可以通过流式细胞术检测抗体与表面表达BCMA的细胞的结合。例如，可以将表达BCMA的H929细胞与系列稀释的抗体在PBS 1％BSA中在冰上孵育一段时间(例如30分钟)。之后与二抗(例如藻胆色素标记的二抗)在PBS 1％BSA中在冰上避光孵育一段时间(例如30分钟)。洗涤细胞后通过流式细胞术分析细胞。流式细胞术可以在Accuri C6***(BD Biosciences)上进行，并利用Graphpad软件计算EC50值。
E.融合物和缀合物
再一方面，本发明提供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，美国专利号5,336,603、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。在再一实施方案中，本发明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。在一个优选的实施方案中，本发明单链scFv抗体用于融合或缀合。在进一步优选的实施方案中，提供包含本发明单链scFv的融合蛋白。这样的融合蛋白可以容易地通过本领域已知的重组方法进行制备。在再一优选的实施方案中，提供包含本发明单链scFv的缀合物，例如，包含本发明的scFv与非蛋白药物分子的缀合物。
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，本发明单链抗体，例如scFv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，本发明单链抗体，例如scFv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。
可以用于形成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如scFv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。
可以用于形成本发明的化学接头，包括，例如，各种偶联剂。偶联剂的实例有N-琥珀酰亚胺-3-(2-吡啶二硫基)丙酸酯(SPDP)、琥珀酰亚胺-4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯(SMCC)、亚氨基硫烷(IT)、亚氨酸酯的双官能衍生物(如己二亚胺酸二甲基酯HCl)、活性酯(如辛二酸二琥珀酰亚胺酯)、醛(如戊二醛)、二叠氮化合物(如二(对叠氮基苯甲酰)己二胺)、双-重氮衍生物(如双-(对二重氮苯甲酰)-乙二胺)、二异氰酸酯(如甲苯2,6-二异氰酸酯)、以及双活性氟化合物(如，1,5-二氟-2,4-二硝基苯)。此外，接头可以是利于多肽在递送至靶位点后释放的“可裂解接头”。例如，可以使用酸不稳定性接头、肽酶敏感性接头、光不稳定性接头、二甲基接头或含二硫化物的接头(Chari等，Cancer Research 52(1992)127-131；US 5,208,020)。
F.用于诊断和检测的方法和组合物
在一方面，本发明提供本发明抗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。
G.用于治疗的方法和组合物
再一方面，本发明涉及治疗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抗体或其抗原结合片段、或本发明的融合物或缀合物。
术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、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，受试者是人。
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。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。
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。
如实施例中所示，本发明人基于筛选自人抗体库的抗体序列而构建本发明的抗体。因此，有利地，在一些实施方案中，本发明抗体是包含全人源的VH区和全人源的VL区氨基酸序列的全人抗体，例如表1中所示的抗体、以及表3所示的单链scFv及由其构建的包含人hFc片段的scFv-Fc抗体。在一些实施方案中，本发明缀合物和融合物是包含全人抗体，例如全人单链scFv的缀合物和融合物。因此，在一个优选的方面，本发明的抗体、融合物和缀合物尤其适用于人的治疗应用。在一些优选的实施方案中，本发明抗体、融合物和缀合物用于治 疗人的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，本发明抗BCMA抗体、融合物和缀合物的抗肿瘤作用，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。
多发性骨髓瘤是成熟浆细胞的B细胞恶性肿瘤。骨髓中克隆性浆细胞异常增生，并且可侵袭邻近的骨，以及有时侵袭血液。多发性骨髓瘤的变体形式包括：显性多发性骨髓瘤、冒烟型多发性骨髓瘤、浆细胞白血病、非分泌型多发性骨髓瘤、IgD型多发性骨髓瘤、骨硬化性骨髓瘤、骨孤立性浆细胞瘤以及髓外浆细胞瘤。(参见，例如Braunwald等人(编辑),Harrison’s Principles of Internal Medicine，第15版(McGraw-Hill 2001))。
存在多种不同类型的非霍奇金淋巴瘤。例如，非霍奇金淋巴瘤可以分为侵袭性的(快速生长的)和惰性的(缓慢生长的)。非霍奇金淋巴瘤(NHL)包括：伯基特淋巴瘤、慢性淋巴细胞白血病/小淋巴细胞淋巴瘤(CLL/SLL)、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、免疫母细胞性大细胞淋巴瘤、前体B淋巴母细胞性淋巴瘤以及套细胞淋巴瘤。发生于骨髓或干细胞移植之后的淋巴瘤通常为B细胞非霍奇金淋巴瘤。
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。
H.组合物和制剂
本发明还考虑包含本文任一种或多种BCMA结合抗体分子、、融合物和缀合物、多核苷酸、载体、或宿主细胞的组合物。组合物包括但不限于药物组合物。药物组合物可以用于向细胞或动物单独施用或者与一种或多种其它治疗形式组合施用。
可以制备本发明抗体、融合物和缀合物的药物制剂，例如通过使具有所需纯度的抗体、融合物和缀合物，与一种或多种任选的药学可接受的载体(Remington’s Pharmaceutical Science，第16版，Osol，A.(编辑)(1980))混合，以冻干制剂或水溶液的形式制备。药学可接受的载体在采用的剂量和浓度下对接受者一般是无毒的，并且包括但不限于：缓冲剂，如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(如十八基二甲基苄基氯化铵；氯己双铵；苯扎氯铵；苄索氯铵；苯酚、丁醇或苯甲醇；对羟基苯甲酸烷基酯例如对羟基苯甲酸甲或丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量(小于约10个残基)多肽；蛋白质，如血清白蛋白、明胶或免疫球蛋白；亲水聚合物，如聚(乙烯吡咯烷酮)；氨基酸，如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；糖，如蔗糖、甘露醇、海藻糖或山梨糖醇；成盐抗衡离子，如钠；金属络合物(例如Zn-蛋白质络合物)；和/或非离子型表面活性剂，如聚乙二醇(PEG)。
示例性冻干抗体制剂描述于US 6,267,958。水性抗体制剂包括US 6,171,586和WO2006/044908中所述的那些，后面的制剂包括组氨酸-醋酸盐缓冲液。
本文中的制剂还可以针对所治疗的特定适应症按照需要含有一种以上的活性成分，优选具有互补活性且不会不利地彼此影响的那些。此类活性成分适于以对于预期目的有效的量组合存在。
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限制本发明的保护范围。
实施例
实施例1.酵母展示技术筛选抗BCMA全人源抗体
利用酵母展示技术，从总的多样性大于1×10 8的6个合成抗体库中筛选特异性结合BCMA的全人源抗体(库的设计和构建可以参见WO 2009036379，WO2010105256，WO2012009568)。简言之，筛选过程如下：首先，第一轮筛选通过磁珠分选方法(MACS)并利用生物素标记的、与Fc融合的重组人BCMA筛选6个不同的合成抗体库来完成；第二轮筛选基本按照Chao et al.Nature Protocols,2006的方法，通过流式细胞分选技术(FACS)并利用与Fc融合的猴源及鼠源的BCMA完成；第三轮筛选中，通过FACS技术，基本按照Xu et al.Protein Engineering,Design&Selection,2013描述的，使用多特异性试剂(PSR)负向选择抗体库种中的抗体，以消弱抗体的非特异性结合及后续的成药性问题；第四轮筛选通过FACS技术利用His标签的人源BCMA单体蛋白完成；最后一轮筛选是通过FACS技术分别富集人源、猴源和鼠源BCMA特异性抗体。
批量优化是初步筛选步骤中的常规环节。简言之，通过分离初始筛选所富集的抗体群体的重链区(本阶段的多样性在10 3到10 4之间)并将其与酵母菌中的天然人轻链序列库重新组合来完成。这一过程被称为轻链批通量重组(light chain batch shuffle，LCBS),最终制备具有人源BCMA结合倾向的、重链/轻链配对多样性在10 7到10 8之间的抗体库。该抗体库进一步经过如上段落中描述的一轮磁珠筛选和四轮流式筛选，进一步富集人源、猴源和鼠源BCMA特异性抗体。如上所述的，此过程中分别使用人源、猴源和鼠源的BCMA与Fc的融合蛋白，以及His标签的人源BCMA单体蛋白作阳性筛选，PSR作阴性筛选。
完成上述筛选过程后，将富集的含有特异抗体序列的酵母群体涂在琼脂平板上，能够得到包含特定抗体基因的酵母单克隆菌落。挑取克隆，利用sanger法对其可变区进行测序，大约有460个序列独特的H3:L3抗体(即，具有独特的重链CDR3区和轻链CDR3区对的抗体)被鉴定出来。然后通过酵母表达并使用蛋白A亲和层析的方法纯化获得了一些抗体。
经进一步测试这些抗体与多种重组BCMA蛋白及BCMA稳定转染的CHO-S细胞系及BCMA阳性肿瘤细胞系NCI-H929的结合能力，最终获得了10株与NCI-H929亲和力较好的克隆株作进一步分析。这些抗体也表现出与猴BCMA具有一定的交叉反应性。此10株抗体分子的氨基酸序列以及对应的核苷酸序列在上表1中给出。
实施例2.酵母表达抗体与NCI-H929细胞的亲和力验证
通过流式细胞术，对上述10株与NCI-H929亲和力较好的酵母表达抗体及一株与NCI-H929无亲和力的抗体(ADI-34819)(作为负对照)作进一步细胞结合验证。具体方法如下：
1.取人NCI-H929(ATCC，CRL-9068)细胞，调整细胞密度为2x10 6/ml,在96孔微孔板中每孔加入100μl,400G离心5min,去上清。
2.将抗BCMA抗体从400nM浓度起始，在含0.1％牛血清白蛋白(BSA)的PBS中以3倍梯度连续稀释共12个点，每孔加入100μl稀释的抗体，4℃孵育30min；
3.400G离心5min,加入PBS洗两次，之后每孔加入100μl稀释在PBS(1％BSA)中的二抗(藻 胆色素蛋白(Phycoerythrin，PE)标记的羊抗人IgG抗体,SoutherBiotech，终浓度5μg/ml)，4℃孵育30min(避光)；
4.400G离心5min,加入PBS洗两次，每孔用100μl PBS重悬细胞。在Accuri C6***(BDBioscience)上进行流式细胞术，检测PE阳性信号，并基于C6软件计算MFI。用GraphPad软件计算EC50值。
检测结果如图1和下表4所示。由实验结果可以确认，10株抗体在400nM浓度下都与NCI-H929细胞有比较强的亲和力，并随着抗体浓度的稀释，其与NCI-H929的亲和力也逐步降低。
表4：10株抗体与NCI-H929细胞结合的EC50值
抗体名称 EC50(nM)
ADI-34832 34.14
ADI-34846 136.1
ADI-34848 24.83
ADI-34849 9.107
ADI-34850 12.12
ADI-34854 83.24
ADI-34857 25.21
ADI-34859 85.85
ADI-34860 143.5
ADI-34861 65.13
ADI-34819 N/A
实施例3.scFv-hFc重组单链抗体表达载体的构建
为验证scFv形式候选抗体与靶标的亲和力，分别构建了上述10株抗体的单链抗体可变区(scFv)与人Fc片段的重组蛋白表达载体。同时，也构建了ADI-34819抗体的重组单链抗体表达载体(作为负对照)、以及基于US20170226216A1中公开的huBCMA-10序列的重组单链抗体表达载体(作为标准品对照)。所构建的这些scFv-Fc重组蛋白的氨基酸序列及其对应的编码核苷酸序列见上表3。
构建表达表3中所列scFv-Fc重组蛋白的表达载体。简言之，通过限制内切酶酶切带有鼠源κ轻链信号肽(METDTLLLWVLLLWVPGSTG，SEQ ID NO:133；编码序列ATGGAGACCGACACCCTGCTGCTCTGGGTCCTGCTGC TGTGGGT GCCCGGATCCACAGGA,SEQ ID NO:134)和人IgG1 Fc编码序列(SEQ ID NO:132)的pDD1-hFc载体(pDD1-hFc载体基于pTT5载体、通过***所述信号肽和hFc编码基因而构建。)，并通过同源重组的方法将合成的scFv序列克隆至轻链信号肽和hFc的编码基因之间形成融合表达。图2中显示了载体构建的示意图。
实施例4.scFv-hFc重组单链抗体的表达
1.根据所需转染体积传代HEK293细胞，转染前一天将细胞密度调整至1.2x10 6/ml。
2.取3mL OptiMEM培养基(Gibco，31985-070)作为转染缓冲液，分别上述相应携带scFv-hFc重组单链抗体编码基因的质粒30μg，混匀后过滤静置5min。
3.加90μL的1mg/mL聚乙烯亚胺(PEI)(Polysciences，23966)到质粒-OptiMEM混合液中，混匀后室温孵育15min。将混合物轻柔倒入细胞，36.5℃，8％CO 2培养。
4.20h后补加0.6mL的200g/L的FEED(大豆蛋白胨Phytone Peptone(BD，211906)与植物蛋白胨Phytone(BD，210931)等比例)，0.3mL的200g/L的葡萄糖母溶液，30μL的2.2M丙戊酸钠盐(VPA)(Sigma，P4543)。
5.继续培养至活力低于60％，收集上清，过滤后作亲和层析纯化。
实施例5.蛋白A法纯化scFv-hFc重组单链抗体
1.用超纯水冲洗填料及重力柱，去除填料保护液。
2.用0.1M NaOH将重力柱及填料浸泡2h。每根重力柱添加300μL蛋白A亲和层析介质(Mabselect sure)(GE Healthcare，17-5438-03)填料。
3.细胞料液以8000r/min离心40min,再使用0.45μm滤器过滤，4℃保存备用。
4.用大量超纯水冲洗重力柱和填料，去除碱液。
5.纯化前用10ml结合/清洗缓冲液(20mM Tris+150mM NaCl(pH 7.2))平衡填料。
6.上样，将需要纯化的上清通过柱子。
7.洗涤，用5～10ml结合/清洗缓冲液(20mM Tris+150mM NaCl(pH 7.2))冲洗填料，去除非特异性结合蛋白。
8.洗脱，用1mL的洗脱缓冲液(100mM柠檬酸钠/柠檬酸缓冲液，pH 3.5)冲洗填料，收集特异性结合蛋白。
9.在收集液中按85μl/ml的比例加入中和缓冲液(2M Tris),调节pH至6～7。
实施例6.scFv-hFc重组单链抗体的Fortebio检测
基于光纤生物传感器的生物膜层光学干涉技术(BLI)，测定抗体分子的动力学常数。
BLI的基本原理是：当生物分子结合到传感器表面就形成了一层生物膜，生物膜对透过传感器的光的波形造成干涉现象，干涉现象以相位移动的方式被检测，从而可以检测结合到传感器分子数量的变化；根据实时响应值的变化拟合出动力学曲线，并计算出结合常数(Kon)、解离常数(Kdis)、亲和力(KD)。
实验中所选用的Fortebio设备型号为Octet Red96，ForteBio亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):p.270-8)进行。具体流程为：
1.实验开始前半个小时，根据样品数量，取合适数量的AHC Sensor浸泡于SD buffer(50mlPBS+0.1％BSA+0.05％Tween-20)中。
2.取100μl的SD buffer、scFv-hFc抗体、人BCMA-His抗原(ACRO BIOSYSTEMS，BCA-H522Y)，分别加入到96孔黑色聚苯乙烯半量微孔板中。
3.根据样品位置布板，选择传感器位置，设置运行步骤及时间：Baseline、Loading～1nm、Baseline、Association和Dissociation时间取决于样品结合、解离速度；转速为1000rpm，温度为30℃。
4.10个scFv-hFc重组单链抗体与人BCMA-His亲和力检测结果详见表5。
表5.scFv-hFc与人BCMA-His亲和力的Fortebio检测结果
Figure PCTCN2019074419-appb-000003
Figure PCTCN2019074419-appb-000004
*参照scFv-Fc为根据US20170226216 A1的BCMA-10序列中scFv构建的scFv-hFc重组蛋白(参见实施例3，SEQ ID NO；131)的亲和力水平。
由上表数据可见，10株单链抗体与单价人源BCMA(BCMA-His)的亲和力(KD值)均与参照单链抗体与BCMA-His的亲和力相当。其中，ADI-34857和ADI-34860两株单链抗体与BCMA-His的亲和力，与参照单链抗体的最为接近。
实施例7.scFv-hFc重组单链抗体与NCI-H929亲和力的检测
scFv-hFc融合抗体制备完成后，进一步对其与NCI-H929的亲和力作了验证。具体方法如下：
1.取人NCI-H929(ATCC，CRL-9068)细胞，调整细胞密度为2x10 6/ml,在96孔微孔板中每孔100μl,400G离心5min,去上清。
2.将scFv-hFc抗体从400nM浓度起始，在含0.1％牛血清白蛋白(BSA)的PBS中以3倍梯度连续稀释共12个点，每孔加入100μl稀释的抗体，4℃孵育30min；
3.400G离心5min,加入PBS洗两次，每孔加入100μl稀释在PBS(1％BSA)中的二抗((藻胆色素蛋白(Phycoerythrin，PE)标记的羊抗人IgG抗体,SoutherBiotech，终浓度5μg/ml)，4℃孵育30min(避光)；
4.400G离心5min,加入PBS洗两次，每孔100μl PBS重悬细胞。在Accuri C6***(BDBioscience)上进行流式细胞术，检测PE阳性信号，并基于C6软件计算MFI。用GraphPad软件计算EC50值。
检测结果如图3和下表6所示。由图中结果可见，除ADI-34846单链抗体外，其它单链抗体与NCI-H929细胞的亲和力(EC50值)都优于参照单链抗体与NCI-H929细胞的亲和力。
表6:单链抗体与NCI-H929细胞的亲和力(EC50值)
抗体名称 EC50(nM)
参照scFv-hc 130.4
ADI-34832 scFv-hc 11.23
ADI-34846 scFv-hc 816.0
ADI-34848 scFv-hc 39.05
ADI-34849 scFv-hc 4.139
ADI-34850 scFv-hc 28.67
ADI-34854 scFv-hc 38.01
ADI-34857 scFv-hc 17.68
ADI-34859 scFv-hc 34.59
ADI-34860 scFv-hc 37.27
ADI-34861 scFv-hc 27.40
ADI-34819 scFv-hc N/A

Claims (27)

  1. 一种分离的特异性结合B细胞成熟抗原(BCMA)的抗体或其抗原结合片段，所述抗体包含：
    (i)如SEQ ID NO:4或5所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:31或41或42所示的轻链可变区的LCDR1、2和3序列，或者
    (ii)如SEQ ID NO:10所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:46所示的轻链可变区的LCDR1、2和3序列，或者
    (iii)如SEQ ID NO:16或17所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:50或58所示的轻链可变区的LCDR1、2和3序列，或者
    (iv)如SEQ ID NO:23所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:64所示的轻链可变区的LCDR1、2和3序列，或者
    (v)如SEQ ID NO:27所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:59或71或72所示的轻链可变区的LCDR1、2和3序列。
  2. 一种分离的特异性结合B细胞成熟抗原(BCMA)的抗体或其抗原结合片段，所述抗体包含3个重链互补决定区HCDR以及3个轻链互补决定区LCDR，其中：
    (a)HCDR1包含SEQ ID NO:1所示的氨基酸序列，HCDR2包含SEQ ID NO:2所示的氨基酸序列，HCDR3包含SEQ ID NO:3所示的氨基酸序列，LCDR1包含SEQ ID NO:28或32或33或34所示的氨基酸序列，LCDR2包含SEQ ID NO:29或35或36或37所示的氨基酸序列，且LCDR3包含SEQ ID NO:30或38或39或40所示的氨基酸序列；或
    (b)HCDR1包含SEQ ID NO:7所示的氨基酸序列，HCDR2包含SEQ ID NO:8所示的氨基酸序列，HCDR3包含SEQ ID NO:9所示的氨基酸序列，LCDR1包含SEQ ID NO:32所示的氨基酸序列，LCDR2包含SEQ ID NO:44所示的氨基酸序列，且LCDR3包含SEQ ID NO:45所示的氨基酸序列；或
    (c)HCDR1包含SEQ ID NO:11所示的氨基酸序列，HCDR2包含SEQ ID NO:12所示的氨基酸序列，HCDR3包含SEQ ID NO:13或14或15所示的氨基酸序列，LCDR1包含SEQ ID NO:47或51所示的氨基酸序列，LCDR2包含SEQ ID NO:48或54所示的氨基酸序列，且LCDR3包含SEQ ID NO:49或55所示的氨基酸序列；或
    (d)HCDR1包含SEQ ID NO:20所示的氨基酸序列，HCDR2包含SEQ ID NO:21所示的氨基酸序列，HCDR3包含SEQ ID NO:22所示的氨基酸序列，LCDR1包含SEQ ID NO:61所示的氨基酸序列，LCDR2包含SEQ ID NO:62所示的氨基酸序列，且LCDR3包含SEQ ID NO:63所示的氨基酸序列；或
    (e)HCDR1包含SEQ ID NO:24所示的氨基酸序列，HCDR2包含SEQ ID NO:25所示的氨基酸序列，HCDR3包含SEQ ID NO:26所示的氨基酸序列，LCDR1包含SEQ ID NO:52或65或66或67所示的氨基酸序列，LCDR2包含SEQ ID NO:62所示的氨基酸序列，且LCDR3包含SEQ ID NO:56或68或69或70所示的氨基酸序列；或
    所述抗体包含(a)-(e)任一项中所述CDR序列组合的变体，其中所述变体在1、2、3、4、 5或优选地6个CDR区上共包含至少一个且不超过10，或不超过5、4、3、2或1个氨基酸改变(优选氨基酸取代，优选保守取代)，优选地重链CDR3保持不变。
  3. 权利要求1或2的抗体或其抗原结合片段，其中所述抗体包含重链可变区VH，所述VH选自：
    (a)包含选自SEQ ID NO:4、5和6的氨基酸序列、或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列、或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VH；
    (b)包含SEQ ID NO:10的氨基酸序列、或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列、或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VH；
    (c)包含选自SEQ ID NO:16、17或19的氨基酸序列、或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列、或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VH；
    (d)包含SEQ ID NO:23的氨基酸序列、或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列、或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VH；
    (e)包含SEQ ID NO:27的氨基酸序列、或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列、或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VH。
  4. 权利要求1或2的抗体或其抗原结合片段，所述抗体包含轻链可变区VL，所述VL选自：
    (a)包含选自SEQ ID NO:31、41、42和43的氨基酸序列、或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列、或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VL；
    (b)包含SEQ ID NO:46的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VL；
    (c)包含选自SEQ ID NO:50和58的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VL；
    (d)包含SEQ ID NO:64的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VL；
    (e)包含选自SEQ ID NO:59、71、72和73的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列或相对于其包含不超过10个(优选不超过5个)氨基酸改变的氨基酸序列的VL。
  5. 权利要求1-4任一项的分离抗体或其抗原结合片段，所述抗体包含重链可变区VH和轻链可变区VL，所述VH和VL选自：
    (a)包含选自SEQ ID NO:4、5和6的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VH，和包含选自SEQ ID NO:31、41、42和43的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VL；
    (b)包含SEQ ID NO:10的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VH，和包含SEQ ID NO:46的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VL；
    (c)包含选自SEQ ID NO:16、17或19的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VH，和包含选自SEQ ID NO:50和58的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VL；
    (d)包含SEQ ID NO:23的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VH，和包含SEQ ID NO:64的氨基酸序列或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VL；
    (e)包含SEQ ID NO:27的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VH，和包含选自SEQ ID NO:59、71、72和73的氨基酸序列或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列的VL。
  6. 前述权利要求任一项的抗体或其抗原结合片段，所述抗体具有以下一个或多个特性：
    (i)显示与表1所列的任一抗体相同或相似的结合亲和力和/或特异性；
    (ii)抑制(例如，竞争性抑制)表1所列的任一抗体与BCMA的结合；
    (iii)与表1所示的任一抗体结合相同或重叠的表位；
    (iv)与表1所示的任一抗体竞争结合BCMA；
    (v)具有表1所列的任一抗体分子的一个或多个生物学特性。
  7. 前述权利要求任一项的抗体或其抗原结合片段，其中所述抗体具有以下一个或多个特性：
    (i)以高亲和力，例如以小于100nM，例如小于50nM，例如5-30nM，优选小于10nM的KD值，与人BCMA(如SEQ ID NO:74的多肽)结合；
    (ii)以高亲和力，例如以小于100nM，例如小于50nM，例如1-40nM，优选小于20nM，更优选小于10或5nM的EC50值，与细胞表面表达的人BCMA(如SEQ ID NO:74的多肽)结合；
    (iii)与人BCMA(如SEQ ID NO:74的多肽)结合的解离速率常数(K d)小于3×10 -2、1.5×10 -2、5×10 -3或3×10 -3s -1，例如约1.46×10 -3s -1；
    (iv)与人BCMA的胞外域ECD上的表位特异性结合；
    (v)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。
  8. 前述权利要求任一项的抗体或其抗原结合片段，其中所述抗体是全人源抗体。
  9. 前述权利要求任一项的抗体或其抗原结合片段，其中所述抗体是单链抗体。
  10. 前述权利要求任一项的抗体，其中所述抗体是单链scFv抗体，优选地所述单链scFv包含：从N端到C端，VL结构域-接头-VH结构域，或VH结构域-接头-VL结构域。
  11. 权利要求10的抗体，其中所述接头包含1个至约25个氨基酸、约5个至约20个氨基酸或约10个至约20个，优选地15-20个氨基酸。
  12. 权利要求11的抗体，其中所述接头包含SEQ ID NO:93的氨基酸序列。
  13. 权利要求10-12任一项的抗体，其中所述单链scFv抗体包含选自SEQ ID NO:99、102、105、108、111、114、117、120、123和126的氨基酸序列、或与其具有至少90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列、或相对于其包含至少一个，两个或三个，但不超过30、20或10个氨基酸改变的氨基酸序列。
  14. 一种分离的特异性结合B细胞成熟抗原(BCMA)的抗体，其中所述抗体包含权利要求10-13任一项的单链scFv抗体和Fc区。
  15. 权利要求14的抗体，其中所述单链scFv通过铰链区与Fc区连接，优选地，铰链区为CD8铰链区，更优选地，铰链区包含SEQ ID NO:95所示的氨基酸序列或相对于SEQ ID NO:95的氨基酸序列包含至少一个，两个或三个，但不超过5个氨基酸改变的氨基酸序列。
  16. 权利要求14-15任一项的抗体，其中所述Fc区是人IgG1或IgG4Fc区，优选地所述Fc区是低或无岩藻糖基化的。
  17. 权利要求14-16任一项的抗体，其中所述抗体包含选自SEQ ID NO:101、104、107、110、113、116、119、122、125、和128的氨基酸序列、或相对于其包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列。
  18. 分离的核酸，其编码权利要求1至17中任一项的分离的抗体或其抗原结合片段。
  19. 包含权利要求18的核酸的载体，优选地所述载体是表达载体。
  20. 包含权利要求19的载体的宿主细胞，优选地，所述宿主细胞选自酵母细胞、哺乳动物细胞。
  21. 制备权利要求1至17中任一项的分离的抗体或其抗原结合片段的方法，包括：在适于表达所述抗体或其抗原结合片段的条件下，培养权利要求20的宿主细胞。
  22. 包含权利要求1至17之任一项的抗体的缀合物或融合物。
  23. 药物组合物，其包含权利要求1至17中任一项的分离的抗体或其抗原结合片段、或权利要求22的缀合物或融合物，以及任选地药用载体。
  24. 一种检测样品中BCMA的方法，包括：
    (a)将所述样品与权利要求1至17中任一项的分离的抗体或其抗原结合片段、或权利要 求22的缀合物或融合物接触；和
    (b)检测所述抗体或其抗原结合片段、或权利要求22的缀合物或融合物和BCMA蛋白之间复合物的形成。
  25. 一种治疗B细胞相关病症的方法，包括向所述受试者施用有效量的权利要求1-17中任一项的分离的抗体或其抗原结合片段、或权利要求22的缀合物或融合物、或权利要求23的组合物。
  26. 权利要求25的方法，其中所述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。
  27. 权利要求26的方法，其中所述B细胞相关病况是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)。
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