WO2021136323A1 - Antibodies binding bcma and uses thereof - Google Patents

Antibodies binding bcma and uses thereof Download PDF

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Publication number
WO2021136323A1
WO2021136323A1 PCT/CN2020/141148 CN2020141148W WO2021136323A1 WO 2021136323 A1 WO2021136323 A1 WO 2021136323A1 CN 2020141148 W CN2020141148 W CN 2020141148W WO 2021136323 A1 WO2021136323 A1 WO 2021136323A1
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antibody
bcma
antigen
seq
nos
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PCT/CN2020/141148
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French (fr)
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John Li
Yong Tang
Shengwei LI
Ming Zhou
Mingjiu Chen
Shukai Xia
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Salubris (Chengdu) Biotech Co., Ltd.
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Priority to KR1020217041996A priority Critical patent/KR20220012314A/en
Publication of WO2021136323A1 publication Critical patent/WO2021136323A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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

Definitions

  • the present disclosure relates generally to an isolated monoclonal antibody, particularly a mouse, chimeric or humanized monoclonal antibody, or the antigen-binding portion thereof, that specifically binds to human BCMA with high affinity and functionality.
  • a nucleic acid molecule encoding the antibody or the antigen-binding portion thereof, an expression vector, a host cell and a method for expressing the antibody or the antigen-binding portion thereof are also provided.
  • the present disclosure further provides an immunoconjugate, a bispecific molecule, a chimeric antigen receptor, an oncolytic virus, and a pharmaceutical composition comprising the antibody or the antigen-binding portion thereof, as well as a diagnostic or treatment method using an anti-BCMA antibody or antigen-binding portion thereof of the disclosure.
  • BCMA tumor necrosis factor receptor superfamily member 17
  • TFRS17 tumor necrosis factor receptor superfamily member 17
  • BCMA B-cell activation factor receptor
  • TACI transmembrane activator and calcium modulator and cyclophilin ligand interactor
  • BCMA is exclusively expressed on the surfaces of plasmablasts and differentiated PCs, and more highly on malignant than normal PCs (Carpenter RO et al., (2013) Clin Cancer Res 19 (8) : 2048–60; O’Connor BP et al., (2004) J Exp Med 199 (1) : 91–8; Benson MJ et al., (2008) J Immunol 180 (6) : 3655–9; Yang M et al., (2005) J Immunol 175 (5) : 2814–24; Avery DT et al., (2003) J Clin Invest 112 (2) : 286–97; Novak AJ et al., (2004) Blood 103 (2) : 689–94; Chiu A et al., (2007) Blood 109 (2) : 729–39; Lee L et al., (2016) Blood 131 (7) : 746–58; Carpenter RO et al., (2013) supra; Tai YT et al., (2014) Blood 123 (20) : 3128–38;
  • BCMA Crohn's disease
  • APRIL and BAFF via binding to BCMA and TACI, further activate NF ⁇ B pathways and upregulate antiapoptotic proteins (Mcl-1, Bcl-2, Bcl-xL) to protect multiple myeloma tumor cells against dexamethasone-and serum deprivation-induced cell death (Moreaux J et al., (2004) Blood 103 (8) : 3148–57; Neri P et al., (2007) Clin Cancer Res 13 (19) : 5903–9; Shen X et al., (2016) Cell Biochem Funct 34 (2) : 104–10) .
  • multiple myeloma is a clonal B-cell malignancy that occurs in multiple sites within the bone marrow before spreading to the circulation; either de novo, or as a progression from monoclonal gammopathy of undetermined significance (MGUS) . It is commonly characterized by increases in paraprotein and osteoclast activity, as well as hypercalcaemia, cytopenia, renal dysfunction, hyperviscosity and peripheral neuropathy. Decreases in both normal antibody levels and numbers of neutrophils are also common, leading to a life-threatening susceptibility to infection.
  • MGUS monoclonal gammopathy of undetermined significance
  • Another anti-BCMA ADC has shown in vitro cytotoxicity against multiple myeloma cells at picomolar range, resulting in significant tumor regression with good toleration.
  • Several anti-BCMA CAR-T cell therapies have also shown impressive clinical outcomes (Shih-Feng Cho et al., (2016) Frontiers in Immunology 9: 1821) .
  • the present disclosure provides an isolated monoclonal antibody, for example, a mouse, human, chimeric or humanized monoclonal antibody, or an antigen-binding portion thereof, that binds to BCMA (e.g., the human BCMA, and monkey BCMA) and has comparable, or higher, binding affinity to BCMA as compared to prior art anti-BCMA antibodies such as the BCMA-binding portion of GSK2857916.
  • BCMA e.g., the human BCMA, and monkey BCMA
  • the antibody or antigen-binding portion of the disclosure can be used for a variety of applications, including detection of the BCMA protein, and treatment and prevention of BCMA associated diseases, such as cancers, autoimmune diseases, and infectious diseases.
  • an isolated monoclonal antibody, or the antigen-binding portion thereof, of the present disclosure comprises a heavy chain and a light chain linked by disulfite bonds, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, the light chain comprising a light chain variable region and a light chain constant region, wherein the C terminus of the heavy chain variable region is linked to the N terminus of the heavy chain constant region, and the C terminus of the light chain variable region is linked to the N terminus of the light chain constant region, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences described above, and the antibody or antigen-binding portion thereof binds to BCMA.
  • the heavy chain constant region may be human IgG1 constant region having an amino acid sequence set forth in e.g., SEQ ID No: 23, and the light chain constant region may be human kappa constant region having an amino acid sequences set forth in e.g., SEQ ID No: 24.
  • the amino acid sequences of SEQ ID NOs.: 23 and 24 may be encoded by nucleotide sequences set forth in SEQ ID NOs.: 28 and 29, respectively.
  • the antibody of the present disclosure in some embodiments comprises or consists of two heavy chains and two light chains, wherein each heavy chain comprises the heavy chain constant region, heavy chain variable region or CDR sequences mentioned above, and each light chain comprises the light chain constant region, light chain variable region or CDR sequences mentioned above, wherein the antibody binds to BCMA.
  • the antibody of the disclosure can be a full-length antibody, for example, of an IgG1, IgG2 or IgG4 isotype.
  • the light chain constant region may be a kappa or lambda constant region.
  • the antibody of the present disclosure in other embodiments may be a single chain variable fragment (scFv) antibody, or antibody fragments, such as Fab or Fab′2 fragments.
  • the antibody, or antigen-binding portion thereof, of the present disclosure has comparable, if not higher, binding affinity/capacity to human BCMA and monkey BCMA than prior art anti-BCMA antibodies such as the BCMA binding portion of GSK2857916, and inhibits the binding of BAFF or APRIL to BCMA.
  • the disclosure also provides an immunoconjugate comprising an antibody or antigen-binding portion thereof of the disclosure, linked to a therapeutic agent, such as a cytotoxin.
  • a bispecific molecule comprising an antibody, or antigen-binding portion thereof, of the disclosure, linked to a second functional moiety (e.g., a second antibody) having a different binding specificity than said antibody, or antigen-binding portion thereof.
  • the antibody or an antigen binding portions thereof of the present disclosure can be made into part of a chimeric antigen receptor (CAR) .
  • an immune cell comprising the chimeric antigen receptor, such as a T cell.
  • the antibody or an antigen binding portion thereof of the present disclosure can also be encoded by or used in conjuction with an oncolytic virus.
  • compositions comprising the antibody, or antigen-binding portion thereof, or immunoconjugate, bispecific molecule, oncolytic virus, CAR or CAR-T cell of the disclosure, and a pharmaceutically acceptable carrier, are also provided.
  • the pharmaceutical composition may further contain an anti-tumor agent and/or a cytokine.
  • Nucleic acid molecules encoding the antibodies, or antigen-binding portions thereof, of the disclosure are also encompassed by the disclosure, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors.
  • a method for preparing an anti-BCMA antibody or an antigen-binding portion thereof using the host cell comprising the expression vector is also provided, comprising steps of (i) expressing the antibody in the host cell and (ii) isolating the antibody from the host cell or its cell culture.
  • the disclosure provides a method for treating a tumor associated with increased BCMA expression, comprising administering to a subject a therapeutically effective amount of the antibody, or antigen-binding portion thereof, of the present disclosure.
  • the tumor may be non-solid tumors, including leukemia, lymphomas, multiple myeloma, etc, or solid tumors.
  • the method comprises administering a composition, a bispecific molecule, an immunnoconjugate such as an antibody-drug conjugate, a CAR-T cell, or an antibody-encoding or antibody-bearing oncolytic virus of the disclosure, or alternatively a nucleic acid molecule or a vector capable of expressing the same in the subject.
  • At least one additional anti-cancer antibody can be administered with the antibody, or an antigen-binding portion thereof, of the disclosure, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, and/or an anti-TIM 3 antibody.
  • an antibody, or an antigen-binding portion thereof, of the disclosure is administered with a cytokine (e.g., IL-2, IL-21, TNF- ⁇ , IFN- ⁇ and/or IL-4) , or a costimulatory antibody (e.g., an anti-CD137 antibody, an anti-OX40 antibody and/or anti-GITR antibody) .
  • the antibodies of the present disclosure can be, for example, mouse, human, chimeric or humanized antibodies.
  • Fig. 1A and 1B show the binding capacities of mouse antibodies B1A1 and B1C1 (A) , and B1G3 (B) to human BCMA.
  • Fig. 2A and 2B show the blocking abilities of mouse antibodies B1A1 and B1C1 (A) , and B1G3 (B) on human BCMA-BAFF interaction.
  • Fig. 3A and 3B show the abilities of mouse antibodies B1A1 and B1C1 (A) , and B1G3 (B) to block Benchmark-human BCMA binding.
  • Fig. 4A-4C show the binding capacities of humanized antibodies huB1G3-V1 to huB1G3-V5 (A) , huB1G3-V6 to huB1G3-V10 (B) and huB1G3-V11 to huB1G3-V15 (C) to human BCMA.
  • Fig. 5A-5C show the binding capacities of humanized antibodies huB1C1-V1 to huB1C1-V7 (A) , huB1C1-V8 to huB1C1-V9 (B) and huB1C1-V11 to huB1C1-V13 (C) to human BCMA.
  • Fig. 6 shows the binding capacity of humanized antibody huB1G3-V13 to human BCMA.
  • Fig. 7 shows the blocking ability of humanized antibody huB1G3-V13 on human BCMA-BAFF interaction.
  • Fig. 8 shows the blocking ability of humanized antibody huB1G3-V13 on Benchmark-human BCMA interaction.
  • Fig. 9 shows the binding capacity of humanized antibody huB1C1-V13 to human BCMA.
  • Fig. 10 shows the blocking ability of humanized antibody huB1C1-V13 on human BCMA-BAFF interaction.
  • Fig. 11 shows the blocking ability of humanized antibody huB1C1-V13 on Benchmark -human BCMA interaction.
  • BCMA refers to B cell maturation antigen, or tumor necrosis factor receptor superfamily member 17.
  • BCMA comprises variants, isoforms, homologs, orthologs and paralogs.
  • an antibody specific for a human BCMA protein may, in certain cases, cross-react with a BCMA protein from a species other than human, such as monkey.
  • an antibody specific for a human BCMA protein may be completely specific for the human BCMA protein and exhibit no cross-reactivity to other species or of other types, or may cross-react with BCMA from certain other species but not all other species.
  • human BCMA refers to a BCMA protein having an amino acid sequence from a human, such as the amino acid sequence of human BCMA having a Genbank accession number of NP_001183.2, or the amino acid sequence set forth in SEQ ID NO.: 25.
  • the terms “monkey or rhesus BCMA” and “mouse BCMA” refer to monkey and mouse BCMA sequences, respectively, e.g. those with the amino acid sequences having Genbank Accession Nos. XP_001106892.1 and NP_035738.1, respectively.
  • antibody as referred to herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion” ) or single chains thereof.
  • Whole antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C H1 , C H2 and C H3 .
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a BCMA protein) . It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a V H domain; (vi) an isolated complementarity determining region (CDR) ; and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains.
  • a Fab fragment a monovalent fragment consisting of the V L
  • the two domains of the Fv fragment, V L and V H are coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) .
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.
  • These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • an “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a BCMA protein is substantially free of antibodies that specifically bind antigens other than BCMA proteins) .
  • An isolated antibody that specifically binds a human BCMA protein may, however, have cross-reactivity to other antigens, such as BCMA proteins from other species.
  • an isolated antibody can be substantially free of other cellular material and/or chemicals.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • mouse antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from mouse germline immunoglobulin sequences.
  • the mouse antibodies of the disclosure can include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) .
  • the term “mouse antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto mouse framework sequences.
  • chimetic antibody refers to an antibody made by combining genetic material from a nonhuman source with genetic material from a human being.
  • a chimetic antibody is an antibody having genetic material from a certain species with genetic material from another species.
  • humanized antibody refers to an antibody from non-human species whose protein sequences have been modified to increase similarity to antibody variants produced naturally in humans.
  • isotype refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.
  • an antibody recognizing an antigen and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen. ”
  • an antibody that “specifically binds to human BCMA” is intended to refer to an antibody that binds to human BCMA protein (and possibly a BCMA protein from one or more non-human species) but does not substantially bind to non-BCMA proteins.
  • the antibody binds to human BCMA protein with “high affinity” , namely with a K D of 5.0 x10 -8 M or less, more preferably 1.0 x10 -8 M or less, and more preferably 7.0 x 10 -9 M or less.
  • does not substantially bind to a protein or cells, as used herein, means does not bind or does not bind with a high affinity to the protein or cells, i.e. binds to the protein or cells with a K D of 1.0 x 10 -6 M or more, more preferably 1.0 x 10 -5 M or more, more preferably 1.0 x 10 -4 M or more, more preferably 1.0 x 10 -3 M or more, even more preferably 1.0 x 10 -2 M or more.
  • high affinity for an IgG antibody refers to an antibody having a K D of 1.0 x 10 -6 M or less, more preferably 5.0 x 10 -8 M or less, even more preferably 1.0 x 10 -8 M or less, even more preferably 7.0 x 10 -9 M or less and even more preferably 1.0 x 10 -9 M or less for a target antigen.
  • “high affinity” binding can vary for other antibody isotypes.
  • “high affinity” binding for an IgM isotype refers to an antibody having a K D of 10 -6 M or less, more preferably 10 -7 M or less, even more preferably 10 -8 M or less.
  • K assoc or “K a ”
  • K dis or “K d ”
  • K D is intended to refer to the dissociation rate of a particular antibody-antigen interaction
  • K D is intended to refer to the dissociation constant, which is obtained from the ratio of K d to K a (i.e., K d /K a ) and is expressed as a molar concentration (M) .
  • K D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K D of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore TM system.
  • EC 50 also known as half maximal effective concentration, refers to the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time.
  • IC 50 also known as half maximal inhibitory concentration, refers to the concentration of an antibody which inhibits a specific biological or biochemical function by 50%relative to the absence of the antibody.
  • subject includes any human or nonhuman animal.
  • nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows and horses.
  • therapeutically effective amount means an amount of the antibody of the present disclosure sufficient to prevent or ameliorate the symptoms associated with a disease or condition (such as a cancer) and/or lessen the severity of the disease or condition.
  • a therapeutically effective amount is understood to be in context to the condition being treated, where the actual effective amount is readily discerned by those of skill in the art.
  • the antibody, or the antigen-binding portion thereof, of the disclosure specifically binds to human BCMA with comparable, if not better, binding affinity/capacity as compared to previously described anti-BCMA antibodies, such as the BCMA-binding portion of GSK2857916.
  • Additional functional properties include the capacity to block BCMA-BAFF interaction or BCMA-APRIL interacion.
  • antibodies of the disclosure are humanized monoclonal antibodies. Additionally or alternatively, the antibodies can be, for example, chimeric monoclonal antibodies.
  • the antibody of the disclosure is the monoclonal antibody structurally and chemically characterized as described below and in the following Examples.
  • the amino acid sequence ID numbers of the heavy/light chain variable regions of the antibodies are summarized in Table 1 below, some antibodies sharing the same V H or V L .
  • the heavy chain constant region for the antibodies may be human IgG1 heavy chain constant region having an amino acid sequence set forth in, e.g., SEQ ID NO: 23, and the light chain constant region for the antibodies may be human kappa constant region having an amino acid sequence set forth in, e.g., SEQ ID NO: 24.
  • These antibodies may also contain mouse IgG1 heavy chain constant region, and mouse kappa constant region.
  • the heavy chain variable region CDRs and the light chain variable region CDRs in Table 1 have been defined by the Kabat numbering system. However, as is well known in the art, CDR regions can also be determined by other systems such as Chothia, and IMGT, AbM, or Contact numbering system/method, based on heavy chain/light chain variable region sequences.
  • V H and V L sequences (or CDR sequences) of other anti-BCMA antibodies which bind to human BCMA can be “mixed and matched” with the V H and V L sequences (or CDR sequences) of the anti-BCMA antibody of the present disclosure.
  • V H and V L chains or the CDRs within such chains
  • a V H sequence from a particular V H /V L pairing is replaced with a structurally similar V H sequence.
  • a V L sequence from a particular V H /V L pairing is replaced with a structurally similar V L sequence.
  • an antibody of the disclosure, or an antigen binding portion thereof comprises:
  • an antibody of the disclosure, or an antigen binding portion thereof comprises:
  • the antibody, or antigen binding portion thereof includes the heavy chain variable CDR2 region of anti-BCMA antibody combined with CDRs of other antibodies which bind human BCMA, e.g., CDR1 and/or CDR3 from the heavy chain variable region, and/or CDR1, CDR2, and/or CDR3 from the light chain variable region of a different anti-BCMA antibody.
  • the CDR3 domain independently from the CDR1 and/or CDR2 domain (s) , alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, e.g., Klimka et al., , British J. of Cancer 83 (2) : 252-260 (2000) ; Beiboer et al., , J. Mol. Biol. 296: 833-849 (2000) ; Rader et al., , Proc. Natl. Acad. Sci. U.S.A.
  • antibodies of the disclosure comprise the CDR2 of the heavy chain variable region of the anti-BCMA antibody and at least the CDR3 of the heavy and/or light chain variable region of the anti-BCMA antibody, or the CDR3 of the heavy and/or light chain variable region of another anti-BCMA antibody, wherein the antibody is capable of specifically binding to human BCMA.
  • These antibodies preferably (a) compete for binding with BCMA; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the anti-BCMA antibody of the present disclosure.
  • the antibodies further may comprise the CDR2 of the light chain variable region of the anti-BCMA antibody, or the CDR2 of the light chain variable region of another anti-BCMA antibody, wherein the antibody is capable of specifically binding to human BCMA.
  • the antibodies of the disclosure may include the CDR1 of the heavy and/or light chain variable region of the anti-BCMA antibody, or the CDR1 of the heavy and/or light chain variable region of another anti-BCMA antibody, wherein the antibody is capable of specifically binding to human BCMA.
  • an antibody of the disclosure comprises a heavy and/or light chain variable region sequences of CDR1, CDR2 and CDR3 sequences which differ from those of the anti-BCMA antibodies of the present disclosure by one or more conservative modifications. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al., (1993) Biochem 32: 1180-8; de Wildt et al., (1997) Prot. Eng. 10: 835-41; Komissarov et al., (1997) J. Biol. Chem. 272: 26864-26870; Hall et al., (1992) J. Immunol.
  • the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:
  • the heavy chain variable region CDR1 sequence comprises a sequence listed in Table 1 above, and/or conservative modifications thereof;
  • the heavy chain variable region CDR2 sequence comprises a sequence listed in Table 1 above, and/or conservative modifications thereof; and/or
  • the heavy chain variable region CDR3 sequence comprises a sequence listed in Table 1 above, and conservative modifications thereof;
  • the light chain variable region CDR1, and/or CDR2, and/or CDR3 sequences comprise the sequence (s) listed in Table 1 above; and/or conservative modifications thereof;
  • the antibody specifically binds human BCMA.
  • the antibody of the present disclosure possesses one or more of the following functional properties described above, such as high affinity binding to human BCMA, and the ability to induce ADCC or CDC against BCMA-expressing cells.
  • the antibody can be, for example, a mouse, human, humanized or chimeric antibody.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • one or more amino acid residues within the CDR regions of an antibody of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth above) using the functional assays described herein.
  • Antibodies of the disclosure can be prepared using an antibody having one or more of the V H /V L sequences of the anti-BCMA antibody of the present disclosure as starting material to engineer a modified antibody.
  • An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., V H and/or V L ) , for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region (s) , for example to alter the effector function (s) of the antibody.
  • CDR grafting can be used to engineer variable regions of antibodies.
  • Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs) . For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs.
  • CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., (1998) Nature 332: 323-327; Jones et al., (1986) Nature 321: 522-525; Queen et al., (1989) Proc. Natl. Acad. Also see U.S.A. 86: 10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370) .
  • another embodiment of the disclosure pertains to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above, and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above. While these antibodies contain the V H and V L CDR sequences of the monoclonal antibody of the present disclosure, they can contain different framework sequences.
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database (available on the Internet at www. mrc-cpe. cam. ac. uk/vbase) , as well as in Kabat et al., (1991) , cited supra; Tomlinson et al., (1992) J. Mol. Biol. 227: 776-798; and Cox et al., (1994) Eur. J. Immunol. 24: 827-836; the contents of each of which are expressly incorporated herein by reference.
  • the germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database.
  • the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG--0010109, NT--024637 &BC070333) , 3-33 (NG--0010109 &NT--024637) and 3-7 (NG--0010109 &NT--024637) .
  • the following heavy chain germline sequences found in the HCo12 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG--0010109, NT--024637 &BC070333) , 5-51 (NG--0010109 &NT--024637) , 4-34 (NG--0010109 &NT--024637) , 3-30.3 (CAJ556644) &3-23 (AJ406678) .
  • Antibody protein sequences are compared against a compiled protein sequence database using one of the sequence similarity searching methods called the Gapped BLAST (Altschul et al., (1997) , supra) , which is well known to those skilled in the art.
  • V H CDR1, CDR2, and CDR3 sequences can be grafted onto framework regions that have the identical sequence (s) as that found in the germline immunoglobulin gene from which the framework sequence derives, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences.
  • the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences.
  • it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370.
  • variable region modification is to mutate amino acid residues within the V H and/or V L CDR1, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest.
  • Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation (s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as known in the art.
  • conservative modifications are introduced.
  • the mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions.
  • typically no more than one, two, three, four or five residues within a CDR region are altered.
  • the disclosure provides isolated anti-BCMA monoclonal antibodies, or antigen binding portions thereof, comprising a heavy chain variable region and a light chain variable region comprising: (a) a V H CDR1 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (b) a V H CDR2 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (c) a V H CDR3 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (d) a V L CDR1 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (e) a V L CDR2 region comprising the sequence of the present disclosure, or an amino acid sequence of the present disclosure, or
  • Engineered antibodies of the disclosure include those in which modifications have been made to framework residues within V H and/or V L , e.g. to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “backmutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043.
  • antibodies of the disclosure can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • modifications within the Fc region typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody of the disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the hinge region of C H1 is modified in such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425.
  • the number of cysteine residues in the hinge region of C H1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C H2 -C H3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcal protein A
  • the glycosylation of an antibody is modified.
  • a deglycosylated antibody can be made (i.e., the antibody lacks glycosylation) .
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the disclosure to thereby produce an antibody with altered glycosylation.
  • the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 ( ⁇ (1, 6) -fucosyltransferase) , such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.
  • the Ms704, Ms705, and Ms709 FUT8-/-cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87: 614-22) .
  • EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the ⁇ -1, 6 bond-related enzyme.
  • EP 1,176,195 also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662) .
  • PCT Publication WO 03/035835 describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn (297) -linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al., (2002) J. Biol. Chem. 277: 26733-26740) .
  • Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06/089231.
  • antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna. Methods for production of antibodies in a plant system are disclosed in the U.S.
  • the fucose residues of the antibody can be cleaved off using a fucosidase enzyme; e.g., the fucosidase ⁇ -L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14: 5516-23) .
  • a fucosidase enzyme e.g., the fucosidase ⁇ -L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14: 5516-23) .
  • An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG) , such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer) .
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C 1 -C 10 ) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See, e.g., EP 0 154 316 and EP 0 401 384.
  • Antibodies of the disclosure can be characterized by their various physical properties, to detect and/or differentiate different classes thereof.
  • antibodies can contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or an alteration of the pK of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41: 673-702; Gala and Morrison (2004) J Immunol 172: 5489-94; Wallick et al (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al (1985) Nature 316: 452-7; Mimura et al., (2000) Mol Immunol 37: 697-706) .
  • Glycosylation has been known to occur at motifs containing an N-X-S/T sequence.
  • an anti-BCMA antibody that does not contain variable region glycosylation. This can be achieved either by selecting antibodies that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation region.
  • the antibodies do not contain asparagine isomerism sites.
  • the deamidation of asparagine may occur on N-G or D-G sequences and result in the creation of an isoaspartic acid residue that introduces a link into the polypeptide chain and decreases its stability (isoaspartic acid effect) .
  • Each antibody will have a unique isoelectric point (pI) , which generally falls in the pH range between 6 and 9.5.
  • the pI for an IgG1 antibody typically falls within the pH range of 7-9.5 and the pI for an IgG4 antibody typically falls within the pH range of 6-8.
  • pI isoelectric point
  • an anti-BCMA antibody that contains a pI value that falls in the normal range. This can be achieved either by selecting antibodies with a pI in the normal range or by mutating charged surface residues.
  • the disclosure provides nucleic acid molecules that encode heavy and/or light chain variable regions, or CDRs, of the antibodies of the disclosure.
  • the nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques.
  • a nucleic acid of the disclosure can be, e.g., DNA or RNA and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • Nucleic acids of the disclosure can be obtained using standard molecular biology techniques.
  • cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.
  • antibodies obtained from an immunoglobulin gene library e.g., using phage display techniques
  • a nucleic acid encoding such antibodies can be recovered from the gene library.
  • Preferred nucleic acids molecules of the disclosure include those encoding the V H and V L sequences of the BCMA monoclonal antibody or the CDRs.
  • DNA fragments encoding V H and V L segments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a V L -or V H -encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term “operatively linked” is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the isolated DNA encoding the V H region can be converted to a full-length heavy chain gene by operatively linking the V H -encoding DNA to another DNA molecule encoding heavy chain constant regions (C H1 , C H2 and C H3 ) .
  • the sequences of human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG4 constant region.
  • the V H -encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain C H1 constant region.
  • the isolated DNA encoding the V L region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the V L -encoding DNA to another DNA molecule encoding the light chain constant region, C L .
  • the sequences of human light chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region.
  • the V H -and V L -encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser) 3, such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H regions joined by the flexible linker (see e.g., Bird et al., (1988) Science 242: 423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., , (1990) Nature 348: 552-554) .
  • a flexible linker e.g., encoding the amino acid sequence (Gly4-Ser) 3, such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H regions joined by the flexible linker (see e.g., Bird
  • Monoclonal antibodies (mAbs) of the present disclosure can be produced using the well-known somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256: 495.
  • Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques.
  • Chimeric or humanized antibodies are also well known in the art. See e.g., U.S. Pat. Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are specifically incorporated herein by reference in their entirety.
  • Antibodies of the disclosure also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202) .
  • DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences.
  • the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody genes.
  • promoters e.g., promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody genes.
  • enhancers e.g., polyadenylation signals
  • polyadenylation signals e.g., polyadenylation signals
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) , Simian Virus 40 (SV40) , adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • nonviral regulatory sequences can be used, such as the ubiquitin promoter or ⁇ -globin promoter.
  • regulatory elements composed of sequences from different sources, such as the SR ⁇ promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472) .
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors.
  • the variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the V H segment is operatively linked to the C H segment (s) within the vector and the V L segment is operatively linked to the C L segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) .
  • the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017) .
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection) .
  • DHFR dihydrofolate reductase
  • the expression vector (s) encoding the heavy and light chains is transfected into a host cell by standard techniques.
  • the various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • Preferred mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker, e.g., as described in R.J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159: 601-621) , NSO myeloma cells, COS cells and SP2 cells.
  • Chinese Hamster Ovary CHO cells
  • dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220
  • a DHFR selectable marker e.g., as described in R.J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159: 601-621
  • another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • Antibodies of the disclosure can be conjugated to a therapeutic agent to form an immunoconjugate such as an antibody-drug conjugate (ADC) .
  • Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents.
  • the antibody and therapeutic agent preferably are conjugated via a linker cleavable such as a peptidyl, disulfide, or hydrazone linker.
  • the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.
  • the ADCs can be prepared as described in U.S. Pat. Nos.
  • bispecific molecules comprising one or more antibodies of the disclosure linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules.
  • another functional molecule e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules.
  • bispecific molecule includes molecules that have two or more specificities.
  • a bispecific molecule has, in addition to an anti-Fc binding specificity and an anti-BCMA binding specificity, a third specificity.
  • the third specificity can be for an anti-enhancement factor (EF) , e.g., a molecule that binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell.
  • EF anti-enhancement factor
  • the anti-enhancement factor can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, or ICAM-1) or other immune cell, resulting in an increased immune response against the target cell.
  • Bispecific molecules may be in many different formats and sizes. At one end of the size spectrum, a bispecific molecule retains the traditional antibody format, except that, instead of having two binding arms of identical specificity, it has two binding arms each having a different specificity. At the other extreme are bispecific molecules consisting of two single-chain antibody fragments (scFv's) linked by a peptide chain, a so-called Bs (scFv) 2 construct. Intermediate-sized bispecific molecules include two different F (ab) fragments linked by a peptidyl linker. Bispecific molecules of these and other formats can be prepared by genetic engineering, somatic hybridization, or chemical methods.
  • an oncolytic virus preferabtially infects and kills cancer cells.
  • Antibodies of the present disclosure can be used in conjunction with oncolytic viruses.
  • oncolytic viruses encoding antibodies of the present disclosure can be introduced into human body.
  • CAR chimeric antigen receptor
  • the anti-BCMA CAR may comprise (a) an extracellular antigen binding domain comprising an anti-BCMA scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.
  • the CAR may contain a signal peptide at the N-terminus of the extracellular antigen binding domain that directs the nascent receptor into the endoplasmic reticulum, and a hinge peptide at the N-terminus of the extracellular antigen binding domain that makes the receptor more available for binding.
  • the CAR preferably comprises, at the intracellular signaling domain, a primary intracellular signaling domain and one or more co-stimulatory signaling domains.
  • the mainly used and most effective primary intracellular signaling domain is CD3-zeta cytoplasmic domain which contains ITAMs, the phosphorylation of which results in T cell activation.
  • the co-stimutory signaling domain may be derived from the co-stimulatory proteins such as CD28, CD137 or OX40.
  • the CARs may further add factors that enhance T cell expansion, persistence, and anti-tumor activity, such as cytokines, and co-stimulatory ligands.
  • the immune effector cell is a T cell, an NK cell, a peripheral blood mononuclear cell (PBMC) , a hematopoietic stem cell, a pluripotent stem cell, or an embryonic stem cell.
  • the immune effector cell is a T cell.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more antibodies (or antigen-binding portion thereof, or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates) of the present disclosure formulated together with a pharmaceutically acceptable carrier.
  • the antibodies (or antigen-binding portion thereof, or or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates) can be dosed separately when the composition contains more than one antibody (or antigen-binding portion thereof, or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates) .
  • the composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug, such as an anti-tumor drug.
  • the pharmaceutical composition can comprise any number of excipients.
  • Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof.
  • the selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams &Wilkins 2003) , the disclosure of which is incorporated herein by reference.
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) .
  • the active ingredient can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • an antibody of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.
  • compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01%to about ninety-nine percent of active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1%to about 30%of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response) .
  • a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • antibody can be administered as a sustained release formulation, in which case less frequent administration is required.
  • the dosage may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg body weight, of the host body weight.
  • dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg body weight.
  • An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.
  • Preferred dosage regimens for an anti-BCMA antibody of the disclosure include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.
  • dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 ⁇ g/ml and in some methods about 25-300 ⁇ g/ml.
  • a “therapeutically effective dosage” of an anti-BCMA antibody, or the antigen-binding portion thereof, or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates of the disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • a “therapeutically effective dosage” preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%relative to untreated subjects.
  • a therapeutically effective amount of a therapeutic antibody can decrease tumor size, or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.
  • the pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556) ; (2) micro-infusion pumps (U.S. Pat. No. 4,487,603) ; (3) transdermal devices (U.S. Pat. No. 4,486,194) ; (4) infusion apparatuses (U.S. Pat. Nos. 4,447,233 and 4,447,224) ; and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196) ; the disclosures of which are incorporated herein by reference.
  • medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413;
  • the monoclonal antibodies of the disclosure can be formulated to ensure proper distribution in vivo.
  • they can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V. V. Ranade (1989) J. Clin. Pharmacol. 29: 685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038; Bloeman et al., (1995) FEBS Lett.
  • composition comprising the antibodies or the antigen-binding portion thereof, or the bispecific, CAR-T cells, oncolytic viruses, immunoconjugates of the present disclosure have numerous in vitro and in vivo utilities involving, for example, treatment of tumors associated with increased BCMA expression.
  • the disclosure provides methods for inhibiting growth of tumor cells in a subject comprising administering to the subject the composition of the disclosure such that growth of the tumor is inhibited in the subject.
  • tumors that can be treated by antibodies of the disclosure include, but not limited to, such as leukemia, lymphomas and multiple myeloma. Additionally, refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the disclosure.
  • the antibodies of the disclosure can be used to enhance an immune response in a subject.
  • the disclosure provides methods of combination therapy in which the anti-BCMA antibodies, or antigen-binding portion thereof, or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates of the present disclosure are co-administered with one or more additional antibodies that are effective in inhibiting tumor growth in a subject.
  • the disclosure provides a method for inhibiting tumor growth in a subject comprising administering to the subject an anti-BCMA antibody (or antigen-binding portion thereof, or the CAR-T cell, oncolytic virus, immunoconjugate) and one or more additional antibodies, such as an anti-OX40 antibody, an anti-TIM-3 antibody, an anti-CD137 antibody, an anti-GITR antibody, an anti-LAG-3 antibody, an anti-PD-L1 antibody, and anti-PD-1 antibody and/or an anti-CTLA-4 antibody.
  • the subject is human.
  • BCMA signaling activation can also be further combined with standard cancer treatments.
  • BCMA signaling activationa can be combined with CTLA-4 and/or LAG-3 and/or PD-1 blockade and also chemotherapeutic regimes.
  • a chemotherapeutic agent can be administered with the anti-BCMA antibodies, which may be a cytotoxic agent.
  • epirubicin, oxaliplatin, and 5-FU are administered to patients receiving anti-BCMA therapy.
  • the combination of anti-BCMA and one or more additional antibodies can be further combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) , and cells transfected with genes encoding immune stimulating cytokines (He et al., (2004) J. Immunol. 173: 4919-28) .
  • an immunogenic agent such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) , and cells transfected with genes encoding immune stimulating cytokines (He et al., (2004) J. Immunol. 173: 4919-28) .
  • Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.
  • IL-2 interleukin-2
  • Other therapies that may be combined with anti-BCMA antibody includes, but not limited to, interleukin-2 (IL-2) administration, radiation, surgery, or hormone deprivation.
  • IL-2 interleukin-2
  • combination of therapeutic agents discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.
  • sequential administration can be reversed or kept in the same order at each time point of administration, sequential administrations can be combined with concurrent administrations, or any combination thereof.
  • the complete Freud's adjuvant and incomplete Freud's adjuvant were used respectively for primary and boost immunizations.
  • adjuvant-antigen mixture was prepared by first gently mixing the adjuvant in a vial using a vortex. The desired amount of adjuvant was transferred to an autoclaved 1.5 mL micro-centrifuge tube. The antigen was prepared in PBS or saline with concentration ranging from 0.5-1.0 mg/ml. The calculated amount of antigen was then added to the micro-centrifuge tube with the adjuvant, and the resulting mixture was mixed by gently vortexing for 2 minutes to generate water-in-oil emulsions.
  • the adjuvant-antigen emulsion was then drawn into the proper syringe for animal injection. A total of 25 ⁇ g of antigen was injected in a volume of 50-100 ⁇ l. Each animal was immunized, and then boosted for 2 to 3 times depending on the anti-sera titer. Animals with good titers were given a final boost by intraperitoneal injection before hybridoma fusion.
  • ELISA-based screening using recombinant human BCMA-his protein. Briefly, ELISA plates were coated with 60 ⁇ l of human BCMA-his (Cat#BCA-H522Y, Acro biosystems, 2.0 ⁇ g/ml in PBS) overnight at 4°C. Plates were washed 4 times with PBST and blocked with 200 ⁇ l blocking buffer (5%w/v non-fatty milk in PBST) . Diluted hybridoma supernatant (60 ⁇ l) was added to each well and incubated at 37°Cfor 40 minutes.
  • Protein A sepharose column (from bestchrom (Shanghai) Biosciences, Cat#AA0273) was washed using PBS buffer in 5 to 10 column volumes. Cell supernatants were passed through the columns, and then the columns were washed using PBS buffer until the absorbance for protein reached the baseline. The columns were eluted with elution buffer (0.1 M Glycine-HCl, pH 2.7) , and immediately collected into 1.5 ml tubes with neutralizing buffer (1 M Tris-HCl, pH 9.0) . Fractions containing immunoglobulins were pooled and dialyzed in PBS overnight at 4°C. Subsequently, the in vitro functional activities of purified monoclonal antibodies were characterized as follows.
  • the purified mouse anti-BCMA monoclonal antibodies (mAbs) generated in Example 1 were characterized for affinities and binding kinetics by Biacore T200 system (GE healthcare, Pittsburgh, PA, USA) .
  • CM5 chip carboxy methyl dextran coated chip
  • Biacore GE healthcare, Pittsburgh, PA, USA
  • BCMA-mab1 or BM an antibody containing BCMA-binding portion of GSK2857916, also referred to as BCMA-mab1 or BM, prepared using the heavy chain and light chain amino acids set forth in SEQ ID NOs.: 26 and 27
  • concentration of 10.0 ⁇ g/ml were flowed onto the chip at a flow rate of 10 ⁇ L/min.
  • the antibodies of the present disclosure specifically bound to human BCMAs, and their binding affinities were comparable to or a bit better than the benchmark.
  • the B1A1, B1G3 and B1C1 antibodies also bound to monkey BCMA specifically.
  • mouse anti-BCMA antibodies were determined by Capture ELISA and Flow Cytometry (FACS) .
  • 96-well micro plates were coated with 2 ⁇ g/ml goat anti-mouse IgG Fc ⁇ fragment specific (Jackson Immuno Research, Cat#115-005-071, 100 ⁇ l/well, for mouse anti-BCMA antibodies of the discolsure) or goat anti-human IgG F (ab’) 2 fragment (Jackson Immuno Research, Cat#109-005-097, 100 ⁇ l/well, for the benchmark BM) in PBS and incubated overnight at 4°C. Plates were washed 4 times with wash buffer (PBS+0.05%Tween-20, PBST) and then blocked with 200 ⁇ l/well blocking buffer (5%w/v non-fatty milk in PBST) for 2 hours at 37°C.
  • wash buffer PBS+0.05%Tween-20, PBST
  • 200 ⁇ l/well blocking buffer 5%w/v non-fatty milk in PBST
  • mice were washed again and incubated with 100 ⁇ l/well purified mouse anti-BCMA antibodies, the benchmark BM, and negative control hIgG (human immunoglobulin (pH4) for intravenous injection, Hualan Biological Engineering Inc. ) (5-fold dilution in 2.5%non-fatty milk in PBST, starting with 10000 ng/ml or 66.67 nM) for 40 minutes at 37°C, and then washed 4 times again.
  • hIgG human immunoglobulin (pH4) for intravenous injection, Hualan Biological Engineering Inc.
  • Plates containing captured anti-BCMA antibodies were incubated with biotin-labeled human BCMA-his protein (Cat#BCA-H522Y, Acro biosystems, 0.28 nM in 2.5%non-fatty milk in PBST, 100 ⁇ l/well) for 40 minutes at 37°C, washed 4 times, and incubated with streptavidin conjugated HRP (1: 10000 dilution in PBST, Jackson Immuno Research, Cat#016-030-084, 100 ⁇ l/well) for 40 minutes at 37°C. After a final wash, plates were incubated with 100 ⁇ l/well ELISA substrate TMB (Innoreagents, Cat#TMB-S-002) .
  • the reaction was stopped in 15 minutes at 25°C with 50 ⁇ l/well 1M H 2 S0 4 , and the absorbance was read at 450-630 nm and plotted against antibody concentration. Data were analyzed using Graphpad Prism software and EC 50 values were reported.
  • TIB-196 TM human myeloma cell line U266 (TIB-196 TM ) were harvested from cell culture flasks, washed two times and resuspended in phosphate buffered saline (PBS) containing 2%v/v Fetal Bovine Serum (FACS buffer) . 2 x 10 5 cells per well in 96 well-plates were incubated with the anti-BCMA antibodies or controls of various concentrations in FACS buffer for 40 minutes on ice.
  • PBS phosphate buffered saline
  • FACS buffer Fetal Bovine Serum
  • the ability of the anti-BCMA antibodies of the present disclosure to block Benchmark BM-human BCMA binding was measured using a competitive ELISA assay. Briefly, the benchmark was coated on 96-well micro plates at 2 ⁇ g/mL in PBS, 100 ⁇ l per well, and incubated overnight at 4°C. The next day, plates were washed with wash buffer, and blocked with 5%w/v non-fatty milk in PBST for 2 hours at 37°C.
  • variable domains of the heavy and light chain of the anti-BCMA mouse mAbs were sequenced, and the sequence ID numbers were summarized in Table 1.
  • variable domains of the heavy and light chain of the anti-BCMA mouse mAbs were cloned in frame to human IgG1 heavy-chain (SEQ ID NO.: 23) and human kappa light-chain constant regions (SEQ ID NO.: 24) , respectively, wherein the C terminus of variable region was linked to the N terminus of the respective constant region.
  • Mouse anti-BCMA antibodies B1G3 and B1C1 were selected for humanization and further investigations. Humanization of the mouse antibody was conducted using the well-established CDR-grafting method as described in detail below.
  • mouse antibodies B1H2, B1G3 and B1C1 To select acceptor frameworks for humanization of mouse antibodies B1H2, B1G3 and B1C1, the light and heavy chain variable region sequences of mouse B1H2, B1G3 and B1C1 were blasted against the human immunoglobulin gene database. The human germlines with the highest homology to mouse B1G3 and B1C1 were selected as the acceptor frameworks for humanization. The mouse antibody heavy/light chain variable region CDRs were inserted into the selected frameworks, and the residue (s) in the frameworks was/were further mutated to obtain more candidate heavy chain/light chain variable regions.
  • a total of 15 humanized B1G3 antibodies (namely huB1G3-V1 to huB1G3-V15) , and 12 humanized B1C1 antibodies (huB1C1-V1 to huB1C1-V9, and huB1C1-V11 to huB1C1-V13) , were obtained whose heavy/light chain variable region sequence ID numbers were in Table 1.
  • the vectors each containing a nucleotide encoding a humanized B1G3/B1C1 heavy chain variable region linked to human IgG1 heavy-chain constant region (SEQ ID NO: 23)
  • the vectors each containing a nucleotide encoding a humanized light chain variable region linked to human kappa light-chain constant region (SEQ ID NO: 24) were transiently transfected into 20 ml of 293F suspension cell cultures in a ratio of 47.62%to 52.38%light to heavy chain construct, with 60 ⁇ g/mL PEI.
  • Cell supernatants were harvested after six days in shaking flasks, spun down to pellet cells, and filtered through 0.22 ⁇ m filters for immunoglubulin separation.
  • the antibodies were purified by protein A affinity chromatography. Briefly, Protein A sepharose column (from bestchrom (Shanghai) Biosciences, Cat#AA0273) was washed 5 to 10 column volumes using PBS buffer. Cell supernatants were passed through the columns, and then the the columns were washed using PBS buffer until the absorbance for protein reached the baseline. The columns were eluted with elution buffer (0.1 M Glycine-HCl, pH 2.7) , and immediately collected into 1.5 ml tubes with neutralizing buffer (1 M Tris-HCl, pH 9.0) . Fractions containing Immunoglobulins were pooled and dialyzed in PBS overnight at 4°C.
  • elution buffer 0.1 M Glycine-HCl, pH 2.7
  • the humanized B1G3 and B1C1 antibodies were also tested for their binding affinities/capacities to human and cynomolgus BCMA and other functional activities by Biacore, capture ELISA, and competitive assays, following the protocols in the foregoing Examples.
  • capture ELISA 96-well micro plates were coated with 2 ⁇ g/ml goat anti-human IgG (AffiniPure Goat Anti-Human IgG, F (ab') 2 fragment specific, Jackson Immunoresearch, Cat#109-005-097) , and a mouse control antibody binding a non-BCMA protein (in house made) was additionally used.
  • Capture ELISA results were summarized in Table 6 above and shown in Fig. 4A-4C and 5A-5C. It can be seen that huB1G3-V8 and huB1C1-V6 did not bind to human BCMA, and the remaining antibodies had comparable or a bit lower binding activities compared to their parent antibodies or the benchmark.
  • the humanized antibodies huB1G3-V13 and huB1C1-V13 were subject to further characterizations. In brief, these two antibodies were tested for their binding capativies to human and monkey BCMAs, and also their capacities of blocking BCMA-BAFF or benchmark-BCMA interaction, following the protocols described above.
  • the antibody huB1G3-V13 showed good binding activity to human BCMA in the Capture ELISA assay, which was comparable to the benchmark (Fig. 6) , and a bit lower binding affinity to human or monkey BCMA than the benchmark. Further, this antibody can effectively block BCMA-BAFF interaction with almost the same blocking capacity compared to the benchmark (Fig. 7) , and can also block benchmark-BCMA interaction (Fig. 8) .
  • the antibody huB1C1-V13 showed good binding activity to human BCMA in the Capture ELISA assay, which was a bit lower than the benchmark (Fig. 9) , and also a bit lower binding affinity to human or monkey BCMA than the benchmark. Further, this antibody can effectively block BCMA-BAFF interaction with almost the same blocking capacity compared to the benchmark (Fig. 10) , and can also block benchmark-BCMA interaction (Fig. 11) .

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Abstract

Provided is an isolated monoclonal antibody that specifically binds human BCMA, or the antigen-binding portion thereof. A nucleic acid molecule encoding the antibody or the antigen-binding portion thereof, an expression vector, a host cell and a method for expressing the antibody or the antigen-binding portion thereof are also provided. Further provided are an immunoconjugate, a bispecific molecule, a chimeric antigen receptor, an oncolytic virus and a pharmaceutical composition comprising the antibody or the antigen-binding portion thereof, as well as a treatment method using an anti-BCMA antibody or the antigen-binding portion thereof.

Description

ANTIBODIES BINDING BCMA AND USES THEREOF FIELD OF THE INVENTION
The present disclosure relates generally to an isolated monoclonal antibody, particularly a mouse, chimeric or humanized monoclonal antibody, or the antigen-binding portion thereof, that specifically binds to human BCMA with high affinity and functionality. A nucleic acid molecule encoding the antibody or the antigen-binding portion thereof, an expression vector, a host cell and a method for expressing the antibody or the antigen-binding portion thereof are also provided. The present disclosure further provides an immunoconjugate, a bispecific molecule, a chimeric antigen receptor, an oncolytic virus, and a pharmaceutical composition comprising the antibody or the antigen-binding portion thereof, as well as a diagnostic or treatment method using an anti-BCMA antibody or antigen-binding portion thereof of the disclosure.
BACKGROUND OF THE INVENTION
B cell maturation antigen (BCMA) , also termed tumor necrosis factor receptor superfamily member 17 (TNFRS17) , is a transmembrane protein having cysteine-rich extracellular domain (Madry C et al., (1998) Int Immunol. 10 (11) : 1693–702; Laabi Y et al., (1994) Nucleic Acids Res 22 (7) : 1147–54; Laabi Y et al., (1992) EMBO J 11 (11) : 3897–904) . It, along with two TNFR superfamily members B-cell activation factor receptor (BAFF-R) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) , regulates humoral immunity, B-cell development and homeostasis, especially B cell proliferation, survival, maturation and differentiation into plasma cells (PC) (Mackay F et al., (2003) Annu Rev Immunol 21: 231–64; Marsters SA et al., (2000) Curr Biol 10 (13) : 785–8; Gross JA et al., (2000) , Nature 404 (6781) : 995–9; Thompson JS et al., (2000) J Exp Med 192 (1) : 129–35) . BCMA is also important for long-lived PC survival.
BCMA is exclusively expressed on the surfaces of plasmablasts and differentiated PCs, and more highly on malignant than normal PCs (Carpenter RO et al., (2013) Clin Cancer Res 19 (8) : 2048–60; O’Connor BP et al., (2004) J Exp Med 199 (1) : 91–8; Benson MJ et al., (2008) J Immunol 180 (6) : 3655–9; Yang M et al., (2005) J Immunol 175 (5) : 2814–24; Avery DT et al., (2003) J Clin Invest 112 (2) : 286–97; Novak AJ et al., (2004) Blood 103 (2) : 689–94; Chiu A et al., (2007) Blood 109 (2) : 729–39; Lee L et al., (2018) Blood 131 (7) : 746–58; Carpenter RO et al., (2013) supra; Tai YT et al., (2014) Blood 123 (20) : 3128–38; Claudio JO et al., (2002) Blood 100 (6) : 2175–86; Seckinger A et al., (2017) Cancer Cell 31 (3) : 396–410) . It has been implicated in leukemia, lymphomas and multiple myeloma. For example, elevated plasma BCMA levels were observed in patients with chronic lymphocytic leukemia, which were correlated with clinical statuses (Kyle A Udd et al., (2015) Blood. 126 (23) : 2931) . Also, BCMA was found to be highly expressed in multiple myeloma tumor cells, and BCMA overexpression or APRIL binding to BCMA in these cells significantly promotes cell growth and survival in vivo (Tai YT et al., (2016) Blood 127 (25) :  3225–36; Matthes T et al., (2011) Blood 118 (7) : 1838–44) . Further, APRIL and BAFF, via binding to BCMA and TACI, further activate NFκB pathways and upregulate antiapoptotic proteins (Mcl-1, Bcl-2, Bcl-xL) to protect multiple myeloma tumor cells against dexamethasone-and serum deprivation-induced cell death (Moreaux J et al., (2004) Blood 103 (8) : 3148–57; Neri P et al., (2007) Clin Cancer Res 13 (19) : 5903–9; Shen X et al., (2016) Cell Biochem Funct 34 (2) : 104–10) .
As the second prevalent hematopoietic malignancy, multiple myeloma is a clonal B-cell malignancy that occurs in multiple sites within the bone marrow before spreading to the circulation; either de novo, or as a progression from monoclonal gammopathy of undetermined significance (MGUS) . It is commonly characterized by increases in paraprotein and osteoclast activity, as well as hypercalcaemia, cytopenia, renal dysfunction, hyperviscosity and peripheral neuropathy. Decreases in both normal antibody levels and numbers of neutrophils are also common, leading to a life-threatening susceptibility to infection. Two monoclonal antibodies targeting CD38 and SLAMF7 were approved in late 2015 for the treatment of relapsed and refractory multiple myoloma, but the wide expression of two antigens in normal tissues limited their long-term clinical use. On contrast, BCMA has emerged as a potential therapeutic target because of its restricted expression pattern and transmembrane structure. The first therapeutic anti-BCMA antibody-drug conjugate (ADC) , GSK2857916, rapidly eliminates multiple myeloma cells in two murine models and significantly prolongs survival of mice (Tai YT et al., (2014) supra) . Another anti-BCMA ADC, HDP-101, has shown in vitro cytotoxicity against multiple myeloma cells at picomolar range, resulting in significant tumor regression with good toleration. Several anti-BCMA CAR-T cell therapies have also shown impressive clinical outcomes (Shih-Feng Cho et al., (2018) Frontiers in Immunology 9: 1821) .
Ongoing efforts are attempting to find more BCMA binding moieties, including antibodies, that are safer and more potent.
SUMMARY OF THE INVENTION
The present disclosure provides an isolated monoclonal antibody, for example, a mouse, human, chimeric or humanized monoclonal antibody, or an antigen-binding portion thereof, that binds to BCMA (e.g., the human BCMA, and monkey BCMA) and has comparable, or higher, binding affinity to BCMA as compared to prior art anti-BCMA antibodies such as the BCMA-binding portion of GSK2857916.
The antibody or antigen-binding portion of the disclosure can be used for a variety of applications, including detection of the BCMA protein, and treatment and prevention of BCMA associated diseases, such as cancers, autoimmune diseases, and infectious diseases.
Accordingly, in one aspect, the disclosure pertains to an isolated monoclonal antibody (e.g., a mouse, chimeric or humanized antibody) , or an antigen-binding portion thereof, that binds BCMA, having a heavy chain variable region that comprises a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and  the CDR3 region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to (1) SEQ ID NOs: 1 (X1=H or F) , 3 and 5, respectively; or (2) SEQ ID NOs: 2, 4 and 6, respectively.
In one aspect, an isolated monoclonal antibody, or an antigen-binding portion thereof, of the present disclosure comprises a heavy chain variable region comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to SEQ ID NOs: 13, 14 (X1=I, X2=A, X3=T, X4=C, X5=F; X1=V, X2=A, X3=T, X4=C, X5=F; X1=I, X2=V, X3=T, X4=C, X5=F; X1=I, X2=A, X3=S, X4=C, X5=F; X1=I, X2=A, X3=Y, X4=C, X5=Y; X1=V, X2=V, X3=S, X4=S, X5=Y; or X1=V, X2=V, X3=S, X4=C, X5=Y) , 15, 16 (X1=I, X2=N, X3=F; X1=V, X2=N, X3=F; X1=I, X2=T, X3=F; X1=I, X2=N, X3=Y; or X1=V, X2=T, X3=Y) , or 17, wherein the antibody or antigen-binding portion thereof binds to BCMA.
In one aspect, an isolated monoclonal antibody, or an antigen-binding portion thereof, of the present disclosure comprises a light chain variable region that comprises a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region, and the CDR3 region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to (1) SEQ ID NOs: 7 (X1=H or F) , 9 (X1=N or T) and 11, respectively; or (2) SEQ ID NOs: 8, 10 and 12, respectively. In some embodiments, the CDR1 region, the CDR2 region, and the CDR3 region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to (1) SEQ ID NOs: 7 (X1=H) , 9 (X1=N) and 11, respectively; (2) SEQ ID NOs: 7 (X1=F) , 9 (X1=T) and 11, respectively; or (3) SEQ ID NOs: 8, 10 and 12, respectively.
In one aspect, an isolated monoclonal antibody, or an antigen-binding portion thereof, of the present disclosure comprises a light chain variable region comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to SEQ ID NOs: 18, 19 (X1=L, X2=P, X3=P, X4=W, X5=Y; X1=M, X2=P, X3=P, X4=W, X5=Y; X1=L, X2=A, X3=P, X4=W, X5=Y; X1=L, X2=P, X3=L, X4=W, X5=Y; X1=L, X2=P, X3=P, X4=L, X5=Y; X1=L, X2=P, X3=P, X4=W, X5=F; or X1=M, X2=A, X3=P, X4=L, X5=F) , 20, 21 (X1=S, X2=P, X3=W, X4=Y, X5=F; X1=A, X2=P, X3=W, X4=Y, X5=F; X1=S, X2=L, X3=W, X4=Y, X5=F; X1=S, X2=P, X3=L, X4=Y, X5=F; X1=S, X2=P, X3=W, X4=F, X5=F; X1=S, X2=P, X3=W, X4=Y, X5=Y; X1=A, X2=P, X3=L, X4=F, X5=Y) or 22, wherein the antibody or antigen-binding portion thereof binds to BCMA.
In one aspect, an isolated monoclonal antibody, or an antigen-binding portion thereof, of the present disclosure comprises a heavy chain variable region and a light chain variable region each comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the heavy chain variable region CDR1, CDR2 and CDR3, and the light chain variable region CDR1, CDR2 and CDR3 comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to (1)  SEQ ID NOs: 1 (X1=H) , 3, 5, 7 (X1=H) , 9 (X1=N) and 11, respectively; (2) SEQ ID NOs: 1 (X1=F) , 3, 5, 7 (X1=F) , 9 (X1=T) and 11, respectively; or (3) SEQ ID NOs: 2, 4, 6, 8, 10 and 12, respectively, wherein the antibody or antigen-binding portion thereof binds to BCMA.
In one embodiment, an isolated monoclonal antibody, or the antigen-binding portion thereof, of the present disclosure comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to (1) SEQ ID NOs: 13 and 18, respectively; (2) SEQ ID NOs: 14 (X1=I, X2=A, X3=T, X4=C, X5=F; X1=V, X2=A, X3=T, X4=C, X5=F; X1=I, X2=V, X3=T, X4=C, X5=F; X1=I, X2=A, X3=S, X4=C, X5=F; X1=I, X2=A, X3=Y, X4=C, X5=Y; X1=V, X2=V, X3=S, X4=S, X5=Y; or X1=V, X2=V, X3=S, X4=C, X5=Y) and 19 (X1=L, X2=P, X3=P, X4=W, X5=Y) , respectively; (3) SEQ ID NOs.: 14 (X1=I, X2=A, X3=T, X4=C, X5=F) and 19 (X1=M, X2=P, X3=P, X4=W, X5=Y; X1=L, X2=A, X3=P, X4=W, X5=Y; X1=L, X2=P, X3=L, X4=W, X5=Y; X1=L, X2=P, X3=P, X4=L, X5=Y; or X1=L, X2=P, X3=P, X4=W, X5=F) , respectively; (4) SEQ ID NOs.: 14 (X1=I, X2=A, X3=T, X4=C, X5=F; or X1=V, X2=V, X3=S, X4=C, X5=Y) and 19 (X1=M, X2=A, X3=P, X4=L, X5=F) , respectively; (5) SEQ ID NOs.: 15 and 20, respectively; (6) SEQ ID NOs.: 16 (X1=I, X2=N, X3=F; X1=V, X2=N, X3=F; X1=I, X2=T, X3=F; X1=I, X2=N, X3=Y; or X1=V, X2=T, X3=Y) and 21 (X1=S, X2=P, X3=W, X4=Y, X5=F) , respectively; (7) SEQ ID NOs.: 16 (X1=I, X2=N, X3=F) and 21 (X1=A, X2=P, X3=W, X4=Y, X5=F; X1=S, X2=L, X3=W, X4=Y, X5=F; X1=S, X2=P, X3=L, X4=Y, X5=F; X1=S, X2=P, X3=W, X4=F, X5=F; or X1=S, X2=P, X3=W, X4=Y, X5=Y) , respectively; (8) SEQ ID NOs.: 16 (X1=I, X2=N, X3=F; or X1=V, X2=T, X3=Y) and 21 (X1=A, X2=P, X3=L, X4=F, X5=Y) , respectively; or (9) SEQ ID NOs.: 17 and 22, respectively.
In one embodiment, an isolated monoclonal antibody, or the antigen-binding portion thereof, of the present disclosure comprises a heavy chain and a light chain linked by disulfite bonds, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, the light chain comprising a light chain variable region and a light chain constant region, wherein the C terminus of the heavy chain variable region is linked to the N terminus of the heavy chain constant region, and the C terminus of the light chain variable region is linked to the N terminus of the light chain constant region, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences described above, and the antibody or antigen-binding portion thereof binds to BCMA. The heavy chain constant region may be human IgG1 constant region having an amino acid sequence set forth in e.g., SEQ ID No: 23, and the light chain constant region may be human kappa constant region having an amino acid sequences set forth in e.g., SEQ ID No: 24. The amino acid sequences of SEQ ID NOs.: 23 and 24 may be encoded by nucleotide sequences set forth in SEQ ID NOs.: 28 and 29, respectively.
The antibody of the present disclosure in some embodiments comprises or consists of two heavy chains and two light chains, wherein each heavy chain comprises the heavy chain constant region, heavy chain variable region or CDR sequences mentioned above, and each light chain comprises the light chain constant region, light chain variable region or CDR sequences mentioned above, wherein the antibody binds to BCMA. The antibody of the disclosure can be a full-length antibody, for example, of an IgG1, IgG2 or IgG4 isotype. The light chain constant region may be a kappa or lambda constant region. The antibody of the present disclosure in other embodiments may be a single chain variable fragment (scFv) antibody, or antibody fragments, such as Fab or Fab′2 fragments.
The antibody, or antigen-binding portion thereof, of the present disclosure has comparable, if not higher, binding affinity/capacity to human BCMA and monkey BCMA than prior art anti-BCMA antibodies such as the BCMA binding portion of GSK2857916, and inhibits the binding of BAFF or APRIL to BCMA.
The disclosure also provides an immunoconjugate comprising an antibody or antigen-binding portion thereof of the disclosure, linked to a therapeutic agent, such as a cytotoxin. The disclosure also provides a bispecific molecule comprising an antibody, or antigen-binding portion thereof, of the disclosure, linked to a second functional moiety (e.g., a second antibody) having a different binding specificity than said antibody, or antigen-binding portion thereof. In another aspect, the antibody or an antigen binding portions thereof of the present disclosure can be made into part of a chimeric antigen receptor (CAR) . Also provided is an immune cell comprising the chimeric antigen receptor, such as a T cell. The antibody or an antigen binding portion thereof of the present disclosure can also be encoded by or used in conjuction with an oncolytic virus.
Pharmaceutical compositions comprising the antibody, or antigen-binding portion thereof, or immunoconjugate, bispecific molecule, oncolytic virus, CAR or CAR-T cell of the disclosure, and a pharmaceutically acceptable carrier, are also provided. In some embodiments, the pharmaceutical composition may further contain an anti-tumor agent and/or a cytokine.
Nucleic acid molecules encoding the antibodies, or antigen-binding portions thereof, of the disclosure are also encompassed by the disclosure, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors. A method for preparing an anti-BCMA antibody or an antigen-binding portion thereof using the host cell comprising the expression vector is also provided, comprising steps of (i) expressing the antibody in the host cell and (ii) isolating the antibody from the host cell or its cell culture.
In yet another aspect, the disclosure provides a method for treating a tumor associated with increased BCMA expression, comprising administering to a subject a therapeutically effective amount of the antibody, or antigen-binding portion thereof, of the present disclosure. The tumor may be non-solid tumors, including leukemia, lymphomas, multiple myeloma, etc, or solid tumors. In some embodiments, the method comprises administering a composition, a bispecific molecule, an immunnoconjugate such as an  antibody-drug conjugate, a CAR-T cell, or an antibody-encoding or antibody-bearing oncolytic virus of the disclosure, or alternatively a nucleic acid molecule or a vector capable of expressing the same in the subject. In some embodiments, at least one additional anti-cancer antibody can be administered with the antibody, or an antigen-binding portion thereof, of the disclosure, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, and/or an anti-TIM 3 antibody. In yet another embodiment, an antibody, or an antigen-binding portion thereof, of the disclosure is administered with a cytokine (e.g., IL-2, IL-21, TNF-α, IFN-γ and/or IL-4) , or a costimulatory antibody (e.g., an anti-CD137 antibody, an anti-OX40 antibody and/or anti-GITR antibody) . The antibodies of the present disclosure can be, for example, mouse, human, chimeric or humanized antibodies.
Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1A and 1B show the binding capacities of mouse antibodies B1A1 and B1C1 (A) , and B1G3 (B) to human BCMA.
Fig. 2A and 2B show the blocking abilities of mouse antibodies B1A1 and B1C1 (A) , and B1G3 (B) on human BCMA-BAFF interaction.
Fig. 3A and 3B show the abilities of mouse antibodies B1A1 and B1C1 (A) , and B1G3 (B) to block Benchmark-human BCMA binding.
Fig. 4A-4C show the binding capacities of humanized antibodies huB1G3-V1 to huB1G3-V5 (A) , huB1G3-V6 to huB1G3-V10 (B) and huB1G3-V11 to huB1G3-V15 (C) to human BCMA.
Fig. 5A-5C show the binding capacities of humanized antibodies huB1C1-V1 to huB1C1-V7 (A) , huB1C1-V8 to huB1C1-V9 (B) and huB1C1-V11 to huB1C1-V13 (C) to human BCMA.
Fig. 6 shows the binding capacity of humanized antibody huB1G3-V13 to human BCMA.
Fig. 7 shows the blocking ability of humanized antibody huB1G3-V13 on human BCMA-BAFF interaction.
Fig. 8 shows the blocking ability of humanized antibody huB1G3-V13 on Benchmark-human BCMA interaction.
Fig. 9 shows the binding capacity of humanized antibody huB1C1-V13 to human BCMA.
Fig. 10 shows the blocking ability of humanized antibody huB1C1-V13 on human BCMA-BAFF interaction.
Fig. 11 shows the blocking ability of humanized antibody huB1C1-V13 on Benchmark -human BCMA interaction.
DETAILED DESCRIPTION OF THE INVENTION
To ensure that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
The term “BCMA” refers to B cell maturation antigen, or tumor necrosis factor receptor superfamily member 17. The term “BCMA” comprises variants, isoforms, homologs, orthologs and paralogs. For example, an antibody specific for a human BCMA protein may, in certain cases, cross-react with a BCMA protein from a species other than human, such as monkey. In other embodiments, an antibody specific for a human BCMA protein may be completely specific for the human BCMA protein and exhibit no cross-reactivity to other species or of other types, or may cross-react with BCMA from certain other species but not all other species.
The term “human BCMA” refers to a BCMA protein having an amino acid sequence from a human, such as the amino acid sequence of human BCMA having a Genbank accession number of NP_001183.2, or the amino acid sequence set forth in SEQ ID NO.: 25. The terms “monkey or rhesus BCMA” and “mouse BCMA” refer to monkey and mouse BCMA sequences, respectively, e.g. those with the amino acid sequences having Genbank Accession Nos. XP_001106892.1 and NP_035738.1, respectively.
The term “antibody” as referred to herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion” ) or single chains thereof. Whole antibodies are glycoproteins comprising two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C H1, C H2 and C H3. Each light chain is comprised of a light chain variable region (abbreviated herein as V L) and a light chain constant region. The light chain constant region is comprised of one domain, C L. The V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each V H and V  L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
The term “antigen-binding portion” of an antibody (or simply “antibody portion” ) , as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a BCMA protein) . It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.  Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L, V H, C L and C H1 domains; (ii) a F (ab')  2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a V H domain; (vi) an isolated complementarity determining region (CDR) ; and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, V L and V H, are coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) . Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
An “isolated antibody” , as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a BCMA protein is substantially free of antibodies that specifically bind antigens other than BCMA proteins) . An isolated antibody that specifically binds a human BCMA protein may, however, have cross-reactivity to other antigens, such as BCMA proteins from other species. Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.
The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
The term “mouse antibody” , as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from mouse germline immunoglobulin sequences. The mouse antibodies of the disclosure can include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) . However, the term “mouse antibody” , as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto mouse framework sequences.
The term “chimeric antibody” refers to an antibody made by combining genetic material from a nonhuman source with genetic material from a human being. Or more  generally, a chimetic antibody is an antibody having genetic material from a certain species with genetic material from another species.
The term “humanized antibody” , as used herein, refers to an antibody from non-human species whose protein sequences have been modified to increase similarity to antibody variants produced naturally in humans.
The term "isotype" refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.
The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen. ”
As used herein, an antibody that “specifically binds to human BCMA” is intended to refer to an antibody that binds to human BCMA protein (and possibly a BCMA protein from one or more non-human species) but does not substantially bind to non-BCMA proteins. Preferably, the antibody binds to human BCMA protein with “high affinity” , namely with a K D of 5.0 x10 -8 M or less, more preferably 1.0 x10 -8 M or less, and more preferably 7.0 x 10 -9 M or less.
The term “does not substantially bind” to a protein or cells, as used herein, means does not bind or does not bind with a high affinity to the protein or cells, i.e. binds to the protein or cells with a K D of 1.0 x 10 -6 M or more, more preferably 1.0 x 10 -5 M or more, more preferably 1.0 x 10 -4 M or more, more preferably 1.0 x 10 -3 M or more, even more preferably 1.0 x 10 -2 M or more.
The term “high affinity” for an IgG antibody refers to an antibody having a K D of 1.0 x 10 -6 M or less, more preferably 5.0 x 10 -8 M or less, even more preferably 1.0 x 10 -8 M or less, even more preferably 7.0 x 10 -9 M or less and even more preferably 1.0 x 10 -9 M or less for a target antigen. However, “high affinity” binding can vary for other antibody isotypes. For example, “high affinity” binding for an IgM isotype refers to an antibody having a K D of 10 -6 M or less, more preferably 10 -7 M or less, even more preferably 10 -8 M or less.
The term “K assoc” or “K a” , as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term “K dis” or “K d” , as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term “K D” , as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of K d to K a (i.e., K d/K a) and is expressed as a molar concentration (M) . K D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K D of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore TM system.
The term “EC 50” , also known as half maximal effective concentration, refers to the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time.
The term “IC 50” , also known as half maximal inhibitory concentration, refers to the concentration of an antibody which inhibits a specific biological or biochemical function by 50%relative to the absence of the antibody.
The term “subject” includes any human or nonhuman animal. The term “nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are preferred, such as non-human primates, sheep, dogs, cats, cows and horses.
The term “therapeutically effective amount” means an amount of the antibody of the present disclosure sufficient to prevent or ameliorate the symptoms associated with a disease or condition (such as a cancer) and/or lessen the severity of the disease or condition. A therapeutically effective amount is understood to be in context to the condition being treated, where the actual effective amount is readily discerned by those of skill in the art.
Various aspects of the disclosure are described in further detail in the following subsections.
Anti-BCMA Antibodies Having Increased Binding Affinity to human BCMA and  Better Anti-tumor Effect
The antibody, or the antigen-binding portion thereof, of the disclosure specifically binds to human BCMA with comparable, if not better, binding affinity/capacity as compared to previously described anti-BCMA antibodies, such as the BCMA-binding portion of GSK2857916.
Additional functional properties include the capacity to block BCMA-BAFF interaction or BCMA-APRIL interacion.
Preferred antibodies of the disclosure are humanized monoclonal antibodies. Additionally or alternatively, the antibodies can be, for example, chimeric monoclonal antibodies.
Monoclonal Anti-BCMA Antibody
The antibody of the disclosure is the monoclonal antibody structurally and chemically characterized as described below and in the following Examples. The amino acid sequence ID numbers of the heavy/light chain variable regions of the antibodies are summarized in Table 1 below, some antibodies sharing the same V H or V L. The heavy chain constant region for the antibodies may be human IgG1 heavy chain constant region having an amino acid sequence set forth in, e.g., SEQ ID NO: 23, and the light chain constant region for the antibodies may be human kappa constant region having an amino acid sequence set forth in, e.g., SEQ ID NO: 24. These antibodies may also contain mouse IgG1 heavy chain constant region, and mouse kappa constant region.
The heavy chain variable region CDRs and the light chain variable region CDRs in Table 1 have been defined by the Kabat numbering system. However, as is well known in the art, CDR regions can also be determined by other systems such as Chothia, and IMGT, AbM, or Contact numbering system/method, based on heavy chain/light chain variable region sequences.
Table 1. Amino acid sequence ID numbers of heavy/light chain variable regions
Figure PCTCN2020141148-appb-000001
Figure PCTCN2020141148-appb-000002
The V H and V L sequences (or CDR sequences) of other anti-BCMA antibodies which bind to human BCMA can be “mixed and matched” with the V H and V L sequences (or CDR sequences) of the anti-BCMA antibody of the present disclosure. Preferably, when V H and V L chains (or the CDRs within such chains) are mixed and matched, a V H sequence from a particular V H/V L pairing is replaced with a structurally similar V H sequence. Likewise, preferably a V L sequence from a particular V H/V L pairing is replaced with a structurally similar V L sequence.
Accordingly, in one embodiment, an antibody of the disclosure, or an antigen binding portion thereof, comprises:
(a) a heavy chain variable region comprising an amino acid sequence listed above in Table 1; and
(b) a light chain variable region comprising an amino acid sequence listed above in Table 1, or the V L of another anti-BCMA antibody, wherein the antibody specifically binds human BCMA.
In another embodiment, an antibody of the disclosure, or an antigen binding portion thereof, comprises:
(a) the CDR1, CDR2, and CDR3 regions of the heavy chain variable region listed above in Table 1; and
(b) the CDR1, CDR2, and CDR3 regions of the light chain variable region listed above in Table 1 or the CDRs of another anti-BCMA antibody, wherein the antibody specifically binds human BCMA.
In yet another embodiment, the antibody, or antigen binding portion thereof, includes the heavy chain variable CDR2 region of anti-BCMA antibody combined with CDRs of other antibodies which bind human BCMA, e.g., CDR1 and/or CDR3 from the heavy chain variable region, and/or CDR1, CDR2, and/or CDR3 from the light chain variable region of a different anti-BCMA antibody.
In addition, it is well known in the art that the CDR3 domain, independently from the CDR1 and/or CDR2 domain (s) , alone can determine the binding specificity of an antibody for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, e.g., Klimka et al., , British J. of Cancer 83 (2) : 252-260 (2000) ; Beiboer et al., , J. Mol. Biol. 296: 833-849 (2000) ; Rader et al., , Proc. Natl. Acad. Sci. U.S.A. 95: 8910-8915 (1998) ; Barbas et al., , J. Am. Chem. Soc. 116: 2161-2162 (1994) ; Barbas et al., , Proc. Natl. Acad. Sci. U.S.A. 92: 2529-2533 (1995) ; Ditzel et al., , J. Immunol. 157: 739-749 (1996) ; Berezov et al., , BIAjournal 8: Scientific Review 8 (2001) ; Igarashi et al., , J. Biochem (Tokyo) 117: 452-7 (1995) ; Bourgeois et al., , J. Virol 72: 807-10 (1998) ; Levi et al., , Proc. Natl. Acad. Sci. U.S.A. 90: 4374-8 (1993) ; Polymenis and Stoller, J. Immunol. 152: 5218-5329 (1994) and Xu and Davis, Immunity 13: 37-45 (2000) . See also, U.S. Pat. Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. Each of these references is hereby incorporated by reference in its entirety.
Accordingly, in another embodiment, antibodies of the disclosure comprise the CDR2 of the heavy chain variable region of the anti-BCMA antibody and at least the CDR3 of the heavy and/or light chain variable region of the anti-BCMA antibody, or the CDR3 of the heavy and/or light chain variable region of another anti-BCMA antibody, wherein the antibody is capable of specifically binding to human BCMA. These antibodies preferably (a) compete for binding with BCMA; (b) retain the functional characteristics; (c) bind to the same epitope; and/or (d) have a similar binding affinity as the anti-BCMA antibody of the present disclosure. In yet another embodiment, the antibodies further may comprise the CDR2 of the light chain variable region of the anti-BCMA antibody, or the CDR2 of the light chain variable region of another anti-BCMA antibody, wherein the antibody is capable of specifically binding to human BCMA. In another embodiment, the antibodies of the disclosure may include the CDR1 of the heavy and/or light chain variable region of the anti-BCMA antibody, or the CDR1 of the heavy and/or light chain variable region of another anti-BCMA antibody, wherein the antibody is capable of specifically binding to human BCMA.
Conservative Modifications
In another embodiment, an antibody of the disclosure comprises a heavy and/or light chain variable region sequences of CDR1, CDR2 and CDR3 sequences which differ from those of the anti-BCMA antibodies of the present disclosure by one or more conservative modifications. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al., (1993) Biochem 32: 1180-8; de Wildt et al., (1997) Prot. Eng. 10: 835-41; Komissarov et al., (1997) J. Biol. Chem. 272: 26864-26870; Hall et al., (1992) J. Immunol. 149: 1605-12; Kelley and O'Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10: 341-6 and Beers et al., (2000) Clin. Can. Res. 6: 2835-43.
Accordingly, in one embodiment, the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:
(a) the heavy chain variable region CDR1 sequence comprises a sequence listed in Table 1 above, and/or conservative modifications thereof; and/or
(b) the heavy chain variable region CDR2 sequence comprises a sequence listed in Table 1 above, and/or conservative modifications thereof; and/or
(c) the heavy chain variable region CDR3 sequence comprises a sequence listed in Table 1 above, and conservative modifications thereof; and/or
(d) the light chain variable region CDR1, and/or CDR2, and/or CDR3 sequences comprise the sequence (s) listed in Table 1 above; and/or conservative modifications thereof; and
(e) the antibody specifically binds human BCMA.
The antibody of the present disclosure possesses one or more of the following functional properties described above, such as high affinity binding to human BCMA, and the ability to induce ADCC or CDC against BCMA-expressing cells.
In various embodiments, the antibody can be, for example, a mouse, human, humanized or chimeric antibody.
As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . Thus, one or more amino acid residues within the CDR regions of an antibody of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth above) using the functional assays described herein.
Engineered and Modified Antibodies
Antibodies of the disclosure can be prepared using an antibody having one or more of the V H/V L sequences of the anti-BCMA antibody of the present disclosure as starting material to engineer a modified antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., V H and/or V L) , for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region (s) , for example to alter the effector function (s) of the antibody.
In certain embodiments, CDR grafting can be used to engineer variable regions of antibodies. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs) . For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., (1998) Nature 332: 323-327; Jones et al., (1986) Nature 321: 522-525; Queen et al., (1989) Proc. Natl. Acad. Also see U.S.A. 86: 10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370) .
Accordingly, another embodiment of the disclosure pertains to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above, and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present disclosure, as described above. While these antibodies contain the V H and V L CDR sequences of the monoclonal antibody of the present disclosure, they can contain different framework sequences.
Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database (available on the Internet at www. mrc-cpe. cam. ac. uk/vbase) , as well as in Kabat et al., (1991) , cited supra; Tomlinson et al., (1992) J. Mol. Biol. 227: 776-798; and Cox et al., (1994) Eur. J. Immunol. 24: 827-836; the contents of each of which are expressly incorporated herein by reference. As another example, the germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in the HCo7 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG--0010109, NT--024637 &BC070333) , 3-33 (NG--0010109 &NT--024637) and 3-7 (NG--0010109 &NT--024637) . As another example, the following heavy chain germline sequences found in the HCo12 HuMAb mouse are available in the accompanying Genbank Accession Nos.: 1-69 (NG--0010109, NT--024637 &BC070333) , 5-51 (NG--0010109 &NT--024637) , 4-34 (NG--0010109 &NT--024637) , 3-30.3 (CAJ556644) &3-23 (AJ406678) .
Antibody protein sequences are compared against a compiled protein sequence database using one of the sequence similarity searching methods called the Gapped BLAST (Altschul et al., (1997) , supra) , which is well known to those skilled in the art.
Preferred framework sequences for use in the antibodies of the disclosure are those that are structurally similar to the framework sequences used by antibodies of the disclosure. The V H CDR1, CDR2, and CDR3 sequences can be grafted onto framework regions that have the identical sequence (s) as that found in the germline immunoglobulin gene from which the framework sequence derives, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370) .
Another type of variable region modification is to mutate amino acid residues within the V H and/or V L CDR1, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation (s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in  vivo assays as known in the art. Preferably conservative modifications (as known in the art) are introduced. The mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.
Accordingly, in another embodiment, the disclosure provides isolated anti-BCMA monoclonal antibodies, or antigen binding portions thereof, comprising a heavy chain variable region and a light chain variable region comprising: (a) a V H CDR1 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (b) a V H CDR2 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (c) a V H CDR3 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (d) a V L CDR1 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; (e) a V L CDR2 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions; and (f) a V L CDR3 region comprising the sequence of the present disclosure, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions.
Engineered antibodies of the disclosure include those in which modifications have been made to framework residues within V H and/or V L, e.g. to improve the properties of the antibody. Typically, such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “backmutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043.
In addition, or as an alternative to modifications made within the framework or CDR regions, antibodies of the disclosure can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
In one embodiment, the hinge region of C H1 is modified in such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of C H1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C H2-C H3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745.
In still another embodiment, the glycosylation of an antibody is modified. For example, a deglycosylated antibody can be made (i.e., the antibody lacks glycosylation) . Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.
Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the disclosure to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α (1, 6) -fucosyltransferase) , such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8-/-cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al., (2004) Biotechnol Bioeng 87: 614-22) . As another example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the α-1, 6 bond-related enzyme. EP 1,176,195 also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662) . PCT Publication WO 03/035835 describes a variant CHO cell line, Lec13 cells, with reduced ability to attach  fucose to Asn (297) -linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al., (2002) J. Biol. Chem. 277: 26733-26740) . Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna. Methods for production of antibodies in a plant system are disclosed in the U.S. patent application corresponding to Alston &Bird LLP attorney docket No. 040989/314911, filed on Aug. 11, 2006. PCT Publication WO 99/54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., β (1, 4) -N-acetylglucosaminyltransferase III (GnTIII) ) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al., (1999) Nat. Biotech. 17: 176-180) . Alternatively, the fucose residues of the antibody can be cleaved off using a fucosidase enzyme; e.g., the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14: 5516-23) .
Another modification of the antibodies herein that is contemplated by this disclosure is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG) , such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer) . As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C 1-C 10) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See, e.g., EP 0 154 316 and EP 0 401 384.
Antibody’s Physical Properties
Antibodies of the disclosure can be characterized by their various physical properties, to detect and/or differentiate different classes thereof.
For example, antibodies can contain one or more glycosylation sites in either the light or heavy chain variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or an alteration of the pK of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41: 673-702; Gala and Morrison (2004) J Immunol 172: 5489-94; Wallick et al (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al (1985) Nature 316: 452-7; Mimura et al., (2000) Mol Immunol 37: 697-706) . Glycosylation has been known to occur at motifs containing an N-X-S/T sequence. In some instances, it is preferred to have an anti-BCMA antibody that does not contain variable region glycosylation. This can be achieved either by selecting antibodies  that do not contain the glycosylation motif in the variable region or by mutating residues within the glycosylation region.
In a preferred embodiment, the antibodies do not contain asparagine isomerism sites. The deamidation of asparagine may occur on N-G or D-G sequences and result in the creation of an isoaspartic acid residue that introduces a link into the polypeptide chain and decreases its stability (isoaspartic acid effect) .
Each antibody will have a unique isoelectric point (pI) , which generally falls in the pH range between 6 and 9.5. The pI for an IgG1 antibody typically falls within the pH range of 7-9.5 and the pI for an IgG4 antibody typically falls within the pH range of 6-8. There is speculation that antibodies with a pI outside the normal range may have some unfolding and instability under in vivo conditions. Thus, it is preferred to have an anti-BCMA antibody that contains a pI value that falls in the normal range. This can be achieved either by selecting antibodies with a pI in the normal range or by mutating charged surface residues.
Nucleic Acid Molecules Encoding Antibodies of the Disclosure
In another aspect, the disclosure provides nucleic acid molecules that encode heavy and/or light chain variable regions, or CDRs, of the antibodies of the disclosure. The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques. A nucleic acid of the disclosure can be, e.g., DNA or RNA and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.
Nucleic acids of the disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below) , cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques) , a nucleic acid encoding such antibodies can be recovered from the gene library.
Preferred nucleic acids molecules of the disclosure include those encoding the V H and V L sequences of the BCMA monoclonal antibody or the CDRs. Once DNA fragments encoding V H and V L segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a V L-or V H-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term “operatively linked” , as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
The isolated DNA encoding the V H region can be converted to a full-length heavy chain gene by operatively linking the V H-encoding DNA to another DNA molecule encoding heavy chain constant regions (C H1, C H2 and C H3) . The sequences of human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, the V H-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain C H1 constant region.
The isolated DNA encoding the V L region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the V L-encoding DNA to another DNA molecule encoding the light chain constant region, C L. The sequences of human light chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region.
To create a scFv gene, the V H-and V L-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser) 3, such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H regions joined by the flexible linker (see e.g., Bird et al., (1988) Science 242: 423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., , (1990) Nature 348: 552-554) .
Production of Monoclonal Antibodies of the Disclosure
Monoclonal antibodies (mAbs) of the present disclosure can be produced using the well-known somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256: 495. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. See e.g., U.S. Pat. Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are specifically incorporated herein by reference in their entirety.
Generation of Transfectomas Producing Monoclonal Antibodies of the Disclosure
Antibodies of the disclosure also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202) . In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody genes. Such regulatory sequences are described, e.g., in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) ) . Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) , Simian Virus 40 (SV40) , adenovirus, e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, nonviral regulatory sequences can be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8: 466-472) . The expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In preferred embodiments, the variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the V H segment is operatively linked to the C H segment (s) within the vector and the V L segment is operatively linked to the C L segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) .
In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017) . For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection) .
For expression of the light and heavy chains, the expression vector (s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and  the like. Although it is theoretically possible to express the antibodies of the disclosure in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
Preferred mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker, e.g., as described in R.J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159: 601-621) , NSO myeloma cells, COS cells and SP2 cells. In particular for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
Immunoconjugates
Antibodies of the disclosure can be conjugated to a therapeutic agent to form an immunoconjugate such as an antibody-drug conjugate (ADC) . Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents. In the ADC, the antibody and therapeutic agent preferably are conjugated via a linker cleavable such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. The ADCs can be prepared as described in U.S. Pat. Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02/096910; WO 07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO 08/103,693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295; the disclosures of which are incorporated herein by reference.
Bispecific Molecules
In another aspect, the present disclosure features bispecific molecules comprising one or more antibodies of the disclosure linked to at least one other functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. Thus, as used herein, “bispecific molecule” includes molecules that have two or more specificities.
In an embodiment, a bispecific molecule has, in addition to an anti-Fc binding specificity and an anti-BCMA binding specificity, a third specificity. The third specificity  can be for an anti-enhancement factor (EF) , e.g., a molecule that binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. For example, the anti-enhancement factor can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, or ICAM-1) or other immune cell, resulting in an increased immune response against the target cell.
Bispecific molecules may be in many different formats and sizes. At one end of the size spectrum, a bispecific molecule retains the traditional antibody format, except that, instead of having two binding arms of identical specificity, it has two binding arms each having a different specificity. At the other extreme are bispecific molecules consisting of two single-chain antibody fragments (scFv's) linked by a peptide chain, a so-called Bs (scFv) 2 construct. Intermediate-sized bispecific molecules include two different F (ab) fragments linked by a peptidyl linker. Bispecific molecules of these and other formats can be prepared by genetic engineering, somatic hybridization, or chemical methods. See, e.g., Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry, 9 (6) , 635-644 (1998) ; and van Spriel et al., Immunology Today, 21 (8) , 391-397 (2000) , and the references cited therein.
Antibody-encoding or Antibody-bearing Oncolytic Virus
An oncolytic virus preferabtially infects and kills cancer cells. Antibodies of the present disclosure can be used in conjunction with oncolytic viruses. Alternatively, oncolytic viruses encoding antibodies of the present disclosure can be introduced into human body.
Chimeric Antigen Receptor
Also provided herein are a chimeric antigen receptor (CAR) containing an anti-BCMA scFv, the anti-BCMA scFv comprising CDRs and heavy/light chain variable regions described herein.
The anti-BCMA CAR may comprise (a) an extracellular antigen binding domain comprising an anti-BCMA scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.
The CAR may contain a signal peptide at the N-terminus of the extracellular antigen binding domain that directs the nascent receptor into the endoplasmic reticulum, and a hinge peptide at the N-terminus of the extracellular antigen binding domain that makes the receptor more available for binding. The CAR preferably comprises, at the intracellular signaling domain, a primary intracellular signaling domain and one or more co-stimulatory signaling domains. The mainly used and most effective primary intracellular signaling domain is CD3-zeta cytoplasmic domain which contains ITAMs, the phosphorylation of which results in T cell activation. The co-stimutory signaling domain may be derived from the co-stimulatory proteins such as CD28, CD137 or OX40.
The CARs may further add factors that enhance T cell expansion, persistence, and anti-tumor activity, such as cytokines, and co-stimulatory ligands.
Also provided are engineered immune effector cells, comprising the CAR provided herein. In some embodiments, the immune effector cell is a T cell, an NK cell, a peripheral  blood mononuclear cell (PBMC) , a hematopoietic stem cell, a pluripotent stem cell, or an embryonic stem cell. In some embodiments, the immune effector cell is a T cell.
Pharmaceutical Compositions
In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more antibodies (or antigen-binding portion thereof, or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates) of the present disclosure formulated together with a pharmaceutically acceptable carrier. The antibodies (or antigen-binding portion thereof, or or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates) can be dosed separately when the composition contains more than one antibody (or antigen-binding portion thereof, or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates) . The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug, such as an anti-tumor drug.
The pharmaceutical composition can comprise any number of excipients. Excipients that can be used include carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams &Wilkins 2003) , the disclosure of which is incorporated herein by reference.
Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) . Depending on the route of administration, the active ingredient can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.
Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a microemulsion, liposome, or other ordered structure suitable to high drug concentration.
The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01%to about ninety-nine percent of active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1%to about 30%of active ingredient in combination with a pharmaceutically acceptable carrier.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response) . For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required.
For administration of the composition, the dosage may range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg body weight, of the host body weight. For example, dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg body weight. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Preferred dosage regimens for an anti-BCMA antibody of the disclosure include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg/ml and in some methods about 25-300 μg/ml.
A “therapeutically effective dosage” of an anti-BCMA antibody, or the antigen-binding portion thereof, or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates of the disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of tumor-bearing subjects, a “therapeutically effective dosage” preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%relative to untreated subjects. A therapeutically effective amount of a therapeutic antibody can decrease tumor size, or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.
The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides,  polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
Therapeutic compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556) ; (2) micro-infusion pumps (U.S. Pat. No. 4,487,603) ; (3) transdermal devices (U.S. Pat. No. 4,486,194) ; (4) infusion apparatuses (U.S. Pat. Nos. 4,447,233 and 4,447,224) ; and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196) ; the disclosures of which are incorporated herein by reference.
In certain embodiments, the monoclonal antibodies of the disclosure can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibody of the disclosure cross the blood-brain barrier, they can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V. V. Ranade (1989) J. Clin. Pharmacol. 29: 685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038; Bloeman et al., (1995) FEBS Lett. 357: 140; M. Owais et al., (1995) Antimicrob. Agents Chemother. 39: 180; Briscoe et al., (1995) Am. J. Physiol. 1233: 134; Schreier et al., (1994) J. Biol. Chem. 269: 9090; Keinanen and Laukkanen (1994) FEBS Lett. 346: 123; and Killion and Fidler (1994) Immunomethods 4: 273.
Uses and Methods of the Disclosure
The composition comprising the antibodies or the antigen-binding portion thereof, or the bispecific, CAR-T cells, oncolytic viruses, immunoconjugates of the present disclosure have numerous in vitro and in vivo utilities involving, for example, treatment of tumors associated with increased BCMA expression.
Given the ability of anti-BCMA antibodies of the disclosure to inhibit proliferation and survival of BCMA-expressing tumor cells, the disclosure provides methods for inhibiting growth of tumor cells in a subject comprising administering to the subject the composition of the disclosure such that growth of the tumor is inhibited in the subject. Non-limiting examples of tumors that can be treated by antibodies of the disclosure include, but not limited to, such as leukemia, lymphomas and multiple myeloma. Additionally, refractory or recurrent malignancies whose growth may be inhibited using the antibodies of the disclosure.
Generally speaking, the antibodies of the disclosure can be used to enhance an immune response in a subject.
Combination Therapy
In another aspect, the disclosure provides methods of combination therapy in which the anti-BCMA antibodies, or antigen-binding portion thereof, or the bispecifics, CAR-T cells, oncolytic viruses, immunoconjugates of the present disclosure are co-administered with one or more additional antibodies that are effective in inhibiting tumor growth in a subject. In one embodiment, the disclosure provides a method for inhibiting tumor growth in a subject comprising administering to the subject an anti-BCMA antibody (or antigen-binding portion  thereof, or the CAR-T cell, oncolytic virus, immunoconjugate) and one or more additional antibodies, such as an anti-OX40 antibody, an anti-TIM-3 antibody, an anti-CD137 antibody, an anti-GITR antibody, an anti-LAG-3 antibody, an anti-PD-L1 antibody, and anti-PD-1 antibody and/or an anti-CTLA-4 antibody. In certain embodiments, the subject is human.
The BCMA signaling activation can also be further combined with standard cancer treatments. For example, BCMA signaling activationa can be combined with CTLA-4 and/or LAG-3 and/or PD-1 blockade and also chemotherapeutic regimes. For example, a chemotherapeutic agent can be administered with the anti-BCMA antibodies, which may be a cytotoxic agent. For example, epirubicin, oxaliplatin, and 5-FU are administered to patients receiving anti-BCMA therapy.
Optionally, the combination of anti-BCMA and one or more additional antibodies (e.g., anti-CTLA-4 and/or anti-LAG-3 and/or anti-PD-1 antibodies) can be further combined with an immunogenic agent, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules) , and cells transfected with genes encoding immune stimulating cytokines (He et al., (2004) J. Immunol. 173: 4919-28) . Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.
Other therapies that may be combined with anti-BCMA antibody includes, but not limited to, interleukin-2 (IL-2) administration, radiation, surgery, or hormone deprivation.
The combination of therapeutic agents discussed herein can be administered concurrently as a single composition in a pharmaceutically acceptable carrier, or concurrently as separate compositions with each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.
Furthermore, if more than one dose of the combination therapy is administered sequentially, the order of the sequential administration can be reversed or kept in the same order at each time point of administration, sequential administrations can be combined with concurrent administrations, or any combination thereof.
The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.
Examples
Example 1 Generation of Mouse Anti-BCMA Monoclonal Antibodies Using  Hybridoma Technology
Immunization
Mice were immunized according to the method as described in E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998. Recombinant human BCMA protein with human IgG1 Fc tag at the C-terminus (Cat#BC7-H5254, Acro biosystems) was used as the immunogen. Human BCMA-his protein  (Cat#BCA-H522Y, Acro biosystems) was used for determining anti-sera titer and for screening hybridomas secreting antigen-specific antibodies. Immunizing dosages contained 25 μg human BCMA-Fc protein/mouse/injection for both primary and boost immunizations. To increase immune response, the complete Freud's adjuvant and incomplete Freud's adjuvant (Sigma, St. Louis, Mo., USA) were used respectively for primary and boost immunizations. Briefly, adjuvant-antigen mixture was prepared by first gently mixing the adjuvant in a vial using a vortex. The desired amount of adjuvant was transferred to an autoclaved 1.5 mL micro-centrifuge tube. The antigen was prepared in PBS or saline with concentration ranging from 0.5-1.0 mg/ml. The calculated amount of antigen was then added to the micro-centrifuge tube with the adjuvant, and the resulting mixture was mixed by gently vortexing for 2 minutes to generate water-in-oil emulsions. The adjuvant-antigen emulsion was then drawn into the proper syringe for animal injection. A total of 25μg of antigen was injected in a volume of 50-100 μl. Each animal was immunized, and then boosted for 2 to 3 times depending on the anti-sera titer. Animals with good titers were given a final boost by intraperitoneal injection before hybridoma fusion.
Hybridoma fusion and screening
Cells of murine myeloma cell line (SP2/0-Ag14, ATCC#CRL-1581) were cultured to reach the log phase stage right before hybridoma fusion. Spleen cells from immunized mice were prepared sterilely and fused with myeloma cells according to the method as described in Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity, " Nature, 256: 495-497 (1975) . Fused "hybrid cells" were subsequently dispensed into 96-well plates in DMEM/20%FCS/HAT media. Surviving hybridoma colonies were observed under the microscope seven to ten days post fusion. After two weeks, the supernatant from each well was subjected to ELISA-based screening using recombinant human BCMA-his protein. Briefly, ELISA plates were coated with 60 μl of human BCMA-his (Cat#BCA-H522Y, Acro biosystems, 2.0 μg/ml in PBS) overnight at 4℃. Plates were washed 4 times with PBST and blocked with 200 μl blocking buffer (5%w/v non-fatty milk in PBST) . Diluted hybridoma supernatant (60 μl) was added to each well and incubated at 37℃for 40 minutes. Plates were then washed 4 times, HRP-goat anti-mouse-IgG (Jackson Immuno research, Cat#115-036-071) was used for detection, and binding ODs were observed at 450 nm. Positive hybridoma secreting antibody that binds to human BCMA-his protein were then selected and transferred to 24-well plates. Hybridoma clones producing antibodies that showed high specific human BCMA binding and BCMA-BAFF ligand blocking activities were subcloned by limiting dilution to ensure the clonality of the cell line, and then monoclonal antibodies were purified. Briefly, Protein A sepharose column (from bestchrom (Shanghai) Biosciences, Cat#AA0273) was washed using PBS buffer in 5 to 10 column volumes. Cell supernatants were passed through the columns, and then the columns were washed using PBS buffer until the absorbance for protein reached the baseline. The columns were eluted with elution buffer (0.1 M Glycine-HCl, pH 2.7) , and immediately collected into 1.5 ml tubes with neutralizing buffer (1 M Tris-HCl, pH 9.0) . Fractions containing  immunoglobulins were pooled and dialyzed in PBS overnight at 4℃. Subsequently, the in vitro functional activities of purified monoclonal antibodies were characterized as follows.
Example 2 Affinity Determination of Mouse Anti-BCMA Monoclonal Antibodies  Using BIACORE Surface Plasmon Resonance Technology
The purified mouse anti-BCMA monoclonal antibodies (mAbs) generated in Example 1 were characterized for affinities and binding kinetics by Biacore T200 system (GE healthcare, Pittsburgh, PA, USA) .
Briefly, goat anti-mouse IgG (GE healthcare, Cat#BR100838, Mouse Antibody Capture Kit) was covalently linked to a CM5 chip (carboxy methyl dextran coated chip) via primary amines, using a standard amine coupling kit provided by Biacore (GE healthcare, Pittsburgh, PA, USA) . Un-reacted moieties on the biosensor surface were blocked with ethanolamine. Then, purified anti-BCMA antibodies and a BCMA benchmark (an antibody containing BCMA-binding portion of GSK2857916, also referred to as BCMA-mab1 or BM, prepared using the heavy chain and light chain amino acids set forth in SEQ ID NOs.: 26 and 27) , at the concentration of 10.0 μg/ml were flowed onto the chip at a flow rate of 10 μL/min. Then, 80 nM, 40 nM, 20 nM or 10 nM recombinant human BCMA-his (Acro biosystems, Cat#BCA-H522Y, MW: 7.8kDa) or cynomolgus monkey BCMA-Fc protein (Acro biosystems, Cat#BCA-C5253, MW: 32.1kDa) in HBS EP buffer (provided by Biacore) was flowed onto the chip at a flow rate of 30 μL/min. The antigen-antibody association kinetics was followed for 2 minutes and the dissociation kinetics was followed for 10 minutes. The association and dissociation curves were fit to a 1: 1 Langmuir binding model using BIA evaluation software. The K D, K a and K d values were determined and summarized in Table 2 below.
Table 2. Binding affinity of mouse anti-BCMA antibodies
Figure PCTCN2020141148-appb-000003
The antibodies of the present disclosure specifically bound to human BCMAs, and their binding affinities were comparable to or a bit better than the benchmark. The B1A1, B1G3 and B1C1 antibodies also bound to monkey BCMA specifically.
Example 3 Binding Activity of Mouse Anti-BCMA Monoclonal Antibodies
The binding activities of mouse anti-BCMA antibodies were determined by Capture ELISA and Flow Cytometry (FACS) .
For the capture ELISA, 96-well micro plates were coated with 2 μg/ml goat anti-mouse IgG Fcγfragment specific (Jackson Immuno Research, Cat#115-005-071, 100 μl/well, for mouse anti-BCMA antibodies of the discolsure) or goat anti-human IgG F (ab’)  2 fragment (Jackson Immuno Research, Cat#109-005-097, 100 μl/well, for the benchmark BM) in PBS and incubated overnight at 4℃. Plates were washed 4 times with wash buffer (PBS+0.05%Tween-20, PBST) and then blocked with 200 μl/well blocking buffer (5%w/v non-fatty milk in PBST) for 2 hours at 37℃. Plates were washed again and incubated with 100 μl/well purified mouse anti-BCMA antibodies, the benchmark BM, and negative control hIgG (human immunoglobulin (pH4) for intravenous injection, Hualan Biological Engineering Inc. ) (5-fold dilution in 2.5%non-fatty milk in PBST, starting with 10000 ng/ml or 66.67 nM) for 40 minutes at 37℃, and then washed 4 times again. Plates containing captured anti-BCMA antibodies were incubated with biotin-labeled human BCMA-his protein (Cat#BCA-H522Y, Acro biosystems, 0.28 nM in 2.5%non-fatty milk in PBST, 100 μl/well) for 40 minutes at 37℃, washed 4 times, and incubated with streptavidin conjugated HRP (1: 10000 dilution in PBST, Jackson Immuno Research, Cat#016-030-084, 100 μl/well) for 40 minutes at 37℃. After a final wash, plates were incubated with 100 μl/well ELISA substrate TMB (Innoreagents, Cat#TMB-S-002) . The reaction was stopped in 15 minutes at 25℃ with 50 μl/well 1M H 2S0 4, and the absorbance was read at 450-630 nm and plotted against antibody concentration. Data were analyzed using Graphpad Prism software and EC 50 values were reported.
For binding of anti-BCMA antibodies to the surface of U266 Cells by flow cytometry (FACS) , human myeloma cell line U266 (
Figure PCTCN2020141148-appb-000004
TIB-196 TM) were harvested from cell culture flasks, washed two times and resuspended in phosphate buffered saline (PBS) containing 2%v/v Fetal Bovine Serum (FACS buffer) . 2 x 10 5 cells per well in 96 well-plates were incubated with the anti-BCMA antibodies or controls of various concentrations in FACS buffer for 40 minutes on ice. Cells were washed twice with FACS buffer, and 100 μL/well R-Phycoerythrin AffiniPure F (ab') 2 Fragment Goat Anti-Mouse IgG (H+L) (1: 1000 dilution in FACS buffer, Jackson Immunoresearch, Cat#115-116-146) were added. Following an incubation of 40 minutes at 4℃ in dark, cells were washed three times and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS equipment. Data were analyzed using Graphpad Prism software and EC 50 values were reported as the antibody concentration to achieve 50%of maximal BCMA antibodies binding to U266 cells.
The results were summarized in Table 3 below and shown in Fig. 1A and 1B.
Table 3. Binding Activity of mouse anti-BCMA antibodies
Figure PCTCN2020141148-appb-000005
The results indicated that the antibodies of the present disclosure bound to human BCMA specifically, with similar or lower EC 50 values compared to the benchmark. Further, as can be seen from Fig. 1A and 1B, the maximum absorbance in B1A1/B1G3/B1C1 group was similar to or a bit lower than that of the benchmark.
Example 4 Functional Assays Using Competittive ELISA
4.1 Ligand Blocking ELISA
The ability of anti-BCMA antibodies to block BCMA-BAFF interaction was measured using a competitive ELISA assay. Briefly, 100 μl human BCMA-his proteins (Acro biosystems, Cat#BCA-H522Y) were coated on 96-well micro plates at 2 μg/mL in PBS and incubated overnight at 4℃. The next day, plates were washed with wash buffer (PBS+0.05%w/v Tween-20, PBST) , and blocked with 5%w/v non-fatty milk in PBST for 2 hours at 37℃. Plates were then washed again using wash buffer.
Serially diluted anti-BCMA antibodies of the present disclosure or controls (starting at 200 nM or 30000 ng/mL with a four-fold serial dilution) in 2.5%w/v non-fatty milk in PBST were added to the plates, 100 μl per well, and incubated with the human BCMA-his proteins at 37℃ for 40 minutes. Plates were washed 4 times using wash buffer, and then 100 μl/well of 0.25 nM biotin-labeled human BAFF-Fc protein (Sino biological inc., Cat#10056-H01H) was added and incubated for 40 minutes at 37℃. Plates were washed again using wash buffer. 100 μl/well of streptavidin conjugated HRP was added and incubated for 40 minutes at 37℃. Plates were washed again using wash buffer. Finally, TMB was added and the reaction was stopped using 1M H 2SO 4, and the absorbance was read at 450-630 nm. Data were analyzed using Graphpad Prism software and IC 50 values were reported.
4.2 Benchmark Blocking ELISA
The ability of the anti-BCMA antibodies of the present disclosure to block Benchmark BM-human BCMA binding was measured using a competitive ELISA assay. Briefly, the benchmark was coated on 96-well micro plates at 2 μg/mL in PBS, 100 μl per well, and incubated overnight at 4℃. The next day, plates were washed with wash buffer, and blocked with 5%w/v non-fatty milk in PBST for 2 hours at 37℃. While blocking, serially diluted anti-BCMA antibodies or controls (starting at 100 nM or 15000 ng/mL with a 4-fold serial dilution) were mixed with biotin labeled human BCMA-his proteins (Cat#BCA-H522Y, Acro biosystems) , 0.22 nM in 2.5%non-fatty milk in PBST, and the mixture was  incubated at 25℃ for 40 minutes. After plate washing, the antibody/biotin labeled human BCMA-his mixtures were added to BM coated plates, 100 μl per well. After incubation at 37℃for 40 minutes, plates were washed using wash buffer. Then 100 μl/well streptavidin conjugated HRP was added and incubated for 40 minutes at 37℃. Plates were washed again using wash buffer. Finally, TMB was added and the reaction was stopped using 1M H 2SO 4, and the absorbance was read at 450-630 nm. Data were analyzed using Graphpad Prism software and IC 50 values were reported.
The results of the two assays were summarized in Table 4 below and shown in Fig. 2A, 2B, 3A and 3B.
Table 4. Anti-BCMA antibodies’ functional assay results
Figure PCTCN2020141148-appb-000006
It can be seen from Table 4 that the anti-BCMA antibodies of the present disclosure were capable of blocking human BCMA-BAFF interaction at lower IC 50 values as compared to the benchmark. Further, as shown in Fig. 2A and 2B, no evident absorbance difference was found beween B1A1/B1C1 and the benchmark, and B1G3 showed better blocking capacity than the benchmark.
The data also showed that the antibodies of the present disclosure were able to block human BCMA-benchmark interaction, suggesting that they bound to the same or similar epitope as the benchmark did.
Example 5 Generation and Characterization of Chimeric Antibodies
The variable domains of the heavy and light chain of the anti-BCMA mouse mAbs were sequenced, and the sequence ID numbers were summarized in Table 1.
The variable domains of the heavy and light chain of the anti-BCMA mouse mAbs were cloned in frame to human IgG1 heavy-chain (SEQ ID NO.: 23) and human kappa light-chain constant regions (SEQ ID NO.: 24) , respectively, wherein the C terminus of variable region was linked to the N terminus of the respective constant region.
The activities of the resulting chimeric antibodies were confirmed in binding capture ELISA, and competitive ELISA assays following the protocols in the foregoing Examples.
The data showed that the chimeric and mouse antibodies had similar binding capacities, as shown in Table 5 below.
Table 5. Binding and functional activities of Chimeric Antibodies
Figure PCTCN2020141148-appb-000007
Example 6 Humanization of Anti-BCMA Mouse Monoclonal Antibodies B1G3 and  B1C1
Mouse anti-BCMA antibodies B1G3 and B1C1 were selected for humanization and further investigations. Humanization of the mouse antibody was conducted using the well-established CDR-grafting method as described in detail below.
To select acceptor frameworks for humanization of mouse antibodies B1H2, B1G3 and B1C1, the light and heavy chain variable region sequences of mouse B1H2, B1G3 and B1C1 were blasted against the human immunoglobulin gene database. The human germlines with the highest homology to mouse B1G3 and B1C1 were selected as the acceptor frameworks for humanization. The mouse antibody heavy/light chain variable region CDRs were inserted into the selected frameworks, and the residue (s) in the frameworks was/were further mutated to obtain more candidate heavy chain/light chain variable regions. A total of 15 humanized B1G3 antibodies (namely huB1G3-V1 to huB1G3-V15) , and 12 humanized B1C1 antibodies (huB1C1-V1 to huB1C1-V9, and huB1C1-V11 to huB1C1-V13) , were obtained whose heavy/light chain variable region sequence ID numbers were in Table 1.
The vectors each containing a nucleotide encoding a humanized B1G3/B1C1 heavy chain variable region linked to human IgG1 heavy-chain constant region (SEQ ID NO: 23) , and the vectors each containing a nucleotide encoding a humanized light chain variable region linked to human kappa light-chain constant region (SEQ ID NO: 24) were transiently transfected into 20 ml of 293F suspension cell cultures in a ratio of 47.62%to 52.38%light to heavy chain construct, with 60 μg/mL PEI. Cell supernatants were harvested after six days in shaking flasks, spun down to pellet cells, and filtered through 0.22 μm filters for immunoglubulin separation. The antibodies were purified by protein A affinity chromatography. Briefly, Protein A sepharose column (from bestchrom (Shanghai) Biosciences, Cat#AA0273) was washed 5 to 10 column volumes using PBS buffer. Cell supernatants were passed through the columns, and then the the columns were washed using PBS buffer until the absorbance for protein reached the baseline. The columns were eluted with elution buffer (0.1 M Glycine-HCl, pH 2.7) , and immediately collected into 1.5 ml tubes with neutralizing buffer (1 M Tris-HCl, pH 9.0) . Fractions containing Immunoglobulins were pooled and dialyzed in PBS overnight at 4℃.
Example 7 Characterization of humanized B1G3 and B1C1 antibodies
The humanized B1G3 and B1C1 antibodies were also tested for their binding affinities/capacities to human and cynomolgus BCMA and other functional activities by Biacore, capture ELISA, and competitive assays, following the protocols in the foregoing Examples. In the capture ELISA, 96-well micro plates were coated with 2 μg/ml goat anti-human IgG (AffiniPure Goat Anti-Human IgG, F (ab')  2 fragment specific, Jackson Immunoresearch, Cat#109-005-097) , and a mouse control antibody binding a non-BCMA protein (in house made) was additionally used.
Table 6. Binding activities to human BCMA of humanized B1G3 and B1C1 antibodies
Figure PCTCN2020141148-appb-000008
The Capture ELISA results were summarized in Table 6 above and shown in Fig. 4A-4C and 5A-5C. It can be seen that huB1G3-V8 and huB1C1-V6 did not bind to human  BCMA, and the remaining antibodies had comparable or a bit lower binding activities compared to their parent antibodies or the benchmark.
The humanized antibodies huB1G3-V13 and huB1C1-V13 were subject to further characterizations. In brief, these two antibodies were tested for their binding capativies to human and monkey BCMAs, and also their capacities of blocking BCMA-BAFF or benchmark-BCMA interaction, following the protocols described above.
The results were summarized in Table 7 below and shown in Fig. 6-11.
The antibody huB1G3-V13 showed good binding activity to human BCMA in the Capture ELISA assay, which was comparable to the benchmark (Fig. 6) , and a bit lower binding affinity to human or monkey BCMA than the benchmark. Further, this antibody can effectively block BCMA-BAFF interaction with almost the same blocking capacity compared to the benchmark (Fig. 7) , and can also block benchmark-BCMA interaction (Fig. 8) .
The antibody huB1C1-V13 showed good binding activity to human BCMA in the Capture ELISA assay, which was a bit lower than the benchmark (Fig. 9) , and also a bit lower binding affinity to human or monkey BCMA than the benchmark. Further, this antibody can effectively block BCMA-BAFF interaction with almost the same blocking capacity compared to the benchmark (Fig. 10) , and can also block benchmark-BCMA interaction (Fig. 11) .
Table 7. Binding and functional activities of humanized antibodies huB1G3-V13 and huB1C1-V13
Figure PCTCN2020141148-appb-000009
While the disclosure has been described above in connection with one or more embodiments, it should be understood that the disclosure is not limited to those embodiments, and the description is intended to cover all alternatives, modifications, and equivalents, as may be included within the spirit and scope of the appended claims. All referenced cited herein are further incorporated by reference in their entirety.
Sequences in the present application are summarized below.
Figure PCTCN2020141148-appb-000010
Figure PCTCN2020141148-appb-000011
Figure PCTCN2020141148-appb-000012
Figure PCTCN2020141148-appb-000013
Figure PCTCN2020141148-appb-000014
Figure PCTCN2020141148-appb-000015

Claims (19)

  1. An isolated monoclonal antibody, or an antigen-binding portion thereof, binding to B cell maturation antigen (BCMA) , comprising a heavy chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to (1) SEQ ID NOs: 1 (X1=H or F) , 3 and 5, respectively; or (2) SEQ ID NOs: 2, 4 and 6, respectively.
  2. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to SEQ ID NOs: 13, 14 (X1=I, X2=A, X3=T, X4=C, X5=F; X1=V, X2=A, X3=T, X4=C, X5=F; X1=I, X2=V, X3=T, X4=C, X5=F; X1=I, X2=A, X3=S, X4=C, X5=F; X1=I, X2=A, X3=Y, X4=C, X5=Y; X1=V, X2=V, X3=S, X4=S, X5=Y; or X1=V, X2=V, X3=S, X4=C, X5=Y) , 15, 16 (X1=I, X2=N, X3=F; X1=V, X2=N, X3=F; X1=I, X2=T, X3=F; X1=I, X2=N, X3=Y; or X1=V, X2=T, X3=Y) , or 17.
  3. The isolated monoclon★al antibody, or the antigen-binding portion thereof, of claim 1, comprising a light chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1 region, the CDR2 region and the CDR3 region comprise amino acid sequences amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to (1) SEQ ID NOs: 7 (X1=H) , 9 (X1=N) and 11, respectively; (2) SEQ ID NOs: 7 (X1=F) , 9 (X1=T) and 11, respectively; or (3) SEQ ID NOs: 8, 10 and 12, respectively.
  4. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 3, wherein the light chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to SEQ ID NOs: 18, 19 (X1=L, X2=P, X3=P, X4=W, X5=Y; X1=M, X2=P, X3=P, X4=W, X5=Y; X1=L, X2=A, X3=P, X4=W, X5=Y; X1=L, X2=P, X3=L, X4=W, X5=Y; X1=L, X2=P, X3=P, X4=L, X5=Y; X1=L, X2=P, X3=P, X4=W, X5=F; or X1=M, X2=A, X3=P, X4=L, X5=F) , 20, 21 (X1=S, X2=P, X3=W, X4=Y, X5=F; X1=A, X2=P, X3=W, X4=Y, X5=F; X1=S, X2=L, X3=W, X4=Y, X5=F; X1=S, X2=P, X3=L, X4=Y, X5=F; X1=S, X2=P, X3=W, X4=F, X5=F; X1=S, X2=P, X3=W, X4=Y, X5=Y; ▲X1=A, X2=P, X3=L, X4=F, X5=Y) or 22.
  5. The isolated monoclonal antibody, or an antigen-binding portion thereof, of claim 1 or claim 3, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to (1) SEQ ID NOs: 1 (X1=H) , 3, 5, 7  (X1=H) , 9 (X1=N) and 11, respectively; (2) SEQ ID NOs: 1 (X1=F) , 3, 5, 7 (X1=F) , 9 (X1=T) and 11, respectively; or (3) SEQ ID NOs: 2, 4, 6, 8, 10 and 12, respectively.
  6. The isolated monoclonal antibody, or an antigen-binding portion thereof, of claim 5, wherein the heavy chain variable region and the light chain variable region comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to (1) SEQ ID NOs: 13 and 18, respectively; (2) SEQ ID NOs: 14 (X1=I, X2=A, X3=T, X4=C, X5=F; X1=V, X2=A, X3=T, X4=C, X5=F; X1=I, X2=V, X3=T, X4=C, X5=F; X1=I, X2=A, X3=S, X4=C, X5=F; X1=I, X2=A, X3=Y, X4=C, X5=Y; X1=V, X2=V, X3=S, X4=S, X5=Y; or X1=V, X2=V, X3=S, X4=C, X5=Y) and 19 (X1=L, X2=P, X3=P, X4=W, X5=Y) , respectively; (3) SEQ ID NOs.: 14 (X1=I, X2=A, X3=T, X4=C, X5=F) and 19 (X1=M, X2=P, X3=P, X4=W, X5=Y; X1=L, X2=A, X3=P, X4=W, X5=Y; X1=L, X2=P, X3=L, X4=W, X5=Y; X1=L, X2=P, X3=P, X4=L, X5=Y; or X1=L, X2=P, X3=P, X4=W, X5=F) , respectively; (4) SEQ ID NOs.: 14 (X1=I, X2=A, X3=T, X4=C, X5=F; or X1=V, X2=V, X3=S, X4=C, X5=Y) and 19 (X1=M, X2=A, X3=P, X4=L, X5=F) , respectively; (5) SEQ ID NOs.: 15 and 20, respectively; (6) SEQ ID NOs.: 16 (X1=I, X2=N, X3=F; X1=V, X2=N, X3=F; X1=I, X2=T, X3=F; X1=I, X2=N, X3=Y; or X1=V, X2=T, X3=Y) and 21 (X1=S, X2=P, X3=W, X4=Y, X5=F) , respectively; (7) SEQ ID NOs.: 16 (X1=I, X2=N, X3=F) and 21 (X1=A, X2=P, X3=W, X4=Y, X5=F; X1=S, X2=L, X3=W, X4=Y, X5=F; X1=S, X2=P, X3=L, X4=Y, X5=F; X1=S, X2=P, X3=W, X4=F, X5=F; or X1=S, X2=P, X3=W, X4=Y, X5=Y) , respectively; (8) SEQ ID NOs.: 16 (X1=I, X2=N, X3=F; or X1=V, X2=T, X3=Y) and 21 (X1=A, X2=P, X3=L, X4=F, X5=Y) , respectively; or (9) SEQ ID NOs.: 17 and 22, respectively.
  7. The isolated monoclonal antibody, or an antigen-binding portion thereof, of claim 6, comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 23, linked to the heavy chain variable region, and a light chain constant region having an amino acid sequence of SEQ ID NO: 24, linked to the light chain variable region.
  8. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 1, which (a) binds human or monkey BCMA; and (b) blocks human BCMA-human BAFF or APRIL interaction.
  9. The isolated monoclon▼al antibody, or the antigen-binding portion thereof, of claim 1, which is a mouse, human, chimeric or humanized antibody.
  10. The isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 1, which is an IgG1, IgG2 or IgG4 isotype.
  11. A pharmaceutical composition comprising the isolated monoclonal antibody, or antigen-binding portion thereof, of claim 1, and a pharmaceutically acceptable carrier.
  12. The pharmaceutical composition of claim 11, further comprising an anti-tumor agent and/or a cytokine.
  13. A method for treating a tumor associated with increased BCMA expression in a subject, comprising administering to the subject a therapeutically effective amount of the isolated monoclonal antibody, or the antigen-binding portion thereof, of claim 1 or the pharmaceutical composition of claim 11.
  14. The method of claim 13, wherein the cancer disease is a non-solid tumor or a solid tumor.
  15. The method of claim 13, wherein the cancer disease is leukemia, lymphomas or multiple myeloma.
  16. The method of claim 13, further comprising administering an immune checkpoint antibody, a costimulatory antibody, and/or a cytokine.
  17. The method of claim 16, wherein the immune checkpoint antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, or the combination thereof.
  18. The method of claim 16, wherein the costimulatory antibody is an anti-CD137 antibody, an anti-OX40 antibody or an anti-GITR antibody.
  19. The method of claim 16, wherein the cytokine is IL-2, IL-21, IFN-γ, TNF-α and/or IL-4.
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