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WO2019110209A1 - Molécules de liaison à l'antigène cd47 et bcma/taci - Google Patents

Molécules de liaison à l'antigène cd47 et bcma/taci Download PDF

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Publication number
WO2019110209A1
WO2019110209A1 PCT/EP2018/079932 EP2018079932W WO2019110209A1 WO 2019110209 A1 WO2019110209 A1 WO 2019110209A1 EP 2018079932 W EP2018079932 W EP 2018079932W WO 2019110209 A1 WO2019110209 A1 WO 2019110209A1
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Prior art keywords
amino acid
acid sequence
seq
antigen
binding
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Inventor
Jerome Douglas BOYD-KIRKUP
Dipti THAKKAR
Piers INGRAM
Zhihao WU
Konrad PASZKIEWICZ
Peter Brauer
Vicente SANCENON
Siyu GUAN
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Hummingbird Bioscience Holdings Ltd
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Hummingbird Bioscience Holdings Ltd
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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
    • C07K16/2803—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 immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2827—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 immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the fields of molecular biology, more specifically antibody technology.
  • the present invention also relates to methods of medical treatment and prophylaxis.
  • CD47 is the“don’t-eat-me” signal and is ubiquitously expressed on normal cells where binding to SIRPa on macrophages inhibits phagocytosis. CD47 is commonly over-expressed in tumors where it correlates with immune evasion and poor prognosis. Blocking CD47-SIRPalpha interaction restores macrophage phagocytosis of tumor cells and anti-CD47 mAbs have shown anti-tumor efficacy in mouse models of solid tumors and hematological malignancies.
  • CD47 Despite the widespread expression of CD47 decreases the bioavailability of anti-CD47 mAbs at the tumor due to antigen sink effects, and also risks off-target toxicities, most substantially from cross-linking of erythrocytes that show high expression of CD47.
  • BCMA is expressed on cells of multiple myeloma, and the anti-BCMA antibody-drug conjugate J6M0- mcMMAF (GSK2857916) has been investigated for the treatment of multiple myeloma (see e.g. Tai et al., Blood. (2014) 123(20): 3128-3138). BCMA is also expressed by cells of B cell malignancies such as Hodgkin’s lymphoma, non-Hodgkin’s lymphoma (e.g.
  • Burkitt lymphoma Burkitt lymphoma
  • lymphocytic leukemia and the BCMA/TACI antagonist Atacicept has been investigated as an agent for use in the treatment of multiple myeloma, B-cell chronic lymphocytic leukemia, and non-Hodgkin's lymphoma (Vasiliou, Drugs Fut 2008, 33(11 ): 921 ).
  • WO 2014/087248 A2 discloses monospecific anti-CD47 antibodies having an affinity for human CD47 as high as -23.6 nM.
  • the high-affinity CD47 antibodies disclosed therein induce substantial
  • MM Multiple myeloma
  • the current treatment landscape for MM patients includes diverse classes of first and second generation agents administered as single therapy or in combination, including alkylating agents, histone deacetylase inhibitors, proteasome inhibitors, immunomodulatory drugs, monoclonal antibodies, and autologous stem cell transplantation.
  • Emerging therapies such as PD1/PD-L1 antibodies have shown severe adverse toxicity effects in clinical trials.
  • the present invention provides an antigen-binding molecule, optionally isolated, which is capable of binding to CD47 and BCMA and/or TACI.
  • the antigen-binding molecule comprises (i) an antigen-binding domain capable of binding to CD47, and (ii) an antigen-binding domain capable of binding to BCMA and/or TACI.
  • an antigen-binding molecule optionally isolated, which is capable of binding to BCMA. Also provided is an antigen-binding molecule, optionally isolated, which is capable of binding to TACI. Also provided is an antigen-binding molecule, optionally isolated, which is capable of binding to BCMA and/or TACI.
  • an antigen-binding molecule optionally isolated, which is capable of binding to CD47 in extracellular region 1.
  • the antigen-binding molecule is capable of binding to the V-type Ig-like domain of CD47. In some embodiments the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:9. In some embodiments the antigen-binding molecule is capable of inhibiting interaction between CD47 and SIRPa. In some embodiments the antigen-binding molecule is capable of increasing phagocytosis of CD47-expressing cells.
  • the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:21. In some embodiments the antigen-binding molecule comprises:
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:220
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:221
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:224; or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC- CDR2 or LC-CDR3 are substituted with another amino acid.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:24
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:25
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:24
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:25
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 190
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • VL light chain variable
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:24
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:25
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • VL light chain variable
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • VL light chain variable
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • VL light chain variable
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:193;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:24
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:25
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26;
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:32
  • LC-CDR2 having the amino acid sequence of SEQ ID NO:33
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:24
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:25
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 190
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 190
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:24
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:25
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 191
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26;
  • VL light chain variable
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26;
  • VL light chain variable
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26;
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 193.
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:23, 39, 229, 178, 180, 181 , 182 or 183;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:31 , 44, 230, 179, 184, 185, 186 or 187.
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:23;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:31 ;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:39;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:44
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:229;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:230;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 178;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:179;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 180;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:179;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 181 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 179;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 182;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:179;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 183;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:179;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 182;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:184;
  • (x) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 183; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:184;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:185;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:185;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:186;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 183;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 187.
  • the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:22.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:50
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:51
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:52,
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:60;
  • VH heavy chain variable region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:50
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:51
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:52;
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:60.
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:49;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:57.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:66
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:67
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:68,
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:76;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:66
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:67
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:68;
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:76.
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:65; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:73.
  • the present invention provides an antigen-binding molecule, optionally isolated, comprising (i) an antigen-binding molecule according to the invention, and (ii) an antigen-binding molecule capable of binding to an antigen other than CD47.
  • the antigen-binding molecule capable of binding to an antigen other than CD47 is capable of binding to BCMA and/or TACI.
  • the present invention provides an antigen-binding molecule, optionally isolated, which is capable of binding to CD47 and BCMA and/or TACI.
  • the antigen-binding molecule comprises (i) an antigen-binding molecule capable of binding to CD47, and (ii) an antigen-binding molecule capable of binding to BCMA and/or TACI.
  • the antigen-binding molecule capable of binding to BCMA is capable of binding to the extracellular domain of BCMA.
  • the antigen-binding molecule capable of binding to TACI is capable of binding to the extracellular domain of TACI.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 104
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 105,
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:1 13;
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 104
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 105;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 1 1 1
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 1 12
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 1 13.
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:102;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 10.
  • the antigen-binding molecule comprises an antigen-binding domain capable of binding to BCMA and/or TACI which comprises or consists of a polypeptide comprising a BCMA- or TACI- binding amino acid sequence.
  • the BCMA- or TACI-binding amino acid sequence is derived from a ligand for BCMA and/or TACI.
  • the BCMA- or TACI-binding amino acid sequence comprises or consists of a BCMA- or TACI-binding amino acid sequence derived from APRIL.
  • the antigen-binding molecule comprises an antigen-binding domain capable of binding to BCMA and/or TACI which comprises or consists of:
  • the antigen-binding molecule comprises an antigen-binding domain capable of binding to BCMA and/or TACI which comprises or consists of:
  • the antigen-binding molecule is capable of binding to cells expressing CD47,
  • BCMA and/or TACI at the cell surface are BCMA and/or TACI at the cell surface.
  • the antigen-binding molecule is capable of inhibiting interaction between CD47 and SIRPa.
  • the antigen-binding molecule is capable of increasing phagocytosis of CD47- expressing cells. In some embodiments the antigen-binding molecule does not induce substantial hemagglutination.
  • the present invention provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to the invention.
  • CAR chimeric antigen receptor
  • the present invention provides an antigen-binding molecule, optionally isolated, which is capable of binding to BCMA and/or TACI, which comprises, or consists of, a polypeptide comprising a BCMA- or TACI-binding amino acid sequence.
  • the BCMA- or TACI-binding amino acid sequence comprises or consists of a BCMA- or TACI-binding amino acid sequence derived from APRIL.
  • the antigen-binding molecule comprises a polypeptide capable of binding to BCMA and/or TACI which comprises or consists of:
  • the antigen-binding molecule comprises a polypeptide capable of binding to BCMA and/or TACI which comprises or consists of:
  • the antigen-binding molecule further comprises an antigen-binding domain capable of binding to an antigen other than BCMA and TACI.
  • the antigen other than BCMA and TACI is CD47.
  • the present invention provides a nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule or a CAR according to the invention.
  • the present invention provides an expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to the invention.
  • the present invention provides a cell comprising an antigen-binding molecule, a CAR, a nucleic acid or a plurality of nucleic acids, or an expression vector or a plurality of expression vectors according to the invention.
  • the present invention provides a method comprising culturing a cell comprising a nucleic acid or a plurality of nucleic acids, or an expression vector or a plurality of expression vectors according to the invention, under conditions suitable for expression of the antigen-binding molecule or CAR from the nucleic acid(s) or expression vector(s).
  • the present invention provides a composition comprising an antigen-binding molecule, a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, or a cell according to the invention.
  • the present invention provides an antigen-binding molecule, a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the invention for use in a method of medical treatment or prophylaxis.
  • the present invention provides an antigen-binding molecule, a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the invention for use in a method of treatment or prevention of a cancer.
  • the present invention provides the use of an antigen-binding molecule, a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the invention in the manufacture of a medicament for use in a method of treatment or prevention of a cancer.
  • the present invention provides a method of treating or preventing a cancer, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the invention.
  • the present invention provides a method for increasing phagocytosis of CD47- expressing cells, comprising contacting CD47-expressing cells with an antigen-binding molecule according to the invention.
  • the present invention provides an in vitro complex, optionally isolated, comprising an antigen-binding molecule according to the invention bound to CD47, BCMA and/or TACI. In another aspect the present invention provides an in vitro complex, optionally isolated, comprising an antigenbinding molecule according to the invention bound to BCMA and/or TACI.
  • the present invention provides a method comprising contacting a sample containing, or suspected to contain, CD47, BCMA and/or TACI with an antigen-binding molecule according to the invention, and detecting the formation of a complex of the antigen-binding molecule with CD47, BCMA and/or TACI.
  • the present invention provides a method comprising contacting a sample containing, or suspected to contain, BCMA and/or TACI with an antigen-binding molecule according to the invention, and detecting the formation of a complex of the antigen-binding molecule with BCMA and/or TACI.
  • the present invention provides a subject for treatment with a CD47-targeted agent, a BCMA-targeted agent and/or a TACI-targeted agent, the method comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to the invention and detecting the formation of a complex of the antigen-binding molecule with CD47, BCMA and/or TACI.
  • the present invention provides a subject for treatment with a BCMA-targeted agent and/or a TACI-targeted agent, the method comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to the invention and detecting the formation of a complex of the antigen-binding molecule with BCMA and/or TACI.
  • the present invention provides the use of an antigen-binding molecule according to the invention as an in vitro or in vivo diagnostic or prognostic agent.
  • the cancer is selected from: a hematologic malignancy, a myeloid hematologic malignancy, a lymphoblastic hematologic malignancy, a B cell malignancy, multiple myeloma (MM), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), lymphocytic leukemia, lymphoma, B cell lymphoma, Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma (NHL), Burkitt lymphoma, bladder cancer, brain cancer, glioblastoma, ovarian cancer, breast cancer, colon cancer, liver cancer, hepatocellular carcinoma, prostate cancer, lung cancer, Non-small Cell Lung Cancer (NSCLC), skin cancer and melanoma.
  • MM myelodysplastic syndrome
  • AML acute myeloid leukemia
  • CML chronic mye
  • the present invention provides antigen-binding molecules having combinations of desirable biophysical and/or functional properties as compared to antigen-binding molecules disclosed in the prior art.
  • MM multiple myeloma
  • CD47, BCMA and TACI are highly co-expressed in all characterized MM cell lines and many patient samples, and play functional roles that reduce risk of antigen loss. Their expression levels are prognostic of the progression and outcome of MM.
  • Overexpression of CD47 is utilized by malignant hematopoietic cells to prevent macrophage clearance and evade immunesurveillance in the bone marrow microenvironment.
  • APRIL secreted by bone marrow osteoclast activates BCMA/TACI receptors in multiple myeloma cells, and induces the upregulation of antiapoptotic, immunosuppressive, and osteoclastogenic genes through the canonical and non-canonical NF-kB pathway.
  • aspects of the present invention relate to antigen-binding molecules capable of binding to both CD47 and BCMA and/or TACI.
  • the bispecific CD47-binding, BCMA/TACI-binding antigen-binding molecules described herein display preferential binding to cells expressing both CD47 and BCMA/TACI, and are therefore useful for targeting myeloid hematologic malignancies, e.g. multiple myeloma. They represent an improved treatment for myeloid hematologic malignancies as compared e.g. to CD47-binding antibodies of the prior art, because the BCMA/TACI-binding arm targets the CD47-binding arm to the cancer cells, minimising off-target effects.
  • antigen-binding molecules which bind to human CD47 with high affinity, are cross-reactive with non-human primate CD47, preferentially bind to CD47- and BCMA/TACI- expressing cells, display potent inhibition of interaction between CD47 and SIRPa, and do not induce hemagglutination.
  • Human CD47 (also known as IAP, MER6 and OA3) is the protein identified by UniProt Q08722.
  • isoform OA3-323 (UniProt: Q08722-1 , v1 ; SEQ ID NO:1 ); isoform OA3-293 (UniProt: Q08722-2; SEQ ID NO:2), which lacks the amino acid sequence corresponding to positions 293 to 323 of SEQ ID NO:1 ; isoform OA3-305 (UniProt: Q08722-3; SEQ ID NO:3), which comprises the substitutions K304N and A305N relative to SEQ ID NO:1 , and which lacks the amino acid sequence corresponding to positions 306 to 323 of SEQ ID NO:1 ; and isoform OA3-312 (UniProt: Q08722-4; SEQ ID NO:4), which lacks the amino acid sequence corresponding to positions 312 to 323 of SEQ ID NO:1.
  • the N-terminal 18 amino acids of SEQ ID NOs:1 to 4 constitute a signal peptide, and so the mature form of isoforms OA3-323, OA3-293, OA3-305 and OA3-312 (i.e. after processing to remove the signal peptide) have the amino acid sequences shown in SEQ ID NOs:5 to 8, respectively.
  • CD47 The structure and function of CD47 is reviewed e.g. in Sick et al., Br J Pharmacol. (2012) 167(7): 1415- 1430 and Willingham et al. Proc Natl Acad Sci U S A. (2012) 109(17): 6662-6667, both of which are hereby incorporated by reference in its entirety.
  • CD47 is a ubiquitously-expressed ⁇ 50 kDa multi-pass membrane receptor that belongs to the immunoglobulin superfamily, comprising an N-terminal extracellular region (SEQ ID NO:10) having a V-type Ig-like domain (SEQ ID NO:9), five transmembrane domains (SEQ ID NOs:11 , 13, 15, 17 and 19), and a short C-terminal intracellular tail (SEQ ID NO:20).
  • CD47 is involved in cell-to-cell communication through ligating to the transmembrane signal-regulatory proteins (SIRPs) SIRPa and SIRPy and integrins (e.g. anb3 integrin), and also mediates cell-extracellular matrix interactions through binding to thrombospondin-1 (TSP-1 ).
  • SIRPs transmembrane signal-regulatory proteins
  • SIRPa and SIRPy and integrins e.g. anb3 integrin
  • TSP-1 thrombospondin-1
  • CD47 is involved in a wide range of cellular processes including adhesion, migration, proliferation and apoptosis, and plays a key role in immune processes and angiogenesis.
  • CD47 is the ligand for SIRPa, which expressed on macrophages and dendritic cells. Binding of CD47 to SIRPa on the surface of phagocytic cells, triggers SIRPa ITIM signalling, inhibiting phagocytosis of the CD47 expressing cell.
  • CD47 is a multi-pass transmembrane protein, whereas SIRPa consists of 4 extracellular domains and an intracellular ITIM-domain. The terminal V-set domain of SIRPa interacts with the Ig V-like domain of CD47.
  • SIRPa Upon binding CD47, SIRPa initiates a signalling cascade that results in the inhibition of phagocytosis of the CD47-expressing cell.
  • This“don't eat me” signal is transmitted by phosphorylation by Src kinases of immunoreceptor tyrosine-based inhibitor motifs (ITIMs) in the cytoplasmic domain of SIRPa.
  • ITIMs immunoreceptor tyrosine-based inhibitor motifs
  • SH2 Src homology-2
  • SHP-1 and SHP-2 blocks phagocytosis, potentially through preventing the accumulation of myosin-IIA at the phagocytic synapse.
  • Disrupting the interaction along the antiparallel beta sheets of CD47 prevents downstream ITIM-mediated signalling, enabling phagocytes to‘eat’ and destroy cancer cells.
  • CD47 expression/activity is implicated in the development and progression of many cancers, and accumulating evidence suggests that cell-surface expression of CD47 is a common mechanism by which cancer cells protect themselves from phagocytosis.
  • CD47 refers to CD47 from any species and includes CD47 isoforms, fragments, variants or homologues from any species.
  • a“fragment”,“variant” or“homologue” of a protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein (e.g. a reference isoform).
  • fragments, variants, isoforms and homologues of a reference protein may be characterised by ability to perform a function performed by the reference protein.
  • A“fragment” generally refers to a fraction of the reference protein.
  • A“variant” generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein.
  • An“isoform” generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein (e.g. OA3-323, OA3-293, OA3-305 and OA3-312 are all isoforms of one another).
  • A“homologue” generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein.
  • human CD47 isoform OA3-323 (Q08722-1 , v1 ; SEQ ID NO:1 ) and Rhesus macaque CD47 (UniProt: F7F5Y9-1 , v2; SEQ ID NO:145) are homologues of one another.
  • Homologues include orthologues.
  • A“fragment” of a reference protein may be of any length (by number of amino acids), although may optionally be at least 25% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.
  • a fragment of CD47 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250 or 300 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250 or 300 amino acids.
  • the CD47 is CD47 from a mammal (e.g. a primate (rhesus, cynomolgous, nonhuman primate or human) and/or a rodent (e.g. rat or murine) CD47).
  • Isoforms, fragments, variants or homologues of CD47 may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature CD47 isoform from a given species, e.g. human.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference CD47 (e.g. human CD47 isoform OA3-323), as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of CD47 may display association with one or more of: SIRPa, SIRPy, TSP-1 and anb3 integrin.
  • the CD47 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NOs:1 to 8.
  • a fragment of CD47 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NO:9 or 10.
  • CD47 is an attractive therapeutic target.
  • CD47 is usually expressed on the surface of normal healthy cells and migrating hematopoietic stem cells to prevent phagocytosis, and is upregulated in nearly all hematological and solid tumors, including MM cells, to evade immune surveillance and escape phagocytosis.
  • Disrupting the interaction between CD47 and SIRPa enables phagocytes to“eat” and destroy cancer cells.
  • CD47 blockade repolarises tumor-associated macrophages into a pro-inflammatory, anti-tumor state, and clearance of malignant cells by phagocytic cells offers an additional route for neoantigen presentation to adaptive immune system.
  • Human B cell maturation antigen (BCMA; also known as TNFRSF17) is the protein identified by UniProt Q02223. Alternative splicing of mRNA encoded by the human TNFRSF17 gene yields two isoforms: isoform 1 (UniProt: Q02223-1 , v2; SEQ ID NO:95) and isoform 2 (UniProt: Q02223-2; SEQ ID NO:96), in which the amino acid sequence corresponding to positions 44 to 93 of SEQ ID NO:95 are substituted with “R”.
  • BCMA Human B cell maturation antigen
  • BCMA is a cell surface receptor of the TNF receptor superfamily.
  • BCMA comprises an N-terminal extracellular domain (SEQ ID NO:97) having a cysteine-rich TNFR repeat region (SEQ ID NO:98).
  • the extracellular domain is connected by a transmembrane domain (SEQ ID NO:99) to a cytoplasmic domain (SEQ ID NO: 100), containing a region which is important for TRAF interaction and activation of NFKB (SEQ ID NO:101 ; Hatzoglou et al., J Immunol. (2000) 165(3): 1322-30).
  • BCMA is expressed by mature B lymphocytes, and plays an important role in differentiation of B cells into plasma cells (see Tai and Anderson, Immunotherapy (2015) 7(11 ): 1 187-1199, which is hereby incorporated by reference in its entirety).
  • BCMA is expressed by B-cell lineage cells, particularly in the interfollicular region of the germinal center, by plasmablasts and by differentiated plasma cells. BCMA expression is selectively induced during plasma cell differentiation. BCMA may enhance humoral immunity by stimulating the survival of normal plasma cells and plasmablasts, but is absent on naive and most memory B cells.
  • BCMA is also expressed by CD138 BDCA-4 + plasmacytoid dendritic cells
  • BCMA B cell activation factor
  • APRIL proliferation-inducing ligand
  • BCMA refers to BCMA from any species and includes BCMA isoforms, fragments, variants or homologues from any species.
  • a fragment of BCMA may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150 or 175 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 125, 150 or 175 amino acids.
  • the BCMA is BCMA from a mammal (e.g. a primate (rhesus, cynomolgous, or human) and/or a rodent (e.g. rat or murine) BCMA).
  • Isoforms, fragments, variants or homologues of BCMA may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature BCMA isoform from a given species, e.g. human.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference BCMA (e.g. human BCMA isoform 1 ), as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of BCMA may display association with e.g. BAFF and/or APRIL.
  • the BCMA comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:95 or 96.
  • a fragment of BCMA comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:97.
  • Transmembrane activator and CAML interactor (TACI; also known as TNFRSF13B) is the protein identified by UniProt 014836.
  • Alternative splicing of mRNA encoded by the human TNFRSF13B gene yields three isoforms: isoform 1 (UniProt: 014836-1 , v1 ; SEQ ID NO:260), isoform 2 (UniProt: 014836-2; SEQ ID NO:261 ), in which the amino acid sequence corresponding to positions 21 to 67 of SEQ ID NO:260 are substituted with“W”, and isoform 3 (UniProt: 014836-3; SEQ ID NO:262), in which the amino acid sequence corresponding to positions 150 to 176 of SEQ ID NO:260 are substituted with a 27 amino acid sequence, and lacking the amino acid sequence corresponding to positions 177 to 293 of SEQ ID NO:260.
  • TACI is a cell surface receptor of the TNF receptor superfamily, and comprises an N-terminal extracellular domain (SEQ ID NO:263) having two cysteine-rich TNFR repeat regions (SEQ ID NOs:264 and 265). The extracellular domain is connected by a transmembrane domain (SEQ ID NO:266) to a cytoplasmic domain (SEQ ID NO:267).
  • TACI is expressed by B lymphocytes, and is the receptor for calcium-modulator and cyclophilin ligand (CAML), BAFF and APRIL (Wu et al., Journal of Biological Chemistry (2000) 275 (45):35478-85).
  • APRIL calcium-modulator and cyclophilin ligand
  • BAFF and APRIL signal through TACI, inducing activation of several transcription factors including NFAT, AP-1 , and NFKB.
  • TACI expression is upregulated in B cell malignancies such as multiple myeloma (MM).
  • MM cell lines and fresh tumor samples from patients have been shown to bind soluble BAFF and express BCMA, TACI, and BAFF-R, and BAFF modulates the proliferative capacity of cytokine-stimulated MM cells, likely through its ability to promote survival via signalling through these receptors (Novak et al., Blood (2004) 103:689- 694).
  • TACI refers to TACI from any species and includes TACI isoforms, fragments, variants or homologues from any species.
  • a fragment of TACI may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200 or 250 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 125, 150, 200 or 250 amino acids.
  • the TACI is TACI from a mammal (e.g. a primate (rhesus, cynomolgous, or human) and/or a rodent (e.g. rat or murine) TACI).
  • Isoforms, fragments, variants or homologues of TACI may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature TACI isoform from a given species, e.g. human.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference TACI (e.g. human TACI isoform 1 ), as determined by analysis by a suitable assay for the functional property/activity.
  • TACI e.g. human TACI isoform 1
  • an isoform, fragment, variant or homologue of TACI may display association with e.g. BAFF and/or APRIL.
  • the TACI comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:260, 261 or 262.
  • a fragment of TACI comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:263.
  • BCMA and TACI are attractive therapeutic targets.
  • BCMA and TACI are differentially expressed during the differentiation of immature B cells to mature plasma cells, and are highly expressed in MM cell lines and CD138+ cells from MM patients.
  • Binding of APRIL to BCMA/TACI promotes cell survival, proliferation, and immunosuppression through the canonical and non-canonical NF-kB signalling pathways. Disrupting the interaction between APRIL and BCMA/TACI with competitive antagonists will inhibit downstream BCMA/TACI-mediated signalling.
  • the present invention provides antigen-binding molecules.
  • the antigen-binding molecules are capable of binding to CD47.
  • the antigen-binding molecules are capable of binding to BCMA and/or TACI.
  • the antigen-binding molecules are capable of binding to CD47 and BCMA and/or TACI.
  • the antigen-binding molecules comprise (i) an antigen-binding domain capable of binding to CD47 and (ii) an antigen-binding domain capable of binding to BCMA and/or TACI.
  • An“antigen-binding molecule” refers to a molecule which is capable of binding to a target antigen, and encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab ⁇ , diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.), as long as they display binding to the relevant target molecule(s).
  • monospecific and multispecific antibodies e.g., bispecific antibodies
  • antibody fragments e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab ⁇ , diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.
  • the antigen-binding molecule of the present invention comprises a moiety or moieties capable of binding to a target antigen(s).
  • the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen.
  • the moiety capable of binding to a target antigen comprises or consists of an aptamer capable of binding to the target antigen, e.g. a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3): 181-202).
  • the moiety capable of binding to a target antigen comprises or consists of a antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e. a singledomain antibody (sdAb)) affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem.
  • a antigen-binding peptide/polypeptide e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e. a singledomain antibody (
  • the antigen-binding molecules of the present invention generally comprise an antigen-binding domain comprising a VH and a VL of an antibody capable of specific binding to the target antigen.
  • the antigenbinding domain formed by a VH and a VL may also be referred to herein as an Fv region.
  • the antigen-binding molecules of the present invention comprise antigen-binding moieties derived from a ligand for the target antigen.
  • An antigen-binding molecule may be, or may comprise, an antigen-binding polypeptide, or an antigenbinding polypeptide complex.
  • An antigen-binding molecule may comprise more than one polypeptide which together form an antigen-binding domain.
  • the polypeptides may associate covalently or non- covalently.
  • the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL).
  • An antigen-binding molecule may refer to a non-covalent or covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, or 8 polypeptides), e.g. an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.
  • polypeptide e.g. 2, 3, 4, 6, or 8 polypeptides
  • IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.
  • the antigen-binding molecules of the present invention may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to CD47, BCMA or TACI.
  • Antigen-binding regions of antibodies such as single chain variable fragment (scFv), Fab and F(ab’)2 fragments may also be used/provided.
  • An“antigen-binding region” is any fragment of an antibody which is capable of binding to the target for which the given antibody is specific.
  • antigen-binding molecules of the present invention may be designed and prepared from the amino acid sequence of a ligand for the target antigen (e.g. BCMA and/or TACI).
  • target antigen e.g. BCMA and/or TACI
  • Antibodies generally comprise six complementarity-determining regions CDRs; three in the heavy chain variable (VH) region: HC-CDR1 , HC-CDR2 and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1 , LC-CDR2, and LC-CDR3.
  • the six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target antigen.
  • VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs.
  • FRs framework regions
  • VH regions comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term.
  • the CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.
  • the antigen-binding molecule comprises the CDRs of an antigen-binding molecule which is capable of binding to CD47. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule which is capable of binding to CD47. In some embodiments, the antigen-binding molecule comprises the CDRs and the FRs of an antigen-binding molecule which is capable of binding to CD47. That is, in some embodiments the antigen-binding molecule comprises the VH region and the VL region of an antigen-binding molecule which is capable of binding to CD47.
  • the antigen-binding molecule comprises a VH region and a VL region which is, or which is derived from, the VH/VL region of a CD47-binding antibody clone described herein (i.e. anti- CD47 antibody clones 1-1-A1_BM, 1-1-A1 , 5-48-A6, 5-48-D2, 11A1 H1 , 11A1 H2, 11A1 H3, 11A1 H4, 11A1 H5, 11A1 H6, 1 1A1 H7, 11A1 H8, 11A1 H9, 1 1A1 H10 or 11A1 H11 ).
  • a CD47-binding antibody clone described herein i.e. anti- CD47 antibody clones 1-1-A1_BM, 1-1-A1 , 5-48-A6, 5-48-D2, 11A1 H1 , 11A1 H2, 11A1 H3, 11A1 H4, 11A1 H5, 11A1 H6, 1 1A1 H7, 11A1 H8,
  • the antigen-binding molecule comprises a VH region according to one of (1 ) to (4) below:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:24
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:25
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:50
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:51
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:52
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:66
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:67
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:68,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:220
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:221
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 188
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 189
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:26,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • the antigen-binding molecule comprises a VH region according to one of (6) to (15) below:
  • HC-FR1 having the amino acid sequence of SEQ ID NO:27
  • HC-FR2 having the amino acid sequence of SEQ ID NO:28
  • HC-FR3 having the amino acid sequence of SEQ ID NO:29
  • HC-FR4 having the amino acid sequence of SEQ ID NO:30,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:40
  • HC-FR2 having the amino acid sequence of SEQ ID NO:41
  • HC-FR3 having the amino acid sequence of SEQ ID NO:42
  • HC-FR4 having the amino acid sequence of SEQ ID NO:43,
  • HC-FR1 having the amino acid sequence of SEQ ID NO:53
  • HC-FR2 having the amino acid sequence of SEQ ID NO:54
  • HC-FR3 having the amino acid sequence of SEQ ID NO:55
  • HC-FR4 having the amino acid sequence of SEQ ID NO:56,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:69
  • HC-FR2 having the amino acid sequence of SEQ ID NO:70
  • HC-FR3 having the amino acid sequence of SEQ ID NO:71
  • HC-FR4 having the amino acid sequence of SEQ ID NO:72,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.
  • HC-FR1 having the amino acid sequence of SEQ ID NO: 194
  • HC-FR2 having the amino acid sequence of SEQ ID NO:225
  • HC-FR3 having the amino acid sequence of SEQ ID NO:226
  • HC-FR4 having the amino acid sequence of SEQ ID NO:227,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR1 having the amino acid sequence of SEQ ID NO: 194
  • HC-FR2 having the amino acid sequence of SEQ ID NO: 195
  • HC-FR3 having the amino acid sequence of SEQ ID NO: 198
  • HC-FR4 having the amino acid sequence of SEQ ID NO:203,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:194
  • HC-FR2 having the amino acid sequence of SEQ ID NO: 195
  • HC-FR3 having the amino acid sequence of SEQ ID NO: 199
  • HC-FR4 having the amino acid sequence of SEQ ID NO:203,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.
  • HC-FR2 having the amino acid sequence of SEQ ID NO: 196
  • HC-FR3 having the amino acid sequence of SEQ ID NO:200
  • HC-FR4 having the amino acid sequence of SEQ ID NO:204,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.
  • HC-FR1 having the amino acid sequence of SEQ ID NO: 194
  • HC-FR2 having the amino acid sequence of SEQ ID NO: 197
  • HC-FR3 having the amino acid sequence of SEQ ID NO:201
  • HC-FR4 having the amino acid sequence of SEQ ID NO:204,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.
  • HC-FR1 having the amino acid sequence of SEQ ID NO: 194
  • HC-FR2 having the amino acid sequence of SEQ ID NO: 197
  • HC-FR3 having the amino acid sequence of SEQ ID NO:202
  • HC-FR4 having the amino acid sequence of SEQ ID NO:203,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.
  • the antigen-binding molecule comprises a VH region comprising the CDRs according to one of (1 ), (2), (3), (4) or (5) above, and the FRs according to one of (6), (7), (8), (9), (10), (1 1 ), (12), (13), (14) or (15) above.
  • the antigen-binding molecule comprises a VH region according to one of (16) to (25) below:
  • VH region comprising the CDRs according to (1 ) and the FRs according to (6).
  • a VH region comprising the CDRs according to (1 ) and the FRs according to (7).
  • the antigen-binding molecule comprises a VH region according to one of (26) to (34) below:
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:23.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:39.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:49.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:65.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:229.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO: 178.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:180.
  • a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO: 181.
  • a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:182.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:183.
  • the antigen-binding molecule comprises a VL region according to one of (36) to (43) below:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:60;
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:76;
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:224;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 190
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34; or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC- CDR2 or LC-CDR3 are substituted with another amino acid.
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:34;
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 193;
  • the antigen-binding molecule comprises a VL region according to one of (44) to (50) below:
  • the antigen-binding molecule comprises a VL region comprising the CDRs according to one of (36), (37), (38), (39), (40), (41 ), (42) or (43) above, and the FRs according to one of (44), (45), (46), (47), (48), (49) or (50) above.
  • the antigen-binding molecule comprises a VL region according to one of (51 ) to (60) below:
  • VL region comprising the CDRs according to (36) and the FRs according to (44).
  • VL region comprising the CDRs according to (38) and the FRs according to (46).
  • the antigen-binding molecule comprises a VL region according to one of (61 ) to (70) below:
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:31.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:44.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:57.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:73.
  • a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • the antigen-binding molecule comprises a VH region according to any one of (1 ) to (35) above, and a VL region according to any one of (36) to (70) above.
  • the present disclosure provides an antigen-binding molecule capable of binding to BCMA.
  • the present disclosure also provides an antigen-binding molecule capable of binding to TACI.
  • the present disclosure also provides an antigen-binding molecule capable of binding to BCMA and/or TACI.
  • the antigen-binding molecule comprises the CDRs of an antigen-binding molecule which is capable of binding to BCMA and/or TACI. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule which is capable of binding to BCMA and/or TACI. In some embodiments, the antigen-binding molecule comprises the CDRs and the FRs of an antigenbinding molecule which is capable of binding to BCMA and/or TACI. That is, in some embodiments the antigen-binding molecule comprises the VH region and the VL region of an antigen-binding molecule which is capable of binding to BCMA and/or TACI.
  • the antigen-binding molecule antigen-binding molecule which is capable of binding to BCMA is also capable of binding to TACI. That is, in some embodiments the antigen-binding molecule of the present invention comprises the CDRs, FRs, or the CDRs and FRs (i.e. the VH and VL region) of an antigen-binding molecule which is cross-reactive for BCMA and TACI. Antibodies which are crossreactive for BCMA and TACI are described e.g. in WO 2002066516 A2, which is hereby incorporated by reference in its entirety.
  • the antigen-binding molecule comprises a VH region and a VL region which is, or which is derived from, the VH/VL region of a BCMA-binding antibody, a TACI-binding antibody, or an antibody capable of binding to BCMA and TACI.
  • the antigen-binding molecule comprises a VH region and a VL region which is, or which is derived from, the VH/VL region of a BCMA- binding antibody clone described herein, i.e. anti-BCMA antibody clone J6M0 (described e.g. in WO 2012/163805 A1 ).
  • the antigen-binding molecule comprises a VH region according to (71 ):
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 103
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 104
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:105,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • the antigen-binding molecule comprises a VH region according to (72):
  • HC-FR1 having the amino acid sequence of SEQ ID NO: 106
  • HC-FR2 having the amino acid sequence of SEQ ID NO: 107
  • HC-FR3 having the amino acid sequence of SEQ ID NO: 108
  • HC-FR4 having the amino acid sequence of SEQ ID NO:109,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 a variant thereof in which one or two or three amino acids in one or more of HC-FR1 , HC-FR2, HC-FR3, or HC-FR4 are substituted with another amino acid.
  • the antigen-binding molecule comprises a VH region according to (73):
  • the antigen-binding molecule comprises a VH region according to (74):
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • the antigen-binding molecule comprises a VL region according to (75):
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 1 13, or a variant thereof in which one or two or three amino acids in one or more of LC-CDR1 , LC- CDR2, or LC-CDR3 are substituted with another amino acid.
  • the antigen-binding molecule comprises a VL region according to (76):
  • the antigen-binding molecule comprises a VL region according to (77):
  • the antigen-binding molecule comprises a VL region according to (78):
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,
  • the antigen-binding molecule comprises a VH region according to any one of (71 ) to (74) above, and a VL region according to any one of (75) to (78) above.
  • the antigen-binding molecule of the present disclosure is capable of binding to BCMA and/or TACI. In some embodiments the antigen-binding molecule comprises an antigen-binding domain capable of binding to BCMA and/or TACI. In some embodiments the antigen-binding molecule or antigen-binding domain comprises a polypeptide comprising, or consisting of, an amino acid sequence which is capable of binding to BCMA and/or TACI.
  • the ability of a given antigen-binding molecule/domain/polypeptide/amino acid sequence to bind to BCMA and/or TACI can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay.
  • ELISA Surface Plasmon Resonance
  • SPR Surface Plasmon Resonance
  • Bio-Layer Interferometry see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507
  • flow cytometry or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent as
  • an antigen-binding molecule/domain/polypeptide/amino acid sequence capable of binding to BCMA and/or TACI binds to BCMA and/or TACI with greater affinity, and/or with greater duration than it binds to a protein other than BCMA and/or TACI.
  • the amino acid sequence which is capable of binding to BCMA and/or TACI is derived from the amino acid sequence of a ligand for BCMA and/or TACI.
  • the amino acid sequence which is capable of binding to BCMA and/or TACI is derived from the BCMA- and/or TACI-binding region of a ligand for BCMA and/or TACI.
  • a BCMA- or TACI-binding amino acid sequence comprises or consists of a BCMA- or TACI-binding amino acid sequence derived from a ligand for BCMA and/or TACI (e.g. APRIL or BAFF).
  • amino acid sequence which is capable of binding to BCMA and/or TACI is derived from the amino acid sequence of APRIL. In some embodiments, amino acid sequence which is capable of binding to BCMA and/or TACI is derived from the amino acid sequence of BAFF.
  • an amino acid sequence which is‘derived from’ a reference amino acid sequence comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference amino acid sequence.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:231.
  • amino acid sequence which is derived from the amino acid sequence of a ligand for BCMA and/or TACI may further comprise one or more modifications (e.g.
  • substitutions/insertions/deletions to the amino acid sequence may provide the amino acid sequence with desired functional and/or structural properties.
  • the amino acid sequence which is derived from the amino acid sequence of a ligand for BCMA and/or TACI comprises modification to increase binding to BCMA as compared to the binding displayed by the corresponding unmodified amino acid sequence. In some embodiments the amino acid sequence which is derived from the amino acid sequence of a ligand for BCMA and/or TACI comprises modification to increase binding to TACI as compared to the binding displayed by the corresponding unmodified amino acid sequence.
  • the amino acid sequence which is derived from the amino acid sequence of a ligand for BCMA and/or TACI comprises modification to decrease the level of BCMA-mediated signalling by a BCMA-expressing cell following binding of an antigen-binding molecule comprising the amino acid sequence as compared to the level of BCMA-mediated signalling by a BCMA-expressing cell following binding of an antigen-binding molecule comprising the corresponding unmodified amino acid sequence.
  • the amino acid sequence which is derived from the amino acid sequence of a ligand for BCMA and/or TACI comprises modification to decrease the level of TACI-mediated signalling by a TACI-expressing cell following binding of an antigen-binding molecule comprising the amino acid sequence as compared to the level of TACI-mediated signalling by a TACI-expressing cell following binding of an antigen-binding molecule comprising the corresponding unmodified amino acid sequence.
  • the amino acid sequence derived from the amino acid sequence of a ligand for BCMA and/or TACI comprises modification to decrease oligomerisation of the antigen-binding polypeptide with itself (i.e. homooligomerisation), or with naturally occurring or non-naturally occurring antigen-binding polypeptide(s) comprising the unmodified amino acid sequence (heterooligomerisation).
  • BCMA-mediated signalling can be analysed using BCMA-expressing cells e.g. in an assay for detecting and/or quantifying BCMA-mediated signalling.
  • TACI-mediated signalling can be analysed using TACI-expressing cells e.g. in an assay for detecting and/or quantifying TACI-mediated signalling.
  • Suitable assays include e.g. NFKB reporter assays, and assays for detecting the
  • the amino acid sequence which is derived from the amino acid sequence of a ligand for BCMA and/or TACI comprises modification to decrease BCMA oligomerisation following binding of an antigen-binding molecule comprising the amino acid sequence as compared to the level of BCMA oligomerisation following binding of an antigen-binding molecule comprising the corresponding unmodified amino acid sequence.
  • the amino acid sequence which is derived from the amino acid sequence of a ligand for BCMA and/or TACI comprises modification to decrease TACI oligomerisation following binding of an antigen-binding molecule comprising the amino acid sequence as compared to the level of TACI oligomerisation following binding of an antigen-binding molecule comprising the corresponding unmodified amino acid sequence.
  • the amino acid sequence which is derived from the amino acid sequence of a ligand for BCMA and/or TACI comprises modification corresponding to one or more of the following modifications: D>A at the position corresponding to position 20 of SEQ ID NO:231 , insertion of‘GGS’ between the positions corresponding to positions 62 and 63 of SEQ ID NO:231 , M>A at the position corresponding to position 90 of SEQ ID NO:231 , R>A at the position corresponding to position 121 of SEQ ID NO:231.
  • the amino acid sequence which is derived from the amino acid sequence of a ligand for BCMA and/or TACI comprises modification corresponding to one or more of the following modifications: R>A at the position corresponding to position 34 of SEQ ID NO:231 , Y>A at the position corresponding to position 56 of SEQ ID NO:231 , S>A at the position corresponding to position 103 of SEQ ID NO:231 , H>A at the position corresponding to position 108 of SEQ ID NO:231 , F>A at the position corresponding to position 134 of SEQ ID NO:231.
  • amino acid sequence is described as comprising modification(s)“corresponding to” reference modification(s)
  • equivalent modification(s) in homologous amino acid sequences are contemplated. That is, the modification(s) identified in the preceding paragraphs are contemplated in amino acid sequences having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:231.
  • Corresponding positions to those identified in the reference sequence i.e. SEQ ID NO:231
  • sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960).
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising one or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10) substitutions/insertions relative to the amino acid sequence of SEQ ID NO:231.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution D>A at the position corresponding to position 20 of SEQ ID NO:231. In some embodiments the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising insertion of‘GGS’ between the positions corresponding to positions 62 and 63 of SEQ ID NO:231. In some embodiments the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution M>A at the position corresponding to position 90 of SEQ ID NO:231.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution R>A at the position corresponding to position 121 of SEQ ID NO:231.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution D>A at the position corresponding to position 20 of SEQ ID NO:231 , and further comprising insertion of‘GGS’ between the positions corresponding to positions 62 and 63 of SEQ ID NO:231 , further comprising the substitution M>A at the position corresponding to position 90 of SEQ ID NO:231 , and further comprising the substitution R>A at the position corresponding to position 121 of SEQ ID NO:231 (that is, the amino acid sequence shown in SEQ ID NO:232).
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution R>A at the position corresponding to position 34 of SEQ ID NO:231. In some embodiments the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution Y>A at the position corresponding to position 56 of SEQ ID NO:231. In some embodiments the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution S>A at the position corresponding to position 103 of SEQ ID NO:231.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution H>A at the position corresponding to position 108 of SEQ ID NO:231. In some embodiments the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution F>A at the position corresponding to position 134 of SEQ ID NO:231.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:231 , further comprising the substitution R>A at the position corresponding to position 34 of SEQ ID NO:231 , further comprising the substitution Y>A at the position corresponding to position 56 of SEQ ID NO:231 , further comprising the substitution S>A at the position corresponding to position 103 of SEQ ID NO:231 , further comprising the substitution H>A at the position corresponding to position 108 of SEQ ID NO:231 , and further comprising the substitution F>A at the position corresponding to position 134 of SEQ ID NO:231 (that is, the amino acid sequence shown in SEQ ID NO:233).
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:232, or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:232.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:233, or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:233.
  • the antigen-binding molecule comprises more than one an amino acid sequence which is capable of binding to BCMA and/or TACI. In some embodiments the antigen-binding molecule comprises one of 1 , 2, 3, 4, 5, 6, 7 or 8 non-overlapping amino acid sequences, which are each independently capable of binding to BCMA and/or TACI. In some embodiments antigen-binding molecule comprises a polypeptide comprising plural amino acid sequences capable of binding to BCMA and/or TACI provided in tandem.
  • the antigen-binding molecule comprises a polypeptide comprising non-overlapping sequences according to (i) and (ii):
  • amino acid sequence according to SEQ ID NO:232, or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:232, and
  • amino acid sequence according to SEQ ID NO:231 , or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:231.
  • the antigen-binding molecule comprises a polypeptide comprising non-overlapping sequences according to (i) to (iii):
  • amino acid sequence (i) an amino acid sequence according to SEQ ID NO:231 , or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:231.
  • amino acid sequence according to SEQ ID NO:231 , or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:231.
  • the antigen-binding molecule comprises a polypeptide comprising non-overlapping sequences according to (i) to (iii):
  • amino acid sequence according to SEQ ID NO:232, or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:232.
  • amino acid sequence according to SEQ ID NO:231 , or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:231.
  • amino acid sequence (iii) an amino acid sequence according to SEQ ID NO:231 , or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:231.
  • the antigen-binding molecule comprises a polypeptide comprising non-overlapping sequences according to (i) to (iii):
  • amino acid sequence according to SEQ ID NO:232, or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:232.
  • amino acid sequence according to SEQ ID NO:232, or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:232.
  • amino acid sequence (iii) an amino acid sequence according to SEQ ID NO:231 , or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:231.
  • linker sequences e.g. linker sequences as described herein.
  • a linker sequence comprises or consists of glycine and serine residues.
  • a linker sequence comprises 1 to 20 amino acids.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:234, or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:234.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:235, or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:235.
  • the antigen-binding molecule comprises a polypeptide having an amino acid sequence according to SEQ ID NO:236, or an amino acid sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:236.
  • the antigen-binding molecule is not a naturally-occurring peptide/polypeptide.
  • the antigen-binding molecule does not consist of the amino acid sequence shown in one of SEQ ID NOs: 153 to 173.
  • the antigen-binding molecule is capable of binding to CD47 and is capable of binding to BCMA. In aspects of the present invention, the antigen-binding molecule is capable of binding to CD47 and is capable of binding to TACI. In aspects of the present invention, the antigenbinding molecule is capable of binding to CD47 and is capable of binding to BCMA and TACI.
  • the antigen-binding molecule comprises the CDRs of an antigen-binding molecule which is capable of binding to CD47 and the CDRs of an antigen-binding molecule which is capable of binding to BCMA and/or TACI. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule which is capable of binding to CD47 and the FRs of an antigen-binding molecule which is capable of binding to BCMA and/or TACI.
  • the antigen-binding molecule comprises the CDRs and the FRs of an antigen-binding molecule which is capable of binding to CD47 and the CDRs and the FRs of an antigen-binding molecule which is capable of binding to BCMA and/or TACI. That is, in some embodiments the antigen-binding molecule comprises the VH region and the VL region of an antigen-binding molecule which is capable of binding to CD47 and the VH region and the VL region of an antigen-binding molecule which is capable of binding to BCMA and/or TACI.
  • the antigen-binding molecule comprises a VH region and a VL region which is, or which is derived from, the VH/VL region of a CD47-binding antibody clone described herein (i.e. anti- CD47 antibody clones 1-1-A1_BM, 1-1-A1 , 5-48-A6, 5-48-D2, 1 1A1 H1 , 1 1A1 H2, 1 1A1 H3, 1 1A1 H4, 1 1A1 H5, 1 1A1 H6, 1 1A1 H7, 1 1A1 H8, 1 1A1 H9, 1 1A1 H10 or 1 1 A1 H1 1 ) and a VH region and a VL region which is, or which is derived from, the VH/VL region of a BCMA and/or TACI-binding antibody clone described herein (i.e. anti-BCMA antibody clone J6M0).
  • a CD47-binding antibody clone described herein i
  • a CD47-binding region comprising a VH region according to any one of (1 ) to (35) above, and a VL region according to any one of (36) to (70) above;
  • BCMA-binding region comprising a VH region according to any one of (71 ) to (74) above, and a VL region according to any one of (75) to (78) above.
  • a CD47-binding region comprising a VH region according to any one of (1 ) to (35) above, and a VL region according to any one of (36) to (70) above;
  • BCMA- and/or TACI-binding region comprising or consisting of (i) an amino acid sequence having at least 70% sequence identity to the amino acid sequence of one of SEQ ID NOs:231 , 232, 233, 234, 235 or 236.
  • substitutions may conservative substitutions, for example according to the following Table.
  • amino acids in the same block in the middle column are substituted.
  • amino acids in the same line in the rightmost column are substituted:
  • substitution(s) may be functionally conservative. That is, in some embodiments the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. target binding) of the antigen-binding molecule comprising the substitution as compared to the equivalent unsubstituted molecule.
  • the VH and VL region of an antigen-binding region of an antibody together constitute the Fv region.
  • the antigen-binding molecule according to the present invention comprises, or consists of, an Fv region which binds to CD47.
  • the antigen-binding molecule comprises an Fv region which binds to BCMA and/or TACI.
  • the antigen-binding molecule according to the present invention comprises, or consists of, an Fv region which binds to CD47 and an Fv region which binds to BCMA and/or TACI.
  • the VH and VL regions of the Fv are provided as single polypeptide joined by a linker region, i.e. a single chain Fv (scFv).
  • the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin heavy chain constant sequence.
  • the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE or IgM.
  • the immunoglobulin heavy chain constant sequence is human immunoglobulin G 1 constant (IGHG1 ; UniProt: P01857-1 , v1 ; SEQ ID NO:146). Positions 1 to 98 of SEQ ID NO:146 form the CH1 region (SEQ ID NO:147). Positions 99 to 110 of SEQ ID NO:146 form a hinge region between CH1 and CH2 regions (SEQ ID NO:148). Positions 111 to 223 of SEQ ID NO:146 form the CH2 region (SEQ ID NO:149). Positions 224 to 330 of SEQ ID NO:146 form the CH3 region (SEQ ID NO:150).
  • the exemplified antigen-binding molecules were prepared using pFUSE-CHIg-hG1 , which comprises the substitutions D356E, L358M (positions numbered according to EU numbering) in the CH3 region relative to SEQ ID NO:146.
  • the amino acid sequence of the CH3 region encoded by pFUSE-CHIg-hG1 is shown in SEQ ID NO: 151. It will be appreciated that CH3 regions may be provided with further substitutions in accordance with modification to an Fc region of the antigen-binding molecule as described herein.
  • a CH1 region comprises or consists of the sequence of SEQ ID NO:147, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%,
  • a CH1-CH2 hinge region comprises or consists of the sequence of SEQ ID NO:148, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:148.
  • a CH2 region comprises or consists of the sequence of SEQ ID NO:149, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:149.
  • a CH3 region comprises or consists of the sequence of SEQ ID NO: 150 or 151 , or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 150 or 151.
  • the antigen-binding molecule comprises a sequence comprising a hinge region comprising the substitution C220S (numbered according to EU numbering).
  • the antigen-binding molecule comprises the amino acid sequence of SEQ ID NO:268, or having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:268.
  • the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin light chain constant sequence.
  • the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; CK; UniProt: P01834-1 , v2; SEQ ID NO: 152).
  • the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CA), e.g. IGLC1 , IGLC2, IGLC3, IGLC6 or IGLC7.
  • a CL region comprises or consists of the sequence of SEQ ID NO: 152, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%,
  • the VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1 ) region of an antigen-binding region of an antibody together constitute the Fab region.
  • the antigen-binding molecule comprises a Fab region comprising a VH, a CH1 , a VL and a CL (e.g. CK or CA).
  • the Fab region comprises a polypeptide comprising a VH and a CH1 (e.g. a VH-CH1 fusion polypeptide), and a polypeptide comprising a VL and a CL (e.g. a VL-CL fusion polypeptide).
  • the Fab region comprises a polypeptide comprising a VH and a CL (e.g. a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH (e.g. a VL-CH1 fusion polypeptide); that is, in some embodiments the Fab region is a CrossFab region.
  • the VH, CH1 , VL and CL regions of the Fab or CrossFab are provided as single polypeptide joined by linker regions, i.e. as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).
  • the antigen-binding molecule of the present invention comprises, or consists of, a Fab region which binds to CD47. In some embodiments, the antigen-binding molecule comprises a Fab region which binds to BCMA and/or TACI. In some embodiments, the antigen-binding molecule of the present invention comprises, or consists of, a Fab region which binds to CD47 and a Fab region which binds to BCMA and/or TACI. In some embodiments, the antigen-binding molecule described herein comprises, or consists of, a whole antibody which binds to CD47.
  • the antigen-binding molecule comprises a whole antibody which binds to a BCMA and/or TACI.
  • the antigen-binding molecule described herein comprises, or consists of, a whole antibody which binds to CD47 and a whole antibody which binds to a BCMA and/or TACI.
  • “whole antibody” refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.
  • Immunoglobulins of type G are -150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chain comprise a VL followed by a CL.
  • immunoglobulins may be classed as IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM.
  • the light chain may be kappa (K) or lambda (l).
  • the antigen-binding molecule described herein comprises, or consists of, an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM which binds to CD47.
  • the antigen-binding molecule described herein comprises an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM which binds to BCMA and/or TACI.
  • the antigen-binding molecule comprises a ligand for BCMA and/or TACI, or a BCMA and/or TACI -binding fragment of a ligand for BCMA and/or TACI.
  • the ligand for BCMA and/or TACI is BAFF.
  • the ligand for BCMA and/or TACI is APRIL.
  • Human A proliferation-inducing ligand (APRIL; also known as TNFSF13) is the protein identified by UniProt 075888.
  • Alternative splicing of mRNA encoded by the human TNFSF13 gene yields 6 isoforms: isoform alpha (UniProt: 075888-1 , v1 ; SEQ ID NO: 153); isoform beta (UniProt: 075888-2; SEQ ID NO:154), in which the amino acid sequence corresponding to positions 113 to 129 of SEQ ID NO:153 are substituted with“N”; isoform gamma (UniProt: 075888-3; SEQ ID NO: 155), which lacks the amino acid sequence corresponding to positions 247 to 249 of SEQ ID NO: 153; isoform 4 (UniProt: 075888-4; SEQ ID NO: 156), which lacks the amino acid sequence corresponding to positions 86 to 113 of SEQ ID NO: 153; isoform TWE-PRIL (
  • APRIL is cleaved in the Golgi between positions 104-105 by a furin convertase to yield the mature, secreted form of the protein (Lopez-Fraga et al. (2001 ) EMBO Rep. 2: 945-951 ).
  • the mature forms of the alpha, beta, gamma and TWE-PRIL isoforms are shown in SEQ ID NOs:159 to 162.
  • APRIL assembles as a homotrimer which establishes contacts with monomeric BCMA and TACI receptors, resulting in receptor trimerisation and activation of the NF-kB pathway.
  • APRIL refers to APRIL from any species and includes APRIL isoforms, fragments, variants or homologues from any species.
  • the APRIL is APRIL from a mammal (e.g. a primate (rhesus, cynomolgous, or human) and/or a rodent (e.g. rat or murine) APRIL).
  • Isoforms, fragments, variants or homologues of APRIL may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature APRIL isoform from a given species, e.g. human.
  • the APRIL, or the fragment of APRIL comprises or consists of an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,
  • Human B cell activating factor (BAFF; also known as TNFSF13B and BLys) is the protein identified by UniProt Q9Y275.
  • Alternative splicing of mRNA encoded by the human TNFSF13B gene yields 3 isoforms: isoform 1 (UniProt: Q9Y275-1 , v1 ; SEQ ID NO:163); isoform 2 (UniProt: Q9Y275-2; SEQ ID NO:164), which lacks the amino acid sequence corresponding to positions 142 to 160 of SEQ ID NO:163; and isoform 3 (UniProt: Q9Y275-3; SEQ ID NO:165), in which positions 162 to 164 of SEQ ID NO:163 are replaced with“FIY”, and which lacks the amino acid sequence corresponding to positions 165 to 285 of SEQ ID NO:163.
  • BAFF comprises an N-terminal cytoplasmic domain (SEQ ID NO:166), a transmembrane domain (SEQ ID NO:167) and an extracellular domain (SEQ ID NOs: 168 to 170), which is cleaved between the positions corresponding to positions 133 and 135 to yield a soluble form (SEQ ID NOs: 171 to 173).
  • BAFF refers to BAFF from any species and includes BAFF isoforms, fragments, variants or homologues from any species.
  • the BAFF is BAFF from a mammal (e.g. a primate (rhesus, cynomolgous, or human) and/or a rodent (e.g. rat or murine) BAFF).
  • Isoforms, fragments, variants or homologues of BAFF may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature BAFF isoform from a given species, e.g. human.
  • the BAFF comprises or consists of an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,
  • BAFF and APRIL display binding to BCMA and TACI.
  • the ligand for BCMA, or BCMA-binding fragment of a ligand for BCMA is capable of binding to transmembrane activator and CAML interactor (TACI).
  • the ligand for TACI, or TACI-binding fragment of a ligand for TACI is capable of binding to BCMA.
  • Aspects of the present invention relate to multispecific antigen-binding molecules. By“multispecific” it is meant that the antigen-binding molecule displays specific binding to more than one target.
  • the antigen-binding molecule is a bispecific antigen-binding molecule.
  • the antigen-binding molecule comprises at least two different antigen-binding domains (i.e. at least two antigen-binding domains, e.g. comprising non-identical VHs and VLs).
  • the antigen-binding molecule binds to CD47 and BCMA and/or TACI, and so is at least bispecific.
  • the term“bispecific” means that the antigen-binding molecule is able to bind specifically to at least two distinct antigenic determinants.
  • the antigen-binding molecule capable of binding to BCMA is also capable of binding to TACI. In some embodiments, the antigen-binding molecule capable of binding to TACI is also capable of binding to BCMA.
  • the antigen-binding molecule comprises an antigen-binding domain capable of binding to BCMA. In some embodiments the antigen-binding domain capable of binding to BCMA is also capable of binding to TACI. In some embodiments the antigen-binding molecule comprises an antigenbinding domain capable of binding to TACI. In some embodiments the antigen-binding domain capable of binding to TACI is also capable of binding to BCMA.
  • the antigen-binding molecule is cross-reactive for BCMA and TACI. In some embodiments the antigen-binding molecule comprises an antigen-binding domain which is cross-reactive for BCMA and TACI.
  • a“cross-reactive” antigen-binding molecule/domain/polypeptide is capable of binding to the target antigens for which the antigen-binding molecule/domain is cross-reactive.
  • an antigen-binding molecule/domain/polypeptide which is cross-reactive for BCMA and TACI is capable of binding to BCMA, and is also capable of binding to TACI.
  • molecules/domains/polypeptides may display specific binding to each of the target antigens.
  • the BCMA-binding region of the antigen-binding molecule/domain is capable of binding to TACI. In some embodiments the TACI-binding region of the antigen-binding molecule/domain is capable of binding to BCMA.
  • an antigen-binding molecule according to the present invention may comprise antigen-binding molecules capable of binding to the targets for which the antigen-binding molecule is specific.
  • an antigen-binding molecule which is capable of binding to CD47 and BCMA and/or TACI may comprise: (i) an antigen-binding molecule which is capable of binding to CD47, and (ii) an antigen-binding molecule which is capable of binding to BCMA and/or TACI.
  • Figure 2 of the present application provides a graphic representation of an antigen-binding molecule which is capable of binding to CD47 and BCMA, comprising (i) an antigen-binding molecule which is capable of binding to CD47, specifically a CD47-binding Fab, and (ii) an antigen-binding molecule which is capable of binding to BCMA, specifically a BCMA-binding scFv.
  • an antigen-binding molecule according to the present invention may comprise antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to the targets for which the antigen-binding molecule is specific.
  • Figure 2 of the present application lower right provides a graphic representation of an antigen-binding molecule which is capable of binding to CD47 and BCMA, comprising (i) an antigenbinding polypeptide complex capable of binding to CD47, comprising a light chain polypeptide
  • a component antigen-binding molecule of a larger antigen-binding molecule may be referred to e.g. as an“antigen-binding domain” or “antigen-binding region” of the larger antigen-binding molecule.
  • the antigen-binding molecule comprises an antigen-binding molecule capable of binding to CD47, and an antigen-binding molecule capable of binding to an antigen other than CD47.
  • the antigen other than CD47 is an immune cell surface molecule.
  • the antigen other than CD47 is a cancer cell antigen.
  • the antigen other than CD47 is a receptor molecule, e.g. a cell surface receptor.
  • the antigen other than CD47 is a cell signalling molecule, e.g. a cytokine, chemokine, interferon, interleukin or lymphokine.
  • the antigen other than CD47 is a growth factor or a hormone.
  • a cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell.
  • a cancer cell antigen may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof.
  • a cancer cell antigen’s expression may be associated with a cancer.
  • a cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell.
  • a cancer cell antigen may be capable of eliciting an immune response.
  • the antigen is expressed at the cell surface of the cancer cell (i.e. the cancer cell antigen is a cancer cell surface antigen).
  • the part of the antigen which is bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (i.e. is extracellular).
  • the cancer cell antigen may be a cancer-associated antigen.
  • the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer.
  • the cancer- associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer.
  • the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and/or protein level) by cells of a cancer, e.g.
  • the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type).
  • the cancer- associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene.
  • the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.
  • An immune cell surface molecule may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed at or on the cell surface of an immune cell.
  • the part of the immune cell surface molecule which is bound by the antigen-binding molecule of the present invention is on the external surface of the immune cell (i.e. is extracellular).
  • the immune cell surface molecule may be expressed at the cell surface of any immune cell.
  • the immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte.
  • the lymphocyte may be e.g. a T cell, B cell, natural killer (NK) cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof (e.g. a thymocyte or pre-B cell).
  • the antigen other than CD47 is an antigen expressed by cells of a hematologic malignancy, a myeloid hematologic malignancy, a lymphoblastic hematologic malignancy,
  • MDS myelodysplastic syndrome
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • NHL non-Hodgkin’s lymphoma
  • multiple myeloma bladder cancer or brain cancer.
  • the antigen other than CD47 is an antigen expressed by cells of AML, e.g. as described in Hoseini and Cheung Blood Cancer J. (2017) 7(2):e522, which is hereby incorporated by reference in its entirety.
  • the antigen other than CD47 is selected from: BCMA, TACI, CD33, CD123, Wilms' tumor protein (WT1 ), CD13, CD15, CD30, CD45, C-type lectin-like molecule 1 (CLL1 ), Fms-like tyrosine kinase 3 (FLT-3), VEGF and angiopoietin-2 (Ang-2).
  • the antigen other than CD47 is BCMA.
  • the antigen other than CD47 is TACI.
  • Multispecific antigen-binding molecules described herein display at least monovalent binding with respect to CD47, and also display at least monovalent binding with respect to the antigen other than CD47.
  • a multispecific antigen-binding molecule according to the present invention displays at least monovalent binding with respect to CD47, and also displays at least monovalent binding with respect to BCMA and/or TACI.
  • the antigen-binding molecule comprises an antigen-binding region (e.g. a polypeptide, Fv, Fab or antibody) capable of binding to CD47, and an antigen-binding region (e.g. a polypeptide, Fv, Fab or antibody) capable of binding to BCMA and/or TACI.
  • the antigen-binding molecule comprises the VH and VL of an antibody capable of binding to CD47, and the VH and VL of an antibody capable of binding to BCMA and/or TACI.
  • Binding valency refers to the number of binding sites in an antigen-binding molecule for a given antigenic determinant.
  • the bispecific anti-CD47/anti-BCMA antibody described herein (e.g. [6] of Example 2.2) is monovalent with respect to binding to CD47, and monovalent with respect to binding to BCMA.
  • the antigen-binding molecule comprises one binding site for CD47, and one binding site for BCMA and/or TACI.
  • Multispecific antigen-binding molecules according to the invention may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann MAbs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety.
  • Suitable formats include those shown in Figure 2 of Brinkmann and Kontermann MAbs (2017) 9(2): 182-212: antibody conjugates, e.g. lgG2, F(ab’)2 or CovX-Body; IgG or IgG-like molecules, e.g. IgG, chimeric IgG, kl-body common HC; CH1/CL fusion proteins, e.g.
  • scFv2-CH1/CL, VHH2-CH1/CL;‘variable domain only’ bispecific antigen-binding molecules e.g. tandem scFv (taFV), triplebodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triple heads, tandem dAb/VHH, tertravalent dAb.VHH;
  • Non-lg fusion proteins e.g.
  • scFv2-albumin scDb- albumin, taFv-albumin, taFv-toxin, miniantibody, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g.
  • Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-lg fusion proteins, e.g. DNL-Fab3, DNL-Fab2-scFv, DNL- Fab2-lgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g.
  • IgGs appended and Fc-modified IgGs, e.g. lgG(kih)-Fv, IgG HA-TF-Fv, lgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-lg, DVI-lg (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, e.g.
  • modified IgGs e.g. DAF (two-in one-lgG), DutaMab, Mab 2 ; and non-lg fusions, e.g. DNL-Fab 4 -lgG.
  • bispecific antigen-binding molecules The skilled person is able to design and prepare bispecific antigen-binding molecules.
  • Methods for producing bispecific antigen-binding molecules include chemically crosslinking of antigen-binding molecules or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1—2.13.16, which is hereby incorporated by reference in its entirety.
  • A/-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink e.g. Fab fragments via hinge region SH- groups, to create disulfide-linked bispecific F(ab)2 heterodimers.
  • bispecific antigen-binding molecules include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14: IV:2.13:2.13.1—2.13.16.
  • Bispecific antigen-binding molecules according to the present invention can also be produced recombinantly, by expression from e.g. a nucleic acid construct encoding polypeptides for the antigenbinding molecules, for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antigen-binding molecules:
  • a DNA construct encoding the light and heavy chain variable domains for the two antigenbinding fragments i.e. the light and heavy chain variable domains for the antigen-binding fragment capable of binding CD47, BCMA and/or TACI, and the light and heavy chain variable domains for the antigen-binding fragment capable of binding to another target protein
  • sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning techniques.
  • Recombinant bispecific antibody can thereafter be produced by expression (e.g. in vitro) of the construct in a suitable host cell (e.g. a mammalian host cell), and expressed recombinant bispecific antibody can then optionally be purified.
  • the antigen-binding molecules of the present invention comprise an Fc region.
  • An Fc region is composed of CH2 and CH3 regions from one polypeptide, and CH2 and CH3 regions from another polypeptide. The CH2 and CH3 regions from the two polypeptides together form the Fc region.
  • the antigen-binding molecule of the present invention comprises an Fc region comprising modification in one or more of the CH2 and CH3 regions promoting association of the Fc region.
  • Recombinant co-expression of constituent polypeptides of an antigen-binding molecule and subsequent association leads to several possible combinations.
  • modification(s) promoting association of the desired combination of heavy chain polypeptides.
  • Modifications may promote e.g. hydrophobic and/or electrostatic interaction between CH2 and/or CH3 regions of different polypeptide chains. Suitable modifications are described e.g. in Ha et al., Front. Immnol (2016) 7:394, which is hereby incorporated by reference in its entirety.
  • the antigen antigen-binding molecule of the present invention comprises an Fc region comprising paired substitutions in the CH3 regions of the Fc region according to one of the following formats, as shown in Table 1 of Ha et al., Front. Immnol (2016) 7:394: KiH, KiH s-s , HA-TF, ZW1 , 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, SEED or A107.
  • the Fc region comprises the“knob-into-hole” or“KiH” modification, e.g. as described e.g. in US 7,695,936 and Carter, J Immunol Meth 248, 7-15 (2001 ).
  • one of the CH3 regions of the Fc region comprises a“knob” modification
  • the other CH3 region comprises a“hole” modification.
  • The“knob” and“hole” modifications are positioned within the respective CH3 regions so that the“knob” can be positioned in the“hole” in order to promote heterodimerisation (and inhibit homodimerisation) of the polypeptides and/or stabilise heterodimers.
  • Knobs are constructed by substituting amino acids having small chains with those having larger side chains (e.g. tyrosine or tryptophan). Holes are created by substituting amino acids having large side chains with those having smaller side chains (e.g. alanine or threonine).
  • one of the CH3 regions of the Fc region of the antigen-binding molecule of the present invention comprises the substitution (numbering of positions/substitutions in the Fc, CH2 and CH3 regions herein is according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 ) T366W, and the other CH3 region of the Fc region comprises the substitution Y407V.
  • one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W, and the other CH3 region of the Fc region comprises the substitutions T366S and L368A. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W, and the other CH3 region of the Fc region comprises the substitutions Y407V, T366S and L368A.
  • the Fc region comprises the“DD-KK” modification as described e.g. in WO 2014/131694 A1.
  • one of the CH3 regions comprises the substitutions K392D and K409D, and the other CH3 region of the Fc region comprises the substitutions E356K and D399K. The modifications promote electrostatic interaction between the CH3 regions.
  • the antigen-binding molecule of the present invention comprises an Fc region modified as described in Labrijn et al., Proc Natl Acad Sci U S A. (2013) 1 10(13):5145-50, referred to as ‘Duobody’ format.
  • one of the CH3 regions comprises the substitution K409R
  • the other CH3 region of the Fc region comprises the substitution F405L.
  • the antigen-binding molecule of the present invention comprises an Fc region comprising the“EEE-RRR” modification as described in Strop et al., J Mol Biol. (2012) 420(3):204-19.
  • one of the CH3 regions comprises the substitutions D221 E, P228E and L368E
  • the other CH3 region of the Fc region comprises the substitutions D221 R, P228R and K409R.
  • the antigen-binding molecule comprises an Fc region comprising the“EW-RVT” modification described in Choi et al., Mol Cancer Ther (2013) 12(12):2748-59.
  • one of the CH3 regions comprises the substitutions K360E and K409W
  • the other CH3 region of the Fc region comprises the substitutions Q347R, D399V and F405T.
  • one of the CH3 regions comprises the substitution S354C
  • the other CH3 region of the Fc region comprises the substitution Y349C.
  • Introduction of these cysteine residues results in formation of a disulphide bridge between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter (2001 ), J Immunol Methods 248, 7-15).
  • the Fc region comprises the“KiHs-s” modification.
  • one of the CH3 regions comprises the substitutions T366W and S354C, and the other CH3 region of the Fc region comprises the substitutions T366S, L368A, Y407V and Y349C.
  • the antigen-binding molecule of the present invention comprises an Fc region comprising the“SEED” modification as described in Davis et al., Protein Eng Des Sel (2010) 23(4): 195- 202, in which b-strand segments of human lgG1 CH3 and IgA CH3 are exchanged.
  • one of the CH3 regions comprises the substitutions S364H and F405A
  • the other CH3 region of the Fc region comprises the substitutions Y349T and T394F (see e.g. Moore et al., MAbs (201 1 ) 3(6):546-57).
  • one of the CH3 regions comprises the substitutions T350V, L351 Y, F405A and Y407V
  • the other CH3 region of the Fc region comprises the substitutions T350V, T366L, K392L and T394W (see e.g. Von Kreudenstein et al., MAbs (2013) 5(5):646-54).
  • one of the CH3 regions comprises the substitutions K360D, D399M and Y407A
  • the other CH3 region of the Fc region comprises the substitutions E345R, Q347R, T366V and K409V (see e.g. Leaver-Fay et al., Structure (2016) 24(4):641-51 ).
  • one of the CH3 regions comprises the substitutions K370E and K409W
  • the other CH3 region of the Fc region comprises the substitutions E357N, D399V and F405T (see e.g. Choi et al., PLoS One (2015) 10(12):e0145349).
  • the present invention also provides polypeptide constituents of antigen-binding molecules.
  • the polypeptides may be provided in isolated or substantially purified form.
  • the antigen-binding molecule of the present invention may be, or may comprise, a complex of polypeptides.
  • a polypeptide comprises more than one domain or region
  • the plural domains/regions are preferably present in the same polypeptide chain. That is, the polypeptide comprising more than one domain or region is a fusion polypeptide comprising the domains/regions.
  • a polypeptide according to the present invention comprises, or consists of, a VH as described herein. In some embodiments a polypeptide according to the present invention comprises, or consists of, a VL as described herein.
  • the polypeptide additionally comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide additionally comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises a CH1 , CH2 region and/or a CH3 region of an immunoglobulin (Ig).
  • CH antibody heavy chain constant regions
  • CL antibody light chain constant regions
  • the polypeptide comprises a CH1 , CH2 region and/or a CH3 region of an immunoglobulin (Ig).
  • the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments the polypeptide comprises a CH1 region as described herein. In some embodiments the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments the polypeptide comprises a CH2 region as described herein. In some embodiments the polypeptide comprises a CH3 region as described herein.
  • the polypeptide comprises a CH3 region comprising any one of the following amino acid substitutions/combinations of amino acid substitutions (shown e.g. in Table 1 of Ha et al., Front. Immnol (2016) 7:394, incorporated by reference hereinabove): T366W; T366S, L368A and Y407V; T366W and S354C; T366S, L368A, Y407V and Y349C; S364H and F405A; Y349T and T394F; T350V, L351Y, F405A and Y407V; T350V, T366L, K392L and T394W; K360D, D399M and Y407A; E345R, Q347R, T366V and K409V; K409D and K392D; D399K and E356K; K360E and K409W; Q347
  • the CH2 and/or CH3 regions of the polypeptide comprise one or more amino acid substitutions for promoting association of the polypeptide with another polypeptide comprising a CH2 and/or CH3 region.
  • polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments the polypeptide comprises a CL region as described herein.
  • the polypeptide according to the present invention comprises a structure from N- to C-terminus according to one of the following: (i) VH
  • antigen-binding molecules composed of the polypeptides of the present invention.
  • the antigen-binding molecule of the present invention comprises one of the following combinations of polypeptides:
  • the antigen-binding molecule comprises more than one of a polypeptide of the combinations shown in (A) to (I) above.
  • the antigen-binding molecule comprises two polypeptides comprising the structure VH- CH 1-CH2-CH3, and two polypeptides comprising the structure VL-CL.
  • the antigen-binding molecule of the present invention comprises one of the following combinations of polypeptides:
  • V VH(anti-CD47)-CH 1 -CH2-CH3 + VL(anti-CD47)-CL + VH(anti-BCMA)-CH1-CH2-CH3 + VL(anti-BCMA)-CL
  • VH(anti-CD47) refers to the VH of an antigen-binding molecule capable of binding to CD47 as described herein, e.g. as defined in one of (1 ) to (35);“VL(anti-CD47)” refers to the VL of an antigenbinding molecule capable of binding to CD47 as described herein, e.g. as defined in one of (36) to (70); “VH(anti-BCMA)” refers to the VH of an antigen-binding molecule capable of binding to BCMA as described herein, e.g.
  • VL(anti-BCMA) refers to the VL of an antigen-binding molecule capable of binding to BCMA as described herein, e.g. as defined in one of (75) to (78).
  • the antigen-binding molecule of the present invention comprises one of the following combinations of polypeptides:
  • VH(anti-CD47) refers to the VH of an antigen-binding molecule capable of binding to CD47 as described herein, e.g. as defined in one of (1 ) to (35);“VL(anti-CD47)” refers to the VL of an antigenbinding molecule capable of binding to CD47 as described herein, e.g. as defined in one of (36) to (70); and“BCMA/TACI-binding polypeptide” refers to a polypeptide comprising, or consisting of, an amino acid sequence capable of binding to BCMA and/or TACI, e.g. as described herein.
  • the polypeptide comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs:23, 31 , 39, 44, 49, 57, 65, 73, 229, 230, 178, 179, 180, 181 , 182, 183, 184, 185, 186 or 187.
  • the polypeptide comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs:231 , 232, 233, 234, 235 or 236.
  • the polypeptide comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs: 130, 131 , 132, 133, 134, 135, 136, 137, 141 , 142, 176, 177, 210, 21 1 , 212, 213, 214, 215, 216, 217, 218, 219 or 259.
  • the polypeptide comprises or consists of an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs:243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257 or 258.
  • the antigen-binding molecules and polypeptides of the present invention comprise a hinge region.
  • a hinge region is provided between a CH 1 region and a CH2 region.
  • a hinge region is provided between a CL region and a CH2 region.
  • the hinge region comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 148.
  • the antigen-binding molecules and polypeptides of the present invention comprise one or more linker sequences between amino acid sequences.
  • a linker sequence may be provided at one or both ends of one or more of a VH, VL, CH1-CH2 hinge region, CH2 region and a CH3 region of the antigen-binding molecule/polypeptide.
  • Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety.
  • a linker sequence may be a flexible linker sequence.
  • Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence.
  • Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.
  • the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids.
  • the antigen-binding molecules and polypeptides of the present invention may additionally comprise further amino acids or sequences of amino acids.
  • the antigen-binding molecules and polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule/polypeptide.
  • the antigen-binding molecule/polypeptide may comprise a sequence encoding a His, (e.g. 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus of the antigen-binding
  • the antigen-binding molecule/polypeptide comprises a detectable moiety, e.g. a fluorescent, lunminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.
  • a detectable moiety e.g. a fluorescent, lunminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.
  • the antigen-binding molecules and polypeptides of the present invention may additionally comprise a signal peptide (also known as a leader sequence or signal sequence).
  • Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.
  • the signal peptide may be present at the N-terminus of the antigen-binding molecule/polypeptide, and may be present in the newly synthesised antigen-binding molecule/polypeptide.
  • the signal peptide provides for efficient trafficking and secretion of the antigen-binding molecule/polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature antigen-binding
  • Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 201 1 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176).
  • SignalP Protein et al., 201 1 Nature Methods 8: 785-786
  • Signal-BLAST Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176.
  • the signal peptide of the antigen-binding molecule/polypeptide of the present invention comprises, or consists of, an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of one of SEQ ID NOs:81 to 86.
  • the antigen-binding molecules of the present invention additionally comprise a detectable moiety.
  • the antigen-binding molecule comprises a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label.
  • a detectable moiety e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label.
  • the antigen-binding molecule may be covalently or non- covalently labelled with the detectable moiety.
  • Fluorescent labels include e.g.
  • GFP green fluorescent protein
  • Radiolabels include radioisotopes such as Iodine 123 , Iodine 125 , Iodine 126 , Iodine 131 , Iodine 133 , Bromine 77 , Technetium” 171 , Indium 111 , Indium 113111 , Gallium 67 , Gallium 68 , Ruthenium 95 , Ruthenium 97 , Ruthenium 103 , Ruthenium 105 , Mercury 207 , Mercury 203 , Rhenium” 111 , Rhenium 101 , Rhenium 105 , Scandium 47 , Tellurium 121111 , Tellurium 122111 , Tellurium 125111 , Thulium 165 , Thuliuml 167 , Thulium 168 , Copper 67 , Fluorine 18 , Yttrium", Palladium 100 , Bismuth 217 and Antimony 211 .
  • Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels.
  • Immuno- detectable labels include haptens, peptides/polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin.
  • Nucleic acid labels include aptamers.
  • Enzymatic labels include e.g. peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase and luciferase.
  • the antigen-binding molecules of the present invention are conjugated to a chemical moiety.
  • the chemical moiety may be a moiety for providing a therapeutic effect.
  • Antibody-drug conjugates are reviewed e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3): 14.
  • the chemical moiety may be a drug moiety (e.g. a cytotoxic agent).
  • the drug moiety may be a chemotherapeutic agent.
  • the drug moiety is selected from calicheamicin, DM1 , DM4, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5 and PBD.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 130; and
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 131.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 132;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 133.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 134; and (ii) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 135.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 136;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 137.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 140;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 139;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 141 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 142.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 176;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 177;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 139.
  • the antigen-binding molecule comprises, or consists of: (i) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:210; and
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:211.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:212; and
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:213.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:213;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:211.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:214;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:211.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:215;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:211.
  • the antigen-binding molecule comprises, or consists of: (i) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:214; and
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:216.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:215;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:216.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:214;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:217.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:215;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:217.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:215;
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:218.
  • the antigen-binding molecule comprises, or consists of: (i) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:215; and
  • polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:219.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:259;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:243.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:259;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:244.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:259;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:245.
  • the antigen-binding molecule comprises, or consists of: (i) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:259;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:246.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 176;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:247.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 176;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:248.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 176;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 176;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:250.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:259;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:251.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:259;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:252.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:259;
  • a polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ; and (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:253.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:259;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:254.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 176;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:255.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 176;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:256.
  • the antigen-binding molecule comprises, or consists of:
  • a polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 176;
  • a polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:257.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 176;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:131 ;
  • polypeptide comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:258.
  • the antigen-binding molecules described herein may be characterised by reference to certain functional properties.
  • the antigen-binding molecule described herein may possess one or more of the following properties:
  • BCMA inhibits interaction between BCMA and a ligand for BCMA (e.g. APRIL/BAFF);
  • a ligand for BCMA e.g. APRIL/BAFF
  • TACI inhibits interaction between TACI and a ligand for TACI (e.g. APRIL/BAFF);
  • a ligand for TACI e.g. APRIL/BAFF
  • phagocytic cells e.g. macrophages
  • the antigen-binding molecules and antigen-binding domains described herein preferably display specific binding to the relevant target antigen(s) (e.g. CD47, BCMA and/or TACI).
  • target antigen e.g. CD47, BCMA and/or TACI.
  • target antigen e.g. CD47, BCMA and/or TACI.
  • specific binding refers to binding which is selective for the antigen, and which can be discriminated from nonspecific binding to non-target antigen.
  • An antigen-binding molecule/domain that specifically binds to a target molecule preferably binds the target with greater affinity, and/or with greater duration than it binds to other, non-target molecules.
  • the ability of a given polypeptide to bind specifically to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:41 1-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015)
  • the extent of binding of the antigen-binding molecule to an non-target molecule is less than about 10% of the binding of the antibody to the target molecule as measured, e.g. by ELISA, SPR, Bio-Layer Interferometry or by RIA.
  • binding specificity may be reflected in terms of binding affinity where the antigen-binding molecule binds with a dissociation constant (KD) that is at least 0.1 order of magnitude (i.e. 0.1 x 10 n , where n is an integer representing the order of magnitude) greater than the KD of the antigen-binding molecule towards a non-target molecule.
  • KD dissociation constant
  • This may optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, or 2.0.
  • the antigen-binding molecule described herein binds to CD47 with a KD of 10 mM or less, preferably one of ⁇ 5 pM, ⁇ 2 pM, ⁇ 1 pM, ⁇ 500 nM, ⁇ 100 nM, ⁇ 75 nM, ⁇ 50 nM, ⁇ 40 nM, ⁇ 30 nM, ⁇ 20 nM, ⁇ 15 nM, ⁇ 12.5 nM, ⁇ 10 nM, ⁇ 9 nM, ⁇ 8 nM, ⁇ 7 nM, ⁇ 6 nM, ⁇ 5 nM, ⁇ 4 nM ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM or ⁇ 500 pM.
  • the antigen-binding molecule described herein binds to BCMA with a KD of 10 mM or less, preferably one of ⁇ 5 mM, ⁇ 2 mM, ⁇ 1 pM, ⁇ 500 nM, ⁇ 100 nM, ⁇ 75 nM, ⁇ 50 nM, ⁇ 40 nM, ⁇ 30 nM, ⁇ 20 nM, ⁇ 15 nM, ⁇ 12.5 nM, ⁇ 10 nM, ⁇ 9 nM, ⁇ 8 nM, ⁇ 7 nM, ⁇ 6 nM, ⁇ 5 nM, ⁇ 4 nM ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM or ⁇ 500 pM.
  • the antigen-binding molecule described herein binds to TACI with a KD of 10 pM or less, preferably one of ⁇ 5 pM, ⁇ 2 pM, ⁇ 1 pM, ⁇ 500 nM, ⁇ 100 nM, ⁇ 75 nM, ⁇ 50 nM, ⁇ 40 nM, ⁇ 30 nM, ⁇ 20 nM, ⁇ 15 nM, ⁇ 12.5 nM, ⁇ 10 nM, ⁇ 9 nM, ⁇ 8 nM, ⁇ 7 nM, ⁇ 6 nM, ⁇ 5 nM, ⁇ 4 nM ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM or ⁇ 500 pM.

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Abstract

L'invention concerne des molécules de liaison à l'antigène CD47 et BCMA/TACI. L'invention concerne également des acides nucléiques et des vecteurs d'expression codant, des compositions comprenant, et des procédés utilisant, les molécules de liaison à l'antigène CD47 et BCMA/TACI.
PCT/EP2018/079932 2017-12-07 2018-11-01 Molécules de liaison à l'antigène cd47 et bcma/taci Ceased WO2019110209A1 (fr)

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WO2022090556A1 (fr) * 2020-11-02 2022-05-05 Hummingbird Bioscience Pte. Ltd. Molécules de liaison à l'antigène bcma/taci
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CN112661855A (zh) * 2019-10-16 2021-04-16 尚健单抗（北京）生物技术有限公司 含SIRPa变体的融合蛋白
CN112661855B (zh) * 2019-10-16 2022-09-13 尚健单抗（北京）生物技术有限公司 含SIRPa变体的融合蛋白
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US12202882B2 (en) 2020-05-08 2025-01-21 Alpine Immune Sciences, Inc. APRIL and BAFF inhibitory immunomodulatory proteins and methods of use thereof
US12304943B2 (en) 2020-05-08 2025-05-20 Alpine Immune Sciences, Inc. April and BAFF inhibitory immunomodulatory proteins and methods of use thereof
CN115885044A (zh) * 2020-09-04 2023-03-31 江苏恒瑞医药股份有限公司 SIRPγ变体及其融合蛋白
WO2022090556A1 (fr) * 2020-11-02 2022-05-05 Hummingbird Bioscience Pte. Ltd. Molécules de liaison à l'antigène bcma/taci

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