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WO2025099434A1 - Bcma binding molecules - Google Patents

Bcma binding molecules Download PDF

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Publication number
WO2025099434A1
WO2025099434A1 PCT/GB2024/052828 GB2024052828W WO2025099434A1 WO 2025099434 A1 WO2025099434 A1 WO 2025099434A1 GB 2024052828 W GB2024052828 W GB 2024052828W WO 2025099434 A1 WO2025099434 A1 WO 2025099434A1
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seq
antigen binding
cdr1
cdr2
cdr3
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Simon BORNSCHEIN
Youlia LAMPI
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Coding Bio Ltd
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Coding Bio 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
    • C07K16/2878—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
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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

Definitions

  • the present invention relates to BCMA (B cell maturation antigen, TNFRSF17) binding molecules, in particular to anti-BCMA antibodies and other BCMA binding molecules, and antigen binding fragments.
  • the present invention further relates to recombinant molecules comprising said BCMA binding molecules, such as, chimeric antigen receptors (CARs), BiTEs and ADCs.
  • Another object of the present invention is an engineered immune cell, suitably, a T cell comprising said CAR. Uses thereof in therapeutic methods of said BCMA binding molecule, said recombinant molecule and said engineered immune cell are further described.
  • BCMA glycoprotein also known as BCMA, BCM, CD269, TNFRSF17
  • BCMA B cell maturation antigen
  • BCM BCM
  • CD269 TNFRSF17
  • TNFRSF17 B cell maturation antigen glycoprotein
  • TNF Receptor-Associated Factor (TRAF) 1 TRAF2 and TRAF3.
  • TNF Receptor Family member BCMA B Cell Maturation Associates with TNF Receptor-Associated Factor (TRAF) 1 , TRAF2 and TRAF3 and Activates NF-KB, Elk-1 , c-Jun N-Terminal Kinase, and p38 Mitogen-Activated Protein Kinase. J. Immunol., 165(3): 1322-1330.).
  • BCMA B Cell Maturation Associates with TNF Receptor-Associated Factor (TRAF) 1 , TRAF2 and TRAF3 and Activates NF-KB, Elk-1 , c-Jun N-Terminal Kinase, and p38 Mitogen-Activated Protein Kinase. J. Immunol., 165(3): 1322-1330.).
  • TNF Receptor Family member BCMA B Cell Maturation
  • BCMA is a member of the tumour necrosis factor receptor family and is highly expressed in mature B- lymphocytes and plasma cells.
  • the N-terminal six-cysteine motif is a common characteristic of all TNFR family members that is repeated in all other members of the TNFR family but is present only once in the BCMA extracellular structure.
  • BCMA promotes B-cell and plasma cell survival through its interaction with A Proliferations-lnducing Ligand (APRIL) and B-cell activating factor (BAFF) and subsequent activation of the nuclear factor kappa-B (NF-KB) and c-Jun N-Terminal Kinase (JNK) signalling cascades.
  • APRIL A Proliferations-lnducing Ligand
  • BAFF B-cell activating factor
  • NF-KB nuclear factor kappa-B
  • JNK c-Jun N-Terminal Kinase
  • Membrane-bound BCMA is directly cleaved from the membrane by y-secretase, and elevated levels of sBCMA are used as a biomarker for Multiple Myeloma (MM), where sBCMA acts as a decoy reducing activation of membrane-bound BCMA by APRIL and BAFF.
  • MM Multiple Myeloma
  • MM typically affects middle aged and older adults and represents approximately 1 .8% of new cancer cases every year in the USA (Cancer Stat Facts: Myeloma. Available online: https://seer.cancer.gov/statfacts/html/mulmy.html (accessed on 20 September 2023).
  • a heterogenous disease MM-affected patients may experience a variety of disease-related symptoms, and the current 5-year survival rate for MM is approximately 62% (Cancer Stat Facts: Myeloma. Most recent data available: 2015. Available online: https://seer.cancer.gov/statfacts/html/mulmy.html (accessed on 20 September 2023).
  • BCMA is also implicated with other cancers and diseases including but not limited to: MM; Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
  • MM Hodgkin’s and non-Hodgkin’s lymphoma
  • various leukaemias glioblastoma
  • Waldentrsom macroglobulinemia multiple sclerosis
  • neuroborreloiosis systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • immunotherapy treatments that make use of the immune system or immune cells are known as immunotherapy treatments.
  • immunotherapy treatment relies on antibodies to an antigen on a target cell triggering or promoting cell death or inhibiting cell-division.
  • Antigens may be targeted using antibody or antigen binding fragments, such as a single chain variable fragment (scFv). Examples of antibodies that target BCMA have been previously described e.g. U.S. Patent No. 9273141 B2.
  • the antigen binding molecule or antigen binding domain can be linked or conjugated to (or be a product of conjugation of) a naturally-occurring or synthetic molecule, such as a naturally-occurring or synthetic molecule that binds to a T-cell antigen, suitably via a flexible linker, such as a flexible peptide linker to form a bi-specific T-cell engager (BiTE), or a dual-affinity retargeting antibody (DART) or other bispecific, tri-specific or multi-specific T-cell engagers.
  • a naturally-occurring or synthetic molecule such as a naturally-occurring or synthetic molecule that binds to a T-cell antigen
  • a flexible linker such as a flexible peptide linker to form a bi-specific T-cell engager (BiTE), or a dual-affinity retargeting antibody (DART) or other bispecific, tri-specific or multi-specific T-cell engagers.
  • antibodies or antigen binding fragments may be linked or conjugated with (or be a product of conjugation of) a naturally-occurring or synthetic molecule such as a label, cytotoxic agent (antibody-drug conjugates or ADCs), or therapeutic isotope.
  • a naturally-occurring or synthetic molecule such as a label, cytotoxic agent (antibody-drug conjugates or ADCs), or therapeutic isotope.
  • CAR T-cell therapy consists of obtaining T-cells from a patient and incorporating an engineered receptor to recognise an antigen found on the cancerous cells. The resulting CAR T-cells are then incorporated back into the patient, where they may then act to mount an immune response against the antigen i.e. against the cancerous cells and kill the cancerous cells.
  • one or more antibodies or antigen binding fragments can be linked or conjugated with (or be a product of conjugation of) a naturally-occurring or synthetic molecule comprising other protein domains, such as hinge domains, transmembrane domains, co-stimulation domains and activation domains to form a CAR that is capable of effecting an immune response towards the antigen-containing cell.
  • This CAR is then expressed in a T-cell.
  • the invention provides an antigen binding molecule that binds BCMA; wherein the antigen binding molecule comprises an antigen binding domain selected from the group consisting of: Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 12 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 15 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 1 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 2 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 3 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 4 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 5 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 10 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 11 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 13 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 16 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 17 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 18 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 19 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 20 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 21 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 22 comprising a single variable domain on a heavy chain comprising, (i) a CDR1 of SEQ ID NO:142,
  • Antigen binding domain 23 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 24 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 25 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 26 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 27 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 29 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 30 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 31 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 32 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 33 comprising:
  • Antigen binding domain 34 comprising:
  • Antigen binding domain 35 comprising:
  • Antigen binding domain 36 comprising:
  • Antigen binding domain 37 comprising:
  • the single variable domain on a heavy chain of any one of the antigen binding domainl to 5 and 10 to 32 is a VHH or wherein, the antigen binding domains 33 to 37 is a ScFv.
  • the VHH is selected from the group consisting of: SEQ ID NOs 3 to 5, SEQ ID NOs 15 to 16, SEQ ID NOs 66 to 71 , SEQ ID NOs 108 to 111 , SEQ ID NOs 136 to 139, and SEQ ID NOs 164 to 172, or the ScFv is selected from the group consisting of SEQ ID Nos 227 to 231 .
  • the antigen binding molecule that binds BCMA comprises antigen binding domain 1 , antigen binding domain 2, antigen binding domain 3, antigen binding domain 4, antigen binding domain 5, antigen binding domain 10, antigen binding domain 11 , antigen binding domain 12, antigen binding domain 13, antigen binding domain 14, antigen binding domain 15, antigen binding domain 16, antigen binding domain 17, antigen binding domain 18, antigen binding domain 19, antigen binding domain 20, antigen binding domain 21 , antigen binding domain 22, antigen binding domain 23, antigen binding domain 24, antigen binding domain 25, antigen binding domain 26, antigen binding domain 27, antigen binding domain 28, antigen binding domain 29, antigen binding domain 30, antigen binding domain 31 , antigen binding domain 32, antigen binding domain 33, antigen binding domain 34, antigen binding domain 35, antigen binding domain 36 or antigen binding domain 37.
  • the antigen binding molecule that binds BCMA is provided, wherein the antigen binding molecule comprises an antigen binding domain is selected from the group consisting of:
  • Antigen binding domain 14 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 15 comprising a single variable domain on a heavy chain comprising,
  • the antigen binding molecule is a chimeric antigen receptor comprising a transmembrane region and an intracellular T-cell receptor signalling domain.
  • the hinge region and the transmembrane region is from CD8A and/or the intracellular T-cell receptor signalling domain is from CD247 (CD3-zeta).
  • antigen binding molecule further comprises an intracellular domain of a costimulatory protein receptor.
  • the antigen binding molecule is a bi-specific T-cell engager (BiTE) comprising a T-cell antigen binding domain.
  • the T-cell antigen binding domain comprises an antibody fragment that specifically binds CD3.
  • the antigen binding molecule is an antibody-drug conjugate (ADC) comprising a cytotoxic drug or a therapeutic radioisotope.
  • ADC antibody-drug conjugate
  • the invention provides a vector encoding the antigen binding molecule of the first aspect of the invention.
  • the vector comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NOs 41 to 45, 102 to 107, 132 to 135, 160 to 163, 218 to 226, and 272 to 276.
  • the invention provides a pharmaceutical composition comprising the antigen binding molecule of the first aspect of the invention and a pharmaceutically acceptable carrier.
  • the invention provides a chimeric antigen receptor that binds BCMA comprising:
  • Antigen binding domain 14 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 15 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 1 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 2 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 3 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 4 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 5 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 10 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 11 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 13 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 16 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 17 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 18 comprising a single variable domain on heavy chain comprising,
  • Antigen binding domain 19 comprising a single variable domain on heavy chain comprising,
  • Antigen binding domain 20 comprising a single variable domain on heavy chain comprising,
  • Antigen binding domain 21 comprising a single variable domain on heavy chain comprising,
  • Antigen binding domain 22 comprising a single variable domain on heavy chain comprising,
  • Antigen binding domain 23 comprising a single variable domain on a heavy chain comprising, (i) a CDR1 of SEQ ID NO:143,
  • Antigen binding domain 24 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 25 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 26 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 27 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 28 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 30 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 31 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 32 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 33 comprising:
  • Antigen binding domain 34 comprising:
  • Antigen binding domain 35 comprising:
  • Antigen binding domain 36 comprising:
  • the chimeric antigen receptor that binds BCMA comprises antigen binding domain 1 , antigen binding domain 2, antigen binding domain 3, antigen binding domain 4, antigen binding domain 5, antigen binding domain 10, antigen binding domain 11 , antigen binding domain 12, antigen binding domain 13, antigen binding domain 14, antigen binding domain 15, antigen binding domain 16, antigen binding domain 17, antigen binding domain 18, antigen binding domain 19, antigen binding domain 20, antigen binding domain 21 , antigen binding domain 22, antigen binding domain 23, antigen binding domain 24, antigen binding domain 25, antigen binding domain 26, antigen binding domain 27, antigen binding domain 28, antigen binding domain 29, antigen binding domain 30, antigen binding domain 31 , antigen binding domain 32, antigen binding domain 33, antigen binding domain 34, antigen binding domain 35, antigen binding domain 36 or antigen binding domain 37.
  • the chimeric antigen receptor that binds BCMA comprises an antigen binding domain selected from the group consisting of:
  • Antigen binding domain 14 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 12 comprising a single variable domain on a heavy chain comprising,
  • the transmembrane region is from CD8A or TNFRSF4, suitably CD8A, and/or the intracellular signalling domain is from CD247 (CD3-zeta).
  • the CAR further comprises an intracellular signalling domain of a costimulatory protein receptor.
  • the CAR comprises a human CD247 (CD3-zeta) cytoplasmic domain (SEQ ID NO:28), a human CD137 co-stimulatory domain (SEQ ID NO:29); a human CD8A hinge/transmembrane domain (SEQ ID NQ:30), the antigen binding molecule of the first aspect; and an CD8A extracellular signal peptide (SEQ ID NO:33).
  • the CAR may comprise two or more antigen binding domains, wherein at least one of the antigen binding domains is a BCMA antigen binding domain of the first aspect of the present invention.
  • the linker has a sequence selected from the group consisting of: SEQ ID NOS: 31 and 32.
  • the CAR may be encoded by a nucleic acid sequence comprising two or more CAR sequences, joined by a sequence encoding a self-cleaving linker.
  • the self-cleaving linker is a T2A self-cleaving linker with a sequence of: SEQ ID NO 34.
  • the CAR has a sequence selected from the group consisting of: SEQ ID NOS: 23 to 29, 90 to 95, 124 to 127, 152 to 155, 200 to 208 and 262 to 266.
  • the invention provides a recombinant T cell comprising the chimeric antigen receptor of the fourth aspect.
  • the invention provides a bispecific T-cell engager that binds BCMA comprising:
  • Antigen binding domain 14 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 12 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 15 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 1 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 2 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 3 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 4 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 5 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 10 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 11 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 13 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 16 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 17 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 18 comprising a single variable domain on a heavy chain comprising, (i) a CDR1 of SEQ ID NO:114,
  • Antigen binding domain 19 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 20 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 21 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 22 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 23 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 25 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 26 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 27 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 28 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 29 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 30 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 31 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 32 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 33 comprising:
  • Antigen binding domain 34 comprising:
  • Antigen binding domain 35 comprising:
  • Antigen binding domain 36 comprising:
  • Antigen binding domain 37 comprising:
  • the bispecific T-cell engager that binds BCMA comprises antigen binding domain 1 , antigen binding domain 2, antigen binding domain 3, antigen binding domain 4, antigen binding domain 5, antigen binding domain 10, antigen binding domain 11 , antigen binding domain 12, antigen binding domain 13, antigen binding domain 14, antigen binding domain 15, antigen binding domain 16, antigen binding domain 17, antigen binding domain 18, antigen binding domain 19, antigen binding domain 20, antigen binding domain 21 , antigen binding domain 22, antigen binding domain 23, antigen binding domain 24, antigen binding domain 25, antigen binding domain 26, antigen binding domain 27, antigen binding domain 28, antigen binding domain 29, antigen binding domain 30, antigen binding domain 31 , antigen binding domain 32, antigen binding domain 33, antigen binding domain 34, antigen binding domain 35, antigen binding domain 36 or antigen binding domain 37.
  • the bispecific T-cell engager that binds BCMA comprises an antigen binding domain selected from the group consisting of:
  • Antigen binding domain 14 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 12 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 15 comprising a single variable domain on a heavy chain comprising,
  • the antigen binding domain which binds to a T-cell antigen comprises an antigen binding fragment that specifically binds CD3.
  • the antigen binding fragment that specifically binds CD3 comprises:
  • the invention provides an antibody-drug conjugate that binds BCMA comprising:
  • Antigen binding domain 14 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 12 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 15 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 2 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 3 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 4 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 5 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 10 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 11 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 13 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 16 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 17 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 18 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 19 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 20 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 21 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 22 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 23 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 24 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 26 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 27 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 28 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 29 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 30 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 31 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 32 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 33 comprising:
  • Antigen binding domain 34 comprising:
  • Antigen binding domain 35 comprising:
  • Antigen binding domain 36 comprising:
  • Antigen binding domain 37 comprising:
  • the chimeric antigen receptor that binds BCMA comprises antigen binding domain 1 , antigen binding domain 2, antigen binding domain 3, antigen binding domain 4, antigen binding domain 5, antigen binding domain 10, antigen binding domain 11 , antigen binding domain 12, antigen binding domain 13, antigen binding domain 14, antigen binding domain 15, antigen binding domain 16, antigen binding domain 17, antigen binding domain 18, antigen binding domain 19, antigen binding domain 20, antigen binding domain 21 , antigen binding domain 22, antigen binding domain 23, antigen binding domain 24, antigen binding domain 25, antigen binding domain 26, antigen binding domain 27, antigen binding domain 28, antigen binding domain 29, antigen binding domain 30, antigen binding domain 31 , antigen binding domain 32, antigen binding domain 33, antigen binding domain 34, antigen binding domain 35, antigen binding domain 36 or antigen binding domain 37.
  • the chimeric antigen receptor that binds BCMA comprises an antigen binding domain selected from the group consisting of:
  • Antigen binding domain 14 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 12 comprising a single variable domain on a heavy chain comprising,
  • Antigen binding domain 15 comprising a single variable domain on a heavy chain comprising,
  • the therapeutic drug substance is a cytotoxic drug.
  • the one or more antigen binding domains of the chimeric antigen receptor of the fourth aspect of the invention, the one or more antigen binding domains of bispecific T-cell engager (BiTE) of the sixth aspect of the invention, or one or more antigen binding domains of the antibodydrug conjugate (ADC) of the seventh aspect of the invention bind BCMA.
  • the VHH is selected from the group consisting of: SEQ ID NOs 3 to 5, SEQ ID NOs 15 to 16 SEQ ID NOs 66 to 71 , SEQ ID NOs 108 to 111 , SEQ ID NOs 136 to 139, and SEQ ID NOs 164 to 172.
  • the ScFv is selected from the group consisting of SEQ ID Nos 227 to 231 .
  • the antigen binding molecule of the first aspect, the pharmaceutical composition of the third aspect, the chimeric antigen receptor of the fourth aspect, the recombinant T-cell of the fifth aspect, the bispecific T-cell engager of the sixth aspect, the antibody-drug conjugate of the seventh aspect may be for use as a medicament or in medicine.
  • the antigen binding molecule of the first aspect, the pharmaceutical composition of the third aspect, the chimeric antigen receptor of the fourth aspect, the recombinant T-cell of the fifth aspect, the bispecific T-cell engager of the sixth aspect, the antibody-drug conjugate of the seventh aspect may be for use in killing or inhibiting the growth of cells expressing BCMA.
  • the antigen binding molecule of the first aspect, the pharmaceutical composition of the third aspect, the chimeric antigen receptor of the fourth aspect, the recombinant T-cell of the fifth aspect, the bispecific T-cell engager of the sixth aspect, the antibody-drug conjugate of the seventh aspect may be for use in the treatment of a disease or disorder selected from the group consisting of: cancers, such as Multiple Myeloma (MM); Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
  • cancers such as Multiple Myeloma (MM); Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Wald
  • the invention provides a method of treating a disease or condition associated with the expression of BCMA, said method comprising administering to a patient in need thereof an effective amount of an antigen binding molecule of the first aspect of the invention, a pharmaceutical composition of the second aspect of the invention, a chimeric antigen receptor of the fourth aspect of the invention, a recombinant T-cell of the fifth aspect of the invention, a bi-specific T-cell engager of the sixth aspect of the invention or the antibody-drug conjugate of the seventh aspect of the invention to a patient in need thereof.
  • the disease or condition is selected from the group consisting of: MM; Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
  • Figure 1 A shows an embodiment of the general structure of the antigen binding molecule of the present invention wherein the antigen binding domain is a VHH comprising CDRs 1 , 2 and 3, within an example single chimeric antigen receptor scaffold comprising a CD8A signal peptide, a CD8A hinge and TM region, CD137 costimulatory domain and CD247 stimulatory domain.
  • the antigen binding domain is a VHH comprising CDRs 1 , 2 and 3, within an example single chimeric antigen receptor scaffold comprising a CD8A signal peptide, a CD8A hinge and TM region, CD137 costimulatory domain and CD247 stimulatory domain.
  • Figure 1 B shows an embodiment of the general structure of the antigen binding molecule of the present invention wherein the antigen binding domain is a scFv comprising CDRs 1 , 2 and 3 of a heavy chain and comprising CDRs 1 , 2 and 3 of a light chain within an example single chimeric antigen receptor scaffold comprising a CD8A signal peptide, a CD8 hinge, a CD8A TM Domain, CD137 costimulatory domain and CD247 stimulatory domain.
  • the antigen binding domain is a scFv comprising CDRs 1 , 2 and 3 of a heavy chain and comprising CDRs 1 , 2 and 3 of a light chain within an example single chimeric antigen receptor scaffold comprising a CD8A signal peptide, a CD8 hinge, a CD8A TM Domain, CD137 costimulatory domain and CD247 stimulatory domain.
  • FIG. 2 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs. Four days after transduction, cells were exposed for 24h to K562 wild-type cells or BCMA- expressing K562 cells. After 24h GFP expression was assessed via flow cytometry.
  • FIG 3 shows cells transduced with different BCMA-CAR constructs and expanded for eight days and stained with BCMA-Fc protein.
  • cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies.
  • Median fluorescence intensity (MFI) is shown for CD34+ CAR- T cells.
  • Figure 4 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on RPMI8226 cells cultured with non-transduced T cells.
  • Figure 5 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (K562_BCMA) at a 1 :1 effector to target cell ratio. 24 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on K562_BCMA cells cultured with non-transduced T cells.
  • K562_BCMA BCMA-expressing cancer cell line
  • Figure 6 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later supernatant was collected and assessed for IFN-gamma production via LegendPlexTM (BiolegendTM) cytokine assay.
  • Figure 7 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later supernatant was collected and assessed for IL-2 production via LegendPlexTM (BiolegendTM) cytokine assay.
  • Figure 8 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later cells were assessed by flow cytometer for the up-regulation of CD25 and CD69 markers.
  • Figure 9 shows T-cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h and 48h with very low BCMA-expressing cancer cell line (Nalm-6) at a 1 :1 effector to target cell ratio. 24 and 48 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on Nalm-6 cells cultured with non-transduced T cells.
  • Figure 10 shows T-cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h and 48h with high BCMA-expressing cancer cell line (RPMI-8226) at a 1 :1 effector to target cell ratio. 24 and 48 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on RPMI-8226 cells cultured with non-transduced T cells.
  • RPMI-8226 BCMA-expressing cancer cell line
  • Figure 11 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 48 hours later supernatant was collected and assessed for IFN-gamma production via LegendPlexTM (BiolegendTM) cytokine assay.
  • Figure 12 shows T-cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h and 48h with very high BCMA-expressing cancer cell line (U266B1) at a 1 :1 effector to target cell ratio. 24 and 48 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on U266B1 cells cultured with non-transduced T cells.
  • U266B1 BCMA-expressing cancer cell line
  • FIG. 13 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs. Four days after transduction, cells were exposed for 24h to BCMA-expressing RPMI-8226 cells. After 24h GFP expression was assessed via flow cytometry.
  • Figure 14 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs, and expanded for 4 days and stained with BCMA-Fc protein.
  • cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies.
  • Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
  • Figure 15 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs based on CAR construct BCMA-3. Four days after transduction, cells were exposed for 24h to BCMA-expressing RPMI-8226 cells. After 24h GFP expression was assessed via flow cytometry.
  • Figure 16 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs based on CAR construct BCMA-3. and expanded for 4 days and stained with BCMA-Fc protein. In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies. Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
  • MFI Median fluorescence intensity
  • Figure 17 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs based on CAR construct BCMA-5. Four days after transduction, cells were exposed for 24h to BCMA-expressing RPMI-8226 cells. After 24h GFP expression was assessed via flow cytometry.
  • Figure 18 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs based on CAR construct BCMA-5. and expanded for 4 days and stained with BCMA-Fc protein. In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies. Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
  • MFI Median fluorescence intensity
  • FIG 19 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs. Four days after transduction, cells were exposed for 24h to BCMA-expressing RPMI-8226 cells. After 24h GFP expression was assessed via flow cytometry.
  • Figure 20 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs, and expanded for 4 days and stained with BCMA-Fc protein.
  • cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies.
  • Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
  • Figure 21 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs. Four days after transduction, cells were exposed for 24h to BCMA-expressing OPM-2 cells. After 24h GFP expression was assessed via flow cytometry.
  • Figure 22 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs, and expanded for 4 days and stained with BCMA-Fc protein.
  • cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies.
  • Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
  • the articles ‘a’, ‘an’ and ‘the’ are used to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.
  • the term ‘comprising’ means any of the recited elements are necessarily included and other elements may optionally be included as well.
  • Consisting essentially of means any recited elements are necessarily included, elements which would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included.
  • Consisting of means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.
  • antibody refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric, polymeric and chimeric forms, unless otherwise specified. Specifically encompassed by the term ‘antibody’ are polyclonal antibodies, monoclonal antibodies (mAbs), single domain antibodies, human (FHVH) or heavy-chain antibodies found in camelids (VHH) and antibodylike polypeptides, such as chimeric antibodies and humanized antibodies.
  • mAbs monoclonal antibodies
  • FHVH single domain antibodies
  • VHH heavy-chain antibodies found in camelids
  • antibodylike polypeptides such as chimeric antibodies and humanized antibodies.
  • the term ‘monoclonal antibody’ refers to an antibody produced by a single clone of cells or cell line and consisting of identical antibodies.
  • antibody or ‘antigen-binding fragments’ or ‘antigen binding molecule’ may more generally encompass any protein or mixed protein/chemical structure that exhibits binding affinity for a particular antigen, or more than one particular antigen.
  • antigen binding molecules include antibodies, chimeric antigen receptors (CARs), bi-specific T-Cell engagers (e.g. BiTEs), dual affinity re-targeting reagent (DARTTM), or antibody-drug conjugates (ADCs).
  • Antigen-binding fragments include those provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments are composed of portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule.
  • antigen-binding fragments may comprise at least one variable region (either a heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind a particular antigen.
  • antigen-binding fragments include, without limitation diabodies, nanobodies and single-chain molecules as well as Fab, F(ab')2, Fc, Fabc, and Fv molecules, single chain (sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in W02007059782, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting essentially of the VH and CH1 domains; a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, a dAb fragment (Ward et al., Nature 341 , 544-546 (1989)), which
  • antigen-binding fragments may include non-antibody proteinaceous frameworks that may successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds.
  • Antigen-binding fragments may be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies.
  • the phrase ‘an antibody or antigenbinding fragment’ may be used to denote that a given antigen binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.
  • antigen binding domain refers to a peptide sequence that is intended or able to bind a target of interest.
  • the antigen binding domain is an antigen-binding fragment as defined above. All types of antigen binding domains are encompassed by the present invention. Examples of some antigen binding domains are scFvs, VHH single domain antibodies or nanobodies, and antigen binding fragments.
  • VHH refers to a single-domain antibody (sdAb), also known as a nanobody, and is an antibody fragment consisting of a single monomeric variable antibody domain located on a heavy chain.
  • sdAb single-domain antibody
  • an scFv is a fusion of the variable regions of the heavy (VH) and light chains (VL) of an antibody for a given target connected by a short linker.
  • the VH and VL regions may be in any order around the linker, for example, the scFv may have an opening signal peptide sequence followed by (i) a VH chain, a linker and a VL chain or (ii) the scFv may have (i) a VL chain, a linker and a VH chain, then followed by a hinge region, a TM domain and an intracellular domain.
  • the scFv may have a first VH chain, a linker and a VL chain.
  • Antigen binding domains may comprise ‘CDRs’ or ‘complementarity determining regions’ which are predominantly responsible fortarget binding. On a typical antibody, antigen binding molecule or antigen binding domain, multiple CDRs exist and may be selected or varied independently to achieve multiple points of diversity.
  • a ‘recognition sequence’ refers to the nucleic acid sequence encoding one or more antigen binding domains.
  • ‘Specific binding’ or ‘immunospecific binding’ or derivatives thereof when used in the context of antibodies, antigen binding molecules or antibody fragments or domains, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopes of a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules.
  • an antibody binds to a cognate antigen with a Kd of less than about 1x1 O' 8 M, as measured by a surface plasmon resonance assay or a cell binding assay.
  • Phrases such as ‘[antigen] -specific’ antibody e.g. BCMA-specific antibody
  • an ‘isolated’ antibody or antigen-binding fragment is intended to refer to an antibody or antigen- binding fragment which is substantially free of other antibodies or antigen-binding fragments having different antigenic specificities (for instance, an isolated antibody that specifically binds to BCMA is substantially free of antibodies that specifically bind antigens other than BCMA ).
  • An isolated antibody that specifically binds to an epitope, isoform or variant of BCMA may, however, have cross-reactivity to other related antigens, for instance from other species (such as BCMA species homologs).
  • chimeric antigen receptor refers to transmembrane receptor that has been engineered to target or bind to a non-native substrate or antigen. In this way the intracellular signalling of the receptor may triggered by binding of a non-native substrate or antigen.
  • CAR refers to a chimeric receptor (i.e. a receptor composed of two or more parts from different sources) that has at least a binding moiety or recognition sequence with a specificity for a target such as an antigen or protein and an intracellular signalling domain that can invoke a signal in the cell in which the CAR is expressed (e.g. a CD247 (CD3 zeta) chain).
  • a ‘chimeric antigen receptor’ or ‘CAR’ is formed of at least three domains: an extracellular antigen binding domain (as defined elsewhere herein), a transmembrane domain and an intracellular domain.
  • a hinge domain between the antigen binding domain and the transmembrane domain is often used to improve recognition of the target.
  • a costimulatory domain may optionally be present in the intracellular domain to modulate the response.
  • the domains of the CAR must be ordered correctly.
  • CARs are often used on T-cells (to produce ‘CAR T-cells’) to effect recognition and elicit an appropriate intracellular response which both binds the T-cell to a target cell and triggers the innate immune response of the T-cell, typically causing lysis of the target cell.
  • T-cells to produce ‘CAR T-cells’
  • Such cells have found use in therapy.
  • the term a ‘tandem CAR’ is used herein to refer to a CAR with one or more, suitably two, antigen binding fragments in a single antigen binding domain, optionally joined by a linker.
  • a ‘parallel CAR’ is used herein to refer to a CAR sequence (or CARs derived from that sequence) that comprises two or more CAR sequences joined by a linker, suitably a self-cleaving peptide linker, such that following translation, two or more CARs are expressed from the separate CAR sequences.
  • a ‘transmembrane domain’ or ‘TM domain’ as used herein is any membrane-spanning protein domain.
  • the TM domain in a CAR is derived from a known transmembrane protein sequence. However, it can also be artificially designed.
  • a ‘transmembrane sequence’ refers to the nucleic acid sequence encoding a transmembrane domain.
  • the term ‘hinge domain’ refers to a peptide sequence that connects the antigen binding domain and transmembrane region of a CAR. The hinge domain is located between the antigen binding fragment and the T cell plasma membrane (Moritz D, et al. Gene Ther. 1995;2(8):539-46)
  • the signalling domain typically comprises a domain derived from a receptor that signals by itself in immune cells, such as the T Cell Receptor (TCR) complex or the Fc receptor or DAP10/DAP12 receptors. Additionally, it may contain a costimulatory domain (i.e. a domain derived from a receptor that is required in addition to the TCR to obtain full activation of T cells).
  • the costimulatory domain can be from an activating costimulatory receptor or from an inhibitory costimulatory receptor.
  • An ‘intracellular domain sequence’ refers to the nucleic acid sequence encoding an intracellular signalling domain.
  • bi-specific refers to an antibody or antigen binding molecule (i.e. an antibody or antigen binding molecule or antigen binding fragment or domain conjugated to, or a product of conjugation of, a naturally-occurring or synthetic molecule) that comprises one or more further antigen binding domains such that the antibody molecule can have specificity for more than one antigen.
  • bispecific T-cell engager or ‘BiTE’ is used synonymously to refer to a single polypeptide chain molecule having two antigen binding domains, one of which binds to a T-cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell (e.g. BCMA ), these two antigen binding domains separated by a linker, specifically a flexible linker, more specifically a flexible protein linker (WO 05/061547; Baeuerle, et al. (2008) Drugs of the Future 33:137-147; Bargou, et al. (2008) Science 321 :974-977).
  • a linker specifically a flexible linker, more specifically a flexible protein linker
  • BiTE antibodies have been constructed to various target antigens including CD19, EpCAM, Her2/neu, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or HMW-MAA) (Baeuerle, et al. (2009) Curr. Opin. Mol. Ther. 1 1 :22-30).
  • Key hallmarks of BiTE antibodies that, in their combination, distinguish them from other bispecific antibody constructs, include a high potency of redirected lysis with EC50 values ranging from 0.1 to 50 pmol/L (2-1 ,000 pg/mL) (Baeuerle, et al. (2009) supra); strict target cell-dependent activation of T cells (Brischwein, et al.
  • BiTE antibodies are typically produced as recombinant, glycosylated proteins secreted by higher eukaryotic cell lines.
  • the terms ‘bi-specific T- Cell engager’ and/or ‘BiTE’ as herein defined encompass, but are not limited to, the specific bi-specific T-Cell engager structure encompassed by the trademark ‘BITE®’ in the name of Amgen Research (Munich) GmbH.
  • DARTTM Dual Affinity Re-Targeting reagent diabody refers to an immunoglobulin molecule that comprises at least two polypeptide chains that associate (for example, through a covalent interaction) to form at least two antigen binding domains, which may recognize the same or different antigens.
  • Each of the polypeptide chains of a DARTTM diabody comprise an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an antigen binding domain. Rather, the immunoglobulin heavy chain variable region of one (e.g., the first) of the DARTTM diabody polypeptide chains interacts with the immunoglobulin light chain variable region of a different (e.g.
  • DARTTM diabodies may be monospecific, bispecific, trispecific, etc., thus being able to simultaneously bind one, two, three or more different antigens (which may be of the same or of different antigens).
  • DARTTM diabodies may additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., thus being able to simultaneously bind one, two, three, four, five, six or more molecules.
  • These two attributes of DARTTM diabodies i.e. degree of specificity and valency, may be combined, for example to produce bispecific antibodies i.e. capable of binding two antigens, which are tetravalent i.e., capable of binding four sets of epitopes, etc.
  • DARTTM diabody molecules are disclosed in PCT applications published as WO 2006/113665, WO 2008/157379, and WO 2010/080538.
  • antibody-drug conjugate means a biopharmaceutical drug that comprises an antibody or antigen binding fragment or domain, and a drug substance, typically a cytotoxic drug substance, connected via a linker, suitably a covalent, chemical linker.
  • high-throughput screening refers to any assay or screening methodology that allows for a higher rate of screening than would be achieved by traditional or previous state of the art techniques.
  • high-throughput screening enables automation to prepare, screen and/or evaluate libraries of test samples in parallel, reproducibly and rapidly.
  • High-throughput screening can also make use of combinatorial or pooled or mixed sample screening strategies, with associated deconvolution of hits.
  • the number of samples for test may be of any size larger than that typically used in prior art non-high-throughput methods. For example, the number of samples may be more than 10. Suitably the number of samples may be more than 11 ,
  • the term ‘recognition sequence library’ refers to a set of one or more antigen binding domain sequences or recognition sequences that may be used for cloning into a CAR construct (wherein a CAR construct comprises all components required for a functioning CAR including one or more antigen binding domains, a hinge domain, a transmembrane domain and an intracellular domain) to prepare a CAR library.
  • An ‘scFv library’ is a recognition sequence library formed of scFv recognition sequences from antibodies.
  • a ‘VHH library’ is a recognition sequence library formed of VHH recognition sequences from antibodies.
  • the number of recognition sequences present in the recognition sequence library may be more than 1 or more than 2.
  • the number of hinge region sequences present in the hinge region sequence library may be more than 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,
  • CAR scaffold refers to the part of the CAR sequence that comprises the components, parts, modules, domains of the CAR excluding the antigen binding domain, that form the sequence of a CAR in a CAR library.
  • the CAR scaffold is formed of the sequences of the individual components, or groups thereof, and combined with the recognition sequence to form a sequence encoding a CAR in any suitable manner, for example, sequentially, convergently, with the recognition sequence being incorporated at any suitable point, i.e. the recognition sequence may be joined to a single unitary sequence of the CAR scaffold, or may be joined to a component part of the CAR scaffold initially and the full CAR sequence of the recognition sequence and scaffold completed subsequently.
  • the component parts of the CAR scaffold may derive from single sequences leading to one or a small number of CAR scaffolds for addition to a recognition sequence, or the component parts of the CAR scaffold may derive from libraries of one or more of the individual components leading to a CAR scaffold library comprising a plurality of sequences.
  • a CAR scaffold library may be formed by combination, suitably in a combinatorial, or directed, manner of the individual components or component libraries.
  • CAR library refers to a set of sequences encoding a functional CAR structure, including at least one recognition domain, such as an scFv or VHH, a hinge domain, a transmembrane domain and intracellular domain(s).
  • the number of CARs present in the CAR library may be more than 10.
  • the number of CARs present in the CAR library may be more than 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200 or more.
  • the term ‘CAR library’ may also be used interchangeably to refer to the plasmid or vector, or otherwise modified sequence of the sequence(s) encoding for a functional CAR structure.
  • CAR-cell library refers to a collection or set of cells expressing CARs on their surface.
  • Each cell in the CAR-cell library may express a single CAR (i.e. express only one CAR encoded by a single sequence), or a single cell in the CAR-cell library may express two or more CARs, each encoded by the same or different sequences.
  • each cell in the CAR-cell library expresses a single CAR or all CARs present in the library.
  • the number of CARs present in the CARcell library may be more than 10.
  • the number of CARs present in the CAR-cell library may be more than 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200 or more.
  • NGS next generation sequencing
  • nucleic acid molecule synonymously referred to as ‘nucleotides’ or ‘nucleic acids’ or ‘polynucleotide’ refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Nucleic acid molecules include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified’ bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications may be made to DNA and RNA; thus, ‘polynucleotide’ embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide’ also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
  • nucleic acid sequence may be inserted into a genome, including but not limited to plasmid or vector transfection, transposition and genome editing. All are contemplated for use in the present invention.
  • a ‘vector’ is a replicon, such as plasmid, phage, cosmid, or virus in which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
  • a ‘transposon’ or ‘transposable elements’ are DNA sequences that can change their position within a genome.
  • Gene editing refers to the ability to edit the genome to insert the required sequence, for example using CRISPR-Cas9 genome editing technology.
  • a ‘clone’ is a population of cells derived from a single cell or common ancestor by mitosis.
  • a ‘cell line’ is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • cells are transformed by transfecting the cells with DNA.
  • express and ‘produce’ are used synonymously herein and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post-translational modifications.
  • subject refers to human and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, goats, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In most particular embodiments of the described methods, the subject is a human.
  • treating refers to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival.
  • the treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations.
  • BMCA BMCA polypeptide
  • terminal residues such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues.
  • Amino acid sequence of an example isoform of BCMA to which the antibodies of the present invention bind is provided as SEQ ID NO: 1 .
  • BCMA The DNA sequence of BCMA (SEQ ID NO: 1) is provided (SEQ ID NO: 2). Splice variants and other isoforms of BCMA are known and are encompassed by the present disclosure.
  • the amino acid sequence of BCMA encompasses a polypeptide that has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity to BCMA (for example SEQ ID NO: 1).
  • the BCMA polynucleotide sequence can comprise a polynucleotide that has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity to BCMA (for example, SEQ ID NO: 2).
  • Sequence identity can also be to a fragment or portion of the full-length polynucleotide or polypeptide.
  • a sequence may have only 50% overall sequence identity with a sequence of the invention but in a particular region, domain or subunit could share 80%, 90%, or as much as 99% sequence identity with sequence of the invention.
  • homology to a nucleic acid sequence is not limited simply to sequence identity. Many nucleic acid sequences can demonstrate biologically significant homology to each other despite having apparently low sequence identity. In the present invention homologous nucleic acid sequences are considered to be those that will hybridise to each other under conditions of low stringency (Sambrook J. et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY).
  • the present application shows a systematic high-throughput screen output of a CAR-T cell library.
  • This allowed identification of multiple novel antigen binding molecules or antigen binding fragments or domains (e.g. VHHs or scFvs) and their associated heavy chain CDRs and light chain CDRs, that bind to BCMA and outperform other known antibody or antigen binding fragments in several experimental models.
  • novel antigen binding molecules or antigen binding fragments or domains e.g. VHHs or scFvs
  • their associated heavy chain CDRs and light chain CDRs that bind to BCMA and outperform other known antibody or antigen binding fragments in several experimental models.
  • the invention provides an antibody or antigen binding fragments or antigen binding molecules having specificity for BCMA (Antigen binding molecule Nos. 1 to 5, 10 to 37), wherein the antibody or antigen binding fragments or antigen binding molecules have an antigen binding domain comprising the amino acid sequences (SEQ ID NOS: 3 to 22, 66 to 89, 108 to 123, 136 to 151 , 164 to 199 and 227 to 261) as shown in Table 1 below:
  • the antigen-binding molecules, or antigen binding domains, of the present invention were identified from a functional high-throughput screen of CAR-T cells, wherein the CAR-T cells express a unique chimeric antigen receptor (CARs), each CAR being or comprising an antigen binding molecule comprising an antigen binding domain as a VHH derived from a diverse VHH library or a scFv from a diverse scFv library.
  • CARs unique chimeric antigen receptor
  • each CAR being or comprising an antigen binding molecule comprising an antigen binding domain as a VHH derived from a diverse VHH library or a scFv from a diverse scFv library.
  • the antigen binding domains as identified in Tables 1 and 2 are those exhibiting surprisingly good functional response (i.e. were positive hits) in various assays against cells expressing BCMA and are therefore deemed to provide superior CAR-T cells overthat known in the art and provide novel and effective antigen binding molecules or
  • the antibody or antigen binding molecule can be an isolated monospecific or bispecific antibody having specificity for BCMA (suitably human BMCA) and can be a full-length antibody or an antibody fragment.
  • the antibody can be polyclonal, monoclonal, recombinant, chimeric, or humanised.
  • the antibody can be of any isotype including without limitation IgA, IgD, IgE, IgG, or IgM.
  • the antibody can be any IgA such as lgA1 or lgA2, or any IgG such as lgG1 , lgG2, lgG3, lgG4, or synthetic IgG.
  • the antibody can also be any antibody fragment having specificity for BCMA, such as F(ab)2, Fv, scFv, F(ab' )2, F(ab), VL, VH, VHH, dsFv, Fv, scFv-Fc, (scFv)2, a diabody, and a bivalent antibody.
  • the antibody can be any modified or synthetic antibody, including, but not limited to, non-depleting IgG antibodies, T-bodies, or other Fe or Fab variants of antibodies.
  • the invention provides an antigen binding molecule or antigen binding domain with avidity for BCMA of about 10 pM or less, 5 pM or less, 2 pM or less, 1 pM or less, 500 nM or less, 400 nM or less, 300 nM or less, or 200 nM or less, or 100 nM or less, or 75 nM or less, or 50 nM or less, or 25 nM or less, or 10 nM or less, or 5 nM or less.
  • Avidity can be measured using art-known techniques, such as ELISA or BIACORE.
  • the antigen binding molecule or antigen binding domain of the invention can be produced by any suitable technique, for example, using any suitable eukaryotic or non-eukaryotic expression system.
  • the antigen binding molecule is produced using a mammalian expression system.
  • the antigen binding molecule or antigen binding domain of the invention can be produced using a suitable non-eukaryotic expression system such as a bacterial expression system.
  • Bacterial expression systems can be used to produce fragments such as a F(ab)2, Fv, scFv, F(ab' )2, F(ab), VL, VH, VHH, dsFv, Fv, scFv-Fc, (scFv)2, and diabodies.
  • the antigen binding molecule can be a product of conjugation to a synthetic molecule, or the antigen binding fragment of the invention can be conjugated to a naturally-occurring or synthetic molecule. Conjugation of, or to produce, the antigen binding molecule or antigen binding domain of the invention to the naturally-occurring or synthetic molecule may be by any suitable method, for example recombinant engineering.
  • the naturally-occurring or synthetic molecule can be any molecule such as a drug targeting a tumour or tumour cells.
  • the naturally-occurring or synthetic molecule can also be a peptide/protein or an antibody or an antigen binding fragment of a different target affinity such that the resultant molecule has specificity for more than one antigen, wherein the resulting fusion protein can be produced by conventional recombinant protein expression systems and methods.
  • CARs are engineered receptors, which confer specificity for a desired antigen onto an immune effector cell, such as a T-cell.
  • CARs may be expressed on the extracellular surface of a cell. Expression may be by any suitable means via retroviral vector expression.
  • the most common form of these molecules are fusions of an antibody or antigen binding molecule or antigen binding fragment or domain, such as an scFv or VHH, to the transmembrane and intracellular domains of a native T-cell activation receptor, such as CD3, typically CD3-zeta.
  • ‘First-generation’ CARs typically have the intracellular domain from the CD3-zeta chain, which is the primary transmitter of signals from endogenous TCRs. ‘Second-generation’ CARs add intracellular signalling domains from various costimulatory protein receptors (e.g., CD28, CD137, 41 BB, ICOS) to the intracellular portion of the CAR to provide additional signals to the T cell (see Figure 1). Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells (Maher, et al. (2002) Nat. Biotechnol.
  • ‘Third-generation’ CARs combine multiple signalling domains, such as CD3z-CD28-41 BB or CD3z-CD28-OX40, to further augment potency (Zhao, et al. (2009) J. Immunol. 183:5563-5574; Pule, et al. (2005) Mol. Ther. 12:933- 941 ; Zhong, et al. (2010) Mol. Ther. 18:413-420).
  • a CAR is provided that is or comprises an anti-BCMA antigen-binding molecule or antibody or antigen binding fragment or domain, such as a VHH fragment, as defined in SEQ ID NOs 3 to 5, 15 to 16, 66 to 71 , 108 to 111 , 136 to 139, and 164 to 172 or a scFv fragment, as defined in SEQ ID Nos 227 to 231 .
  • an anti-BCMA antigen-binding molecule or antibody or antigen binding fragment or domain such as a VHH fragment, as defined in SEQ ID NOs 3 to 5, 15 to 16, 66 to 71 , 108 to 111 , 136 to 139, and 164 to 172 or a scFv fragment, as defined in SEQ ID Nos 227 to 231 .
  • CARs of this invention can be prepared using standard recombinant protein techniques using sequences of CD3, e.g. CD247 (CD3-zeta) and optionally other costimulatory molecules known in the art.
  • CD247 (CD3-zeta) sequence is available under GENBANK accession number NP_932170
  • the human CD28 sequence is available under GENBANK accession number NP 006130
  • the human CD8A sequence is available under GENBANK accession number AAH25715
  • the human CD137 sequence is available under GENBANK accession number NP_001552.
  • the CARs of the present invention include, but are not necessarily limited to: a human CD247 (CD3-zeta) cytoplasmic domain (SEQ ID NO:28), a human CD137 co-stimulatory domain (SEQ ID NO:29); a human CD8A hinge/transmembrane domain (SEQ ID NQ:30); one or more antigen binding fragments or domains of the present invention (SEQ ID NOS: 3 to 5, 15 and 16, 66 to 71 , 108 to 111 , 136 to 139, 164 to 172 and 227 to 231)); and a CD8A extracellular signal peptide (SEQ ID NO: 33).
  • the CARs of the present invention are represented by the SEQ ID NOS: 23 to 27, 90 to 95, 124 to 127, 152 to 155, 200 to 208 and 262 to 266.
  • the CARs of the present invention have a single BCMA VHH antigen binding domain, with a sequence as defined in SEQ ID NOS: 3 to 5, 15, 16, 66 to 71 , 108 to 111 , 136 to 139, and 164 to 172.
  • the CARs of the present invention have a scFv BCMA antigen binding domain, with a sequence as defined in SEQ ID NOS: 227 to 231 .
  • the CARs as defined in SEQ ID NOS: 23 to 27, 90 to 95, 124 to 127, 152 to 155, 200 to 208 and 262 to 266 are encoded by DNA sequences represented by the SEQ ID NOS: 41 to 45, 102 to 107, 132 to 135, 160 to 163, 218 to 226, and 272 to 276.
  • the antigen binding molecule can be the product of conjugation to a naturally- occurring or synthetic molecule, or the antigen binding fragment or domain can be conjugated to a naturally-occurring or synthetic molecule that can confer specificity for one or more antigens in addition to BCMA.
  • the antigen binding molecule of the invention can be engineered (e.g. as a bivalent diabody or a conjugated Fab dimer or trimer) to have specificity for BCMA and another tumour antigen, e.g., an antigen associated with a disease or disorder as disclosed herein.
  • the antigen binding molecule can be engineered to have specificity for BCMA and an antigen that promotes activation or targeting of other cells, such as cytotoxic effector cells or T cells.
  • the invention also includes bispecific, tri-specific and multi-specific molecules such as BiTEs (bi-specific T-cell engagers) and DARTTMs (dual affinity retargeting reagents).
  • a bi-specific T-cell engager refers to a single polypeptide chain molecule having two antigen binding domains, one of which binds to a T-cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell (WO 05/061547; Baeuerle, et al. (2008) Drugs of the Future 33:137-147; Bargou, et al. (2008) Science 321 :974-977).
  • a T-cell antigen e.g., CD3
  • BiTE antibodies have been constructed to various target antigens including CD19, EpCAM, Her2/neu, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or HMW-MAA) (Baeuerle, et al. (2009) Curr. Opin. Mol. Ther. 1 1 :22-30).
  • an anti-BCMA antigen binding molecule or antigen binding domain e.g. a scFv or VHH
  • BiTE bi-specific T-cell engager
  • the bi-specific T-cell engager (BiTE) of this invention is composed of an anti- BCMA antigen binding molecule or antigen binding fragment or domain and an anti-CD3 antibody fragment fused together by a linker, e.g., a flexible protein linker. See, for example, US 5,929,212.
  • a linker e.g., a flexible protein linker.
  • the term ‘BiTE’ as used herein may encompass but is not limited to the bispecific T-cell engager named BiTE® available from Amgen®.
  • the antibody fragment that specifically binds CD3 comprises:
  • the invention provides the antigen binding molecule or the antigen binding fragment or domain of the invention coupled to a naturally-occurring or synthetic molecule as defined herein to provide a DARTTM.
  • DARTTM refers to an immunoglobulin molecule that includes at least two polypeptide chains that associate (especially through a covalent interaction) to form at least two antigen binding sites, which may recognize the same or different antigens.
  • Each of the polypeptide chains of a DARTTM include a VHH heavy chain region, or an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope binding site.
  • the immunoglobulin heavy chain variable region of one (e.g., the first) of the DARTTM polypeptide chains interacts with the immunoglobulin light chain variable region of a different (e.g. the second) DARTTM polypeptide chain to form an epitope binding site.
  • the immunoglobulin light chain variable region of one (e.g. the first) of the DARTTM polypeptide chains interacts with the immunoglobulin heavy chain variable region of a different (e.g., the second) DARTTM polypeptide chain to form an epitope binding site.
  • DARTTMs may be monospecific, bi-specific, tri-specific, etc., thus being able to simultaneously bind one, two, three or more different antigens (which may be of the same or of different antigens).
  • DARTTMs may additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., thus being able to simultaneously bind one, two, three, four, five, six or more molecules.
  • These two attributes of DARTTMs i.e., degree of specificity and valency may be combined, for example to produce bispecific antibodies (i.e., capable of binding two epitopes) that are tetravalent (i.e. capable of binding four sets of antigens/epitopes), etc.
  • the anti-BCMA antigen binding molecule is a DARTTM or the antigen binding fragment or domain is included in a DARTTM.
  • the antigen binding molecule or antigen binding fragment or domain of the present invention is conjugated, linked or joined to other naturally occurring or synthetic molecules including therapeutic agents (or ‘payloads’) such as cytotoxic, cytostatic, or anti-angiogenic agents and radioisotopes, labels or nanoparticles.
  • therapeutic agents or ‘payloads’
  • Such molecules are terms antibody-drug conjugates or ADCs.
  • ADCs include: Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Trastuzumab emtansine and Trastuzumab emtansine.
  • the invention provides a method of inhibiting or killing cells that express BCMA (BCMA cells) by contacting the cells with an antigen binding molecule, antigen binding fragment or domain or antibody, antigen-binding molecule or antigen binding domain conjugated to a naturally-occurring or synthetic molecule (e.g. a CAR, a bispecific T-cell engager such as a BiTE, DARTTM, or ADC), all of which are aspects of the invention.
  • the method can be used to inhibit BCMA cells in vitro or in a subject (i.e., in vivo).
  • the contacted BCMA cells can be in, for example, a cell culture or animal model of a disorder associated with aberrant expression or levels of BCMA.
  • the method is useful, for example, to measure and/or rank (relative to another antibody or antigen binding molecule) the antigen binding molecule's inhibitory activity for a BCMA cell type.
  • Inhibiting BCMA cells can include blocking or reducing the activity or growth of BCMA. Inhibiting can also include the killing of BCMA.
  • Cytotoxicity of an antigen binding molecule, antibody, antigen binding fragment, antigen binding domain or antigen binding molecule of antigen binding domain conjugated to a naturally-occurring or synthetic molecule, such as a CAR, BiTE, DARTTM, or ADC ofthe invention can be assessed using any conventional assay including, e.g.
  • the invention provides an antigen binding molecule, antigen binding fragment or domain or antibody conjugated to, or as a product of conjugation of, a naturally-occurring or synthetic molecule (e.g. a bispecific T-cell engager such as a CAR, BiTE, DARTTM, or ADC) of the invention for use in inhibiting and killing cells expressing BCMA.
  • a naturally-occurring or synthetic molecule e.g. a bispecific T-cell engager such as a CAR, BiTE, DARTTM, or ADC
  • the invention also provides a method of treating a subject that has, is suspected to have, or is at risk for a disorder associated with aberrant levels of BCMA cells.
  • the term ‘aberrant’ is intended to include increased or decreased BCMA expression on a cell as compared to expression of BCMA in normal or healthy cells, or an increase in cells expressing BCMA.
  • the method of treatment includes administering a therapeutically effective amount of an isolated antigen binding molecule, antibody, antigen binding fragment or domain, or fusion protein of the invention to the subject.
  • the antibody can be any anti-BCMA antigen binding molecule or antibody, antigen binding fragment or domain (e.g.
  • a bispecific T-cell engager such as a CAR, BiTE, DARTTM, or ADC
  • the invention provides an antibody, antigen binding fragment or antibody conjugated to, or a product of conjugation of, a naturally-occurring or synthetic molecule (e.g., a bispecific T-cell engager such as a CAR, BiTE, DARTTM, or ADC) of the invention for use in medicine, and for the treatment of disease and disorders associated with aberrant or elevated BCMA expression.
  • a naturally-occurring or synthetic molecule e.g., a bispecific T-cell engager such as a CAR, BiTE, DARTTM, or ADC
  • disorders that can be treated include, for example, cancers, such as Multiple myeloma (MM); Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
  • cancers such as Multiple myeloma (MM); Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
  • MM Multiple myeloma
  • the invention also provides a method of treating a subject that has, is suspected to have, or is at risk for a disorder associated with elevated levels of BCMA on a cell, or by elevated levels of BCMA cells by adoptive transfer of the recombinant host cells, e.g. T-cells described herein, which express an antigen binding molecule, antibody or antigen binding fragment or domain, optionally conjugated to, or as a product of conjugation to, a naturally-occurring or synthetic molecule of the invention as a CAR that selectively binds BCMA.
  • the recombinant host cells e.g. T-cells described herein, which express an antigen binding molecule, antibody or antigen binding fragment or domain, optionally conjugated to, or as a product of conjugation to, a naturally-occurring or synthetic molecule of the invention as a CAR that selectively binds BCMA.
  • the invention provides recombinant cells expressing an antigen binding molecule or antigen binding fragment or domain that is conjugated to, or a product of conjugation of, a naturally-occurring or synthetic molecule (e.g. CAR) of the invention for use in medicine.
  • a naturally-occurring or synthetic molecule e.g. CAR
  • Recombinant technology can be used to introduce CAR-encoding genetic material into any suitable T- cells, e.g. effector memory T-cells from the subject to be treated.
  • the recombinant T-cells are transferred, typically by infusion, to the patient.
  • the transferred T-cells of the invention can then mount an immune response against BCMA expressing cells (BCMA cells) in the patient.
  • BCMA cells BCMA expressing cells
  • the adoptive transfer method can be used, for example, to treat subjects that have or are suspected to have cancers, such as Multiple myeloma (MM); Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
  • cancers such as Multiple myeloma (MM); Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
  • SLE systemic
  • the invention also provides a pharmaceutical composition containing an antigen binding molecule or antigen binding fragment or domain or antibody conjugated to, or a product of conjugation of, a naturally-occurring or synthetic molecule (e.g., a CAR, a bispecific T-cell engager such as a BiTE, DARTTM, or ADC) as described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition can be prepared from any of the antigen binding molecules, antibody, antigen binding fragment or domain (e.g.
  • a naturally-occurring or synthetic molecule e.g., a CAR, a bispecific T-cell engager such as a BiTE, DARTTM, or ADC
  • composition of the invention can include a carrier for the antigen binding molecule or antigen binding fragment, or antigen binding molecule conjugated to, or a product of, conjugation of, a naturally- occurring or synthetic molecule, desirably a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances, which are suitable for administration into a human or veterinary patient (e.g. a physiologically acceptable carrier or a pharmacologically acceptable carrier).
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the pharmaceutically acceptable carrier can be comingled with one or more of the active components, e.g., a hybrid molecule, and with each other, when more than one pharmaceutically acceptable carrier is present in the composition in a manner so as not to substantially impair the desired pharmaceutical efficacy.
  • ‘Pharmaceutically acceptable’ materials typically are capable of administration to a patient without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset.
  • the pharmaceutical composition can contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • suitable buffering agents including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • suitable preservatives such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
  • the pharmaceutical composition can be presented in unit dosage form and can be prepared by any suitable method, many of which are well-known in the pharmaceutical arts. Such methods include the step of bringing the antigen binding molecule of the invention into association with a carrier that constitutes one or more accessory ingredients.
  • the composition is prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • VHH human single domain heavy chain phage library
  • R&D BiosystemsTM recombinant human BCMA protein
  • each library was grown to log phase, and then rescued with M13KO7 helper phage (Antibody Design LabTM, PH010L) before being amplified overnight at 32°C in a shaker.
  • M13KO7 helper phage Antibody Design LabTM, PH010L
  • Each phage library was subsequently precipitated with PEG/NaCI, re-suspended in PBS and stored at -80°C.
  • Protein G coated magnetic beads were coated with BCMA-Fc or Fc recombinant protein in PBS and subsequently blocked in PBS + BSA. Phage particles were incubated for 30 minutes with negative magnetic particles (Fc protein).
  • VHH sequences were PCR amplified from the eluted phages or the isolated plasmids using Q5 DNA polymerase (NEB) and VHH-specific forward and reverse primers.
  • Forward and reverse primers contained overhang sequences, including protein linker sequences and Esp3l restriction sites.
  • Resulting amplicons were cleaned by PCR clean-up and PCR amplicons were directly used as inserts for golden gate assembly.
  • Golden gate assembly was performed with NEB Golden Gate kit and incubated, cycling between 16C and 42C for 40x cycles.
  • the resulting CAR-library was electroporated into electrocompetent bacteria and grown overnight at 30°C, followed by plasmid maxi-prep isolation.
  • the CAR sequences from the resulting plasmid library were briefly PCR amplified and sequenced on an Oxford Nanopore TechnologyTM MinlONTM, using the amplicon sequencing kit.
  • the CAR library was used to produce lentiviral vector particles and was then transduced into human T cells or Jurkat cell line.
  • the resulting CAR-T cell library was expanded for four more days after transduction. Transduction efficiency was assessed by flow cytometry through staining with APC-conjugated anti- CD34 and transduced cells were enriched through CD34 microbeads (MiltenyiTM). Enriched cells were then assessed for BCMA binding.
  • BCMA CAR-T cell library cells were stained with BCMA-Fc fusion protein (recombinant Fc, R&D systemsTM).
  • cells were stained with APC-conjugated anti-Fc and Pacific Blue-conjugated anti-CD34 antibody to detect transduced cells.
  • the expression of CARs on the cell surface was assessed via flow cytometry.
  • BCMA-positive cells were sorted by flow cytometry using a SH800S cell sorter (Sony Biotechnology). Genomic DNA was isolated and PCR amplicons were sequenced using Illumina NextSeqI OOO.
  • a CAR-T cell library was produced by activation of PBMCs with TransActTM (MiltenyiTM) in the presence of human IL-2 (100 lU/ml) and transduced at day two with the lentiviral BCMA CAR library. The next day, cells were washed and further expanded until day 8 of the process. Transduction was assessed by flow cytometry and transduced cells were enriched through CD34 microbeads (MiltenyiTM). Purity was assessed by flow cytometry.
  • the CAR-T-cell library was co-cultured with BCMA-positive (BCMA- expressing) cells (K562 cells genetically engineered to express BCMA or RPMI8226 cell line) or K562 wild-type cells.
  • CAR-T cells were prepared for single cell sequencing analysis.
  • CAR-T cell libraries from different donors were prepared for 10x genomics single cell gene expression analysis (1 Ox genomics 3’ sequencing kit V3 or Single Cell 5’ Kit v2) and sequenced on a NovaSeqTM 6000 (IlluminaTM).
  • Cell rangerTM software (10x GenomicsTM) was used for downstream processing and alignment of reads.
  • CAR sequences were amplified using long PCR with a forward read 1 primer and CAR-specific reverse primer. After 10 cycles of PCR, the product was cleaned using SPRI bead clean-up and a second, nested PCR was performed. PCR products were barcoded and an Oxford NanoporeTM library was prepared using the Oxford Nanopore ligation sequencing kit. Libraries were then sequenced on a MinlON® flow cell and CAR sequences and 10x cell barcodes were determined.
  • Single CAR constructs were selected based on best activation propensity from the single cell gene expression analysis, and CAR sequences were synthesised by Integrated DNA technologiesTM (IDTTM). Primary T cells expressing the different CAR constructs were generated and used for functional assays.
  • IDTTTM Integrated DNA technologiesTM
  • Comparative antibodies 6 to 9 are prior art BCMA CARs (SEQ ID NOS: 56 to 59, encoded by DNA sequences represented by the SEQ ID NOS: 61 to 64). Comparative antibody 6 is used as an “antibody targeting B-cell maturation antigen and methods of use (US201801 18842A1). Comparative antibody 6 is used as an “antibody targeting B-cell maturation antigen and methods of use (US20180118842A1). Comparative antibody 7 is obtained from US20170281766A1 . Comparative antibody 8 has been demonstrated in a “method for treating tumor using immune effector cell (W02020020210A1)”. Comparative antibody 9 has been demonstrated use in cancer chemotherapy in WO2018028647A1 .
  • Comparative antibodies 6 to 8 comprise ScFv antigen binding fragments (SEQ ID NOS: 46-48 respectively, encoded by the DNA SEQ ID NOS: 51 -53).
  • Comparative antibody 9 comprises a dual VHH antigen binding fragment (SEQ ID NO: 49, encoded by the DNA SEQ ID NO: 54).
  • the CAR labelled CD19 (SEQ ID NO: 60, encoded by DNA SEQ ID NO: 65) relates to a CAR with a CD19 antigen binding domain and acts as a negative control.
  • CD19 CAR has been shown efficacy in “Biomarkers predictive of therapeutic responsiveness to chimeric antigen receptor therapy and uses thereof’ (W02016057705A1). CD19 is used as a negative control in these BCMA assays.
  • the activation level of the BCMA CAR T cells was assessed by quantifying the percentage change in GFP expression in Jurkat NFAT_reporter cell line upon exposure to the BCMA-expressing cells for 24 hours. The activation was calculated by comparing the GFP fluorescence before the exposure to the BCMA-expressing cells against the GFP fluorescence following 24 h exposure to the BCMA-expressing cells.
  • each of the CARs showed similar activation level against BCMA compared to the prior art CARs.
  • the activation level of the BCMA CAR T cells transduced with BCMA CAR 10-15 (SEQ ID NOs 90 to 95) ( Figure 13) or 24 to 32 (SEQ ID NOs 200 to 208) ( Figure 19) are comparable to that of the comparative antibody 9
  • BCMA CAR T cells transduced with CAR 16 to 19 constructs (SEQ ID NOs 124 to 127) are comparable to that of the BCMA CAR-3 transduced T cells ( Figure 15)
  • BCMA CAR T cells transduced with CAR 20 to 23 are comparable to that of the BCMA CAR-5 transduced T cells ( Figure 17).
  • %GFP+ denotes percentage activation in a NFAT_GFP reporter cell line.
  • %AGFP percentage delta GFP
  • Figure 2 shows both (specific plus non-specific activation); whereas Figure 13 is the value of specific activation minus tonic activation.
  • the affinity/avidity of the BCMA CAR T cells against BCMA was assessed by quantifying the median fluorescence intensity from PE (phycoerythrin) conjugated anti-Fc that detects BCMA bound to BCMA CAR-T cells transduced with CAR constructs.
  • PE phytoerythrin conjugated anti-Fc conjugated anti-Fc that detects BCMA bound to BCMA CAR-T cells transduced with CAR constructs.
  • the BCMA CAR-T cells transduced with different CAR constructs were stained with BCMA-Fc.
  • the cells were stained with PE-conjugated anti-Fc.
  • APC conjugated anti-CD34 antibodies The median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
  • BCMA CAR-T cells transduced with CAR-1 through CAR-5 constructs exhibit a degree of BCMA binding comparable to comparative antibodies 6 to 9.
  • BCMA CAR-T cells transduced with CAR- 10 to CAR-15 constructs demonstrate a comparable or higher degree of BCMA binding (Figure 14), while those transduced with CAR-16 through CAR-19 constructs show a significantly higher degree of BCMA binding relative to the comparative antibodies 6 to 9 ( Figure 16).
  • BCMA CAR-T cells transduced with CAR-20 to CAR-23 ( Figure 18) and CAR-24 to CAR-32 ( Figure 20) demonstrate a comparable or higher degree of BCMA binding.
  • CAR-T cell activation was assessed via upregulation of activation markers, such as CD69 and CD25 on CD3+, CD34+ CAR-T cells after a 24h co-culture with RPMI8226 cells.
  • activation markers such as CD69 and CD25 on CD3+, CD34+ CAR-T cells
  • CAR-T cells were cultured in the absence of target cells for 24h. After 24h cells were assessed for activation markers via flow cytometry (Figure 8).
  • the supernatant of the cell co-cultures was used for IFNg ELISA assay. IFNg was quantified using human IFN-gamma and IL-2 Legendplex (Biolegend) ( Figure 6, Figure 7, Figure 11).
  • Figure 8 shows cells transduced with BCMA CAR-1 to BCMA CAR-5 constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later cells were assessed by flow cytometer for the up-regulation of CD25 and CD69 markers. It demonstrates that BCMA CAR-1 to BCMA CAR-5 demonstrate comparable CD25 and CD69 upregulation compared to that of the prior art CARs 6, 7 and 9.
  • Figure 6 demonstrates that BCMA CAR T cells transduced with BCMA CAR-1 to CAR-5 constructs produce comparable IFN-gamma level to that of the comparative antibodies 6,7 and 9.
  • Figure 7 illustrates that BCMA CAR T cells transduced with BCMA CAR-1 or CAR-3 produce comparable IL-2 level to that of the comparative antibody 9, and higher than that of the comparative antibody 8.
  • FIG 11 illustrates that BCMA CAR T cells transduced with BCMA CAR-10 to BCMA CAR-15 constructs produce higher or comparable IFN-gamma to that of the comparative antibody 9.
  • BCMA-CAR T-cells were evaluated for cellular toxicity and activity against cancer cells.
  • BCMA CAR T cells transduced with BCMA CAR-1 to CAR-5 constructs demonstrate a higher cell killing level relative to that of the comparative antibody 8, and similar level to that of the comparative antibodies 6,7 and 9 (Figure 4).
  • the killing percentage of BCMA CAR T cells for with each CAR construct was calculated relative to the killing percentage observed in RPMI8226 cells cultured with nontransduced T cells.
  • BCMA CAR T cells transduced with BCMA CAR-1 to CAR-5 constructs demonstrate a higher cell killing level relative to that of the comparative antibody 8 and comparative antibody CD19 CAR, and similar level to that of the comparative antibodies 6,7 and 9 (Figure 5).
  • the killing percentage of BCMA CAR T cells with each CAR construct was calculated relative to the killing percentage observed in K562_BCMA cells cultured with non-transduced T cells.
  • BCMA CAR T cells transduced with BCMA CAR-10 to BCMA-15 constructs demonstrate a comparable cell killing level relative to that of the comparative antibody 9 (Figure 9).
  • the killing percentage of BCMA CAR T cells with each CAR construct was calculated relative to the killing percentage observed in Nalm-6 cells cultured with non-transduced T cells.
  • BCMA CAR T cells transduced with BCMA CAR-10 to BCMA-15 constructs demonstrate a comparable cell killing level relative to that of the comparative antibody 9 (Figure 10).
  • the killing percentage of BCMA CAR T cells with each CAR construct was calculated relative to the killing percentage observed in RPMI8226 cells cultured with non-transduced T cells.
  • BCMA CAR T cells transduced with BCMA CAR-10 to CAR-15 constructs demonstrate a higher level relative to that of the comparative antibody 9 ( Figure 12).
  • BCMA CAR-T cells transduced with BCMA CAR 13 demonstrate comparable cell killing level compared to that of the comparative antibody 9. The killing percentage was calculated relative to that of U266B1 cells cultured with non-transduced T cells.
  • CD19 CAR of Figures 1 to 8 is synonymous (mans the same as) “negative control” in Figures 9 to 14, 19 and 20.
  • each of the BCMA CARs 1 to 5 (corresponding to CAR SEQ ID NOs 23 to 27 comprising the VHHs SEQ ID NOs 3, 4, 5, 15, 16) and each of the CARs 10-15 (corresponding to SEQ ID NOs 90 to 95 comprising the VHHs SEQ ID Nos 66 to 71), and the associated CDRs, as detailed in Table 1 , showed enhanced activity against BCMA-expressing cells than the prior art CARs with regards to anti-cancer killing activity.

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Abstract

The present disclosure relates to a novel class of BCMA (B cell maturation antigen, TNFRSF17) binding molecules, in particular to anti-BCMA antibodies and other BCMA binding molecules, and antigen binding fragments. The present invention further relates to recombinant molecules comprising said BCMA binding molecules, such as, chimeric antigen receptors (CARs), BiTEs and ADCs. Another object of the present invention is an engineered immune cell, suitably, a T cell comprising said CAR. Uses thereof in therapeutic methods of said BCMA binding molecule, said recombinant molecule and said engineered immune cell are further described.

Description

BCMA BINDING MOLECULES
FIELD OF THE INVENTION
The present invention relates to BCMA (B cell maturation antigen, TNFRSF17) binding molecules, in particular to anti-BCMA antibodies and other BCMA binding molecules, and antigen binding fragments. The present invention further relates to recombinant molecules comprising said BCMA binding molecules, such as, chimeric antigen receptors (CARs), BiTEs and ADCs. Another object of the present invention is an engineered immune cell, suitably, a T cell comprising said CAR. Uses thereof in therapeutic methods of said BCMA binding molecule, said recombinant molecule and said engineered immune cell are further described.
BACKGROUND OF THE INVENTION
The B cell maturation antigen (BCMA) glycoprotein (also known as BCMA, BCM, CD269, TNFRSF17) is a cell membrane type III non-tyrosine kinase receptor glycoprotein comprising an extracellular ligandbinding domain containing a single six-cysteine motif, a transmembrane domain, and a cytoplasmic tail that associates with TNF Receptor-Associated Factor (TRAF) 1 , TRAF2 and TRAF3. (https://www.uniprot.org/uniprotkb/Q02223/entry; Hatzoglou et al, 2000). TNF Receptor Family member BCMA (B Cell Maturation) Associates with TNF Receptor-Associated Factor (TRAF) 1 , TRAF2 and TRAF3 and Activates NF-KB, Elk-1 , c-Jun N-Terminal Kinase, and p38 Mitogen-Activated Protein Kinase. J. Immunol., 165(3): 1322-1330.).
BCMA is a member of the tumour necrosis factor receptor family and is highly expressed in mature B- lymphocytes and plasma cells. The N-terminal six-cysteine motif is a common characteristic of all TNFR family members that is repeated in all other members of the TNFR family but is present only once in the BCMA extracellular structure. BCMA promotes B-cell and plasma cell survival through its interaction with A Proliferations-lnducing Ligand (APRIL) and B-cell activating factor (BAFF) and subsequent activation of the nuclear factor kappa-B (NF-KB) and c-Jun N-Terminal Kinase (JNK) signalling cascades.
Membrane-bound BCMA is directly cleaved from the membrane by y-secretase, and elevated levels of sBCMA are used as a biomarker for Multiple Myeloma (MM), where sBCMA acts as a decoy reducing activation of membrane-bound BCMA by APRIL and BAFF.
MM typically affects middle aged and older adults and represents approximately 1 .8% of new cancer cases every year in the USA (Cancer Stat Facts: Myeloma. Available online: https://seer.cancer.gov/statfacts/html/mulmy.html (accessed on 20 September 2023). A heterogenous disease, MM-affected patients may experience a variety of disease-related symptoms, and the current 5-year survival rate for MM is approximately 62% (Cancer Stat Facts: Myeloma. Most recent data available: 2015. Available online: https://seer.cancer.gov/statfacts/html/mulmy.html (accessed on 20 September 2023). Currently, MM is not considered curable. Relapse is often inevitable, leading to relapsed/refractory MM (RRMM) with a progressively worse prognosis (Kumar et al, 2017. “Multiple Myeloma”. Nat. Rev. Dis. Primers., 3:17046).
BCMA is also implicated with other cancers and diseases including but not limited to: MM; Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
Therapeutic treatments that make use of the immune system or immune cells are known as immunotherapy treatments. In some cases, immunotherapy treatment relies on antibodies to an antigen on a target cell triggering or promoting cell death or inhibiting cell-division. Antigens may be targeted using antibody or antigen binding fragments, such as a single chain variable fragment (scFv). Examples of antibodies that target BCMA have been previously described e.g. U.S. Patent No. 9273141 B2. The antigen binding molecule or antigen binding domain can be linked or conjugated to (or be a product of conjugation of) a naturally-occurring or synthetic molecule, such as a naturally-occurring or synthetic molecule that binds to a T-cell antigen, suitably via a flexible linker, such as a flexible peptide linker to form a bi-specific T-cell engager (BiTE), or a dual-affinity retargeting antibody (DART) or other bispecific, tri-specific or multi-specific T-cell engagers.
Alternatively, such antibodies or antigen binding fragments may be linked or conjugated with (or be a product of conjugation of) a naturally-occurring or synthetic molecule such as a label, cytotoxic agent (antibody-drug conjugates or ADCs), or therapeutic isotope.
A specific type of immunotherapy is chimeric antigen receptor (CAR) T-cell therapy. CAR T-cell therapy consists of obtaining T-cells from a patient and incorporating an engineered receptor to recognise an antigen found on the cancerous cells. The resulting CAR T-cells are then incorporated back into the patient, where they may then act to mount an immune response against the antigen i.e. against the cancerous cells and kill the cancerous cells.
In this case, one or more antibodies or antigen binding fragments can be linked or conjugated with (or be a product of conjugation of) a naturally-occurring or synthetic molecule comprising other protein domains, such as hinge domains, transmembrane domains, co-stimulation domains and activation domains to form a CAR that is capable of effecting an immune response towards the antigen-containing cell. This CAR is then expressed in a T-cell.
Examples of CARs that target BCMA have been previously described e.g. International Patent No.
WO 2018/028647 A1 ; US Patent No. US 2020/0138865 A1 ; International Patent No. WO 2020/221873 A1.
A recent report found that BCMA-targeted CAR-T cell therapy was highly efficacious even in advanced MM, despite considerable observed toxicity (Roex et al, 2020. Safety and clinical efficacy of BCMA CAR-T-cell therapy in multiple myeloma. (J. Hematol. Oncol., 13:164) thereby highlighting emerging potential for use in MM and other cancers.
The nature of the binding between the CAR and the target antigen, and the associated activation of the T-cell as a result of this binding via other functional domains, is critical to the effectiveness of the CAR T-cell therapy.
Producing CARs that demonstrate high activation from low levels of BCMA is important due to the known variable level of BCMA expression MM, and thus the potential for antigen escape during immunotherapy treatment (Brudno et al, 2018. T Cells Genetically Modified to Express an Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor Cause Remissions of Poor-Prognosis Relapsed Multiple Myeloma. J Clinic. Oncol., 36(22): 2267-2280).
In addition to these known mechanisms of antigen escape, there is also evidence of CAR-driven acquired mutations that could reduce the long-term efficacy of CAR-T treatment (Song et al, 2019. Resistance Mechanisms to CAR T-Cell Therapy and Overcoming Strategy in B-Cell Hematologic Malignancies. Int. J. Mol. Sci., 20(20):5010). For CARs, for example, the combination of two VHHs directed against two different epitopes of the target antigen into a single CAR can be considered to mitigate the challenge of antigen escape through synergistic targeting of mutated and alternative target antigen structures.
Therefore, there exists a need for novel and improved antibodies or antigen binding fragments, suitable for treating BCMA-associated diseases and disorders.
SUMMARY OF THE INVENTION
In a first aspect, the invention provides an antigen binding molecule that binds BCMA; wherein the antigen binding molecule comprises an antigen binding domain selected from the group consisting of: Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89; and,
Antigen binding domain 1 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:6,
(ii) a CDR2 of SEQ ID NO:9, and
(iii) a CDR3 of SEQ ID NO:12; and,
Antigen binding domain 2, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:7,
(ii) a CDR2 of SEQ ID NQ:10, and
(iii) a CDR3 of SEQ ID NO:13; and,
Antigen binding domain 3, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:8,
(ii) a CDR2 of SEQ ID NO:11 , and
(iii) a CDR3 of SEQ ID NO:14; and,
Antigen binding domain 4, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:17,
(ii) a CDR2 of SEQ ID NO:19, and
(iii) a CDR3 of SEQ ID NO:21 ; and,
Antigen binding domain 5, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:18,
(ii) a CDR2 of SEQ ID NQ:20, and
(iii) a CDR3 of SEQ ID NO:22;and,
Antigen binding domain 10, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:72,
(ii) a CDR2 of SEQ ID NO:78, and
(iii) a CDR3 of SEQ ID NO:84; and,
Antigen binding domain 11 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:73,
(ii) a CDR2 of SEQ ID NO:79, and
(iii) a CDR3 of SEQ ID NO:85; and,
Antigen binding domain 13, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:75,
(ii) a CDR2 of SEQ ID NO:81 , and
(iii) a CDR3 of SEQ ID NO:87; and,
Antigen binding domain 16, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:112,
(ii) a CDR2 of SEQ ID NO:116, and (iii) a CDR3 of SEQ ID NO:120; and,
Antigen binding domain 17, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 13,
(ii) a CDR2 of SEQ ID NO:117, and
(iii) a CDR3 of SEQ ID NO:121 ; and,
Antigen binding domain 18, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:114,
(ii) a CDR2 of SEQ ID NO:118, and
(iii) a CDR3 of SEQ ID NO:122; and,
Antigen binding domain 19, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 15,
(ii) a CDR2 of SEQ ID NO:119, and
(iii) a CDR3 of SEQ ID NO:123; and,
Antigen binding domain 20, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:140,
(ii) a CDR2 of SEQ ID NO:144, and
(iii) a CDR3 of SEQ ID NO:148; and,
Antigen binding domain 21 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:141 ,
(ii) a CDR2 of SEQ ID NO:145, and
(iii) a CDR3 of SEQ ID NO:149; and,
Antigen binding domain 22, comprising a single variable domain on a heavy chain comprising, (i) a CDR1 of SEQ ID NO:142,
(ii) a CDR2 of SEQ ID NO:146, and
(Hi) a CDR3 of SEQ ID NO:150; and,
Antigen binding domain 23, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:143,
(ii) a CDR2 of SEQ ID NO:147, and
(iii) a CDR3 of SEQ ID NO:151 ; and,
Antigen binding domain 24, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:173,
(ii) a CDR2 of SEQ ID NO:182, and
(iii) a CDR3 of SEQ ID NO:191 ; and,
Antigen binding domain 25, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:174,
(ii) a CDR2 of SEQ ID NO:183, and
(iii) a CDR3 of SEQ ID NO:192; and,
Antigen binding domain 26, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:175,
(ii) a CDR2 of SEQ ID NO:184, and
(iii) a CDR3 of SEQ ID NO:193; and,
Antigen binding domain 27, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:176,
(ii) a CDR2 of SEQ ID NO:185, and
(iii) a CDR3 of SEQ ID NO:194; and, Antigen binding domain 28, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:177,
(ii) a CDR2 of SEQ ID NO:186, and
(iii) a CDR3 of SEQ ID NO:195; and,
Antigen binding domain 29, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:178,
(ii) a CDR2 of SEQ ID NO:187, and
(iii) a CDR3 of SEQ ID NO:196; and,
Antigen binding domain 30, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:179,
(ii) a CDR2 of SEQ ID NO:188, and
(iii) a CDR3 of SEQ ID NO:197; and,
Antigen binding domain 31 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:180,
(ii) a CDR2 of SEQ ID NO:189, and
(iii) a CDR3 of SEQ ID NO:198; and,
Antigen binding domain 32, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:181 ,
(ii) a CDR2 of SEQ ID NQ:190, and
(iii) a CDR3 of SEQ ID NO:199; and,
Antigen binding domain 33, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:232, (ii) a CDR2 of SEQ ID NO:237, and
(iii) a CDR3 of SEQ ID NO:242; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:247,
(ii) a CDR2 of SEQ ID NO:252, and
(iii) a CDR3 of SEQ ID NO:257; and,
Antigen binding domain 34, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:233,
(ii) a CDR2 of SEQ ID NO:238, and
(iii) a CDR3 of SEQ ID NO:243, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:248,
(ii) a CDR2 of SEQ ID NO:253, and
(iii) a CDR3 of SEQ ID NO:258; and,
Antigen binding domain 35, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:234,
(ii) a CDR2 of SEQ ID NO:239,
(iii) a CDR3 of SEQ ID NO:244, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:249,
(ii) a CDR2 of SEQ ID NO:254, and
(iii) a CDR3 of SEQ ID NO:259; and,
Antigen binding domain 36, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:235,
(ii) a CDR2 of SEQ ID NQ:240, (iii) a CDR3 of SEQ ID NO:245, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:250,
(ii) a CDR2 of SEQ ID NO:255, and
(iii) a CDR3 of SEQ ID NQ:260; and,
Antigen binding domain 37, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:236,
(ii) a CDR2 of SEQ ID NO:241 ,
(iii) a CDR3 of SEQ ID NO:246, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:251 ,
(ii) a CDR2 of SEQ ID NO:256, and
(iii) a CDR3 of SEQ ID NO:261 .
In embodiments, the single variable domain on a heavy chain of any one of the antigen binding domainl to 5 and 10 to 32 is a VHH or wherein, the antigen binding domains 33 to 37 is a ScFv. Suitably, the VHH is selected from the group consisting of: SEQ ID NOs 3 to 5, SEQ ID NOs 15 to 16, SEQ ID NOs 66 to 71 , SEQ ID NOs 108 to 111 , SEQ ID NOs 136 to 139, and SEQ ID NOs 164 to 172, or the ScFv is selected from the group consisting of SEQ ID Nos 227 to 231 .
In embodiments, the antigen binding molecule that binds BCMA comprises antigen binding domain 1 , antigen binding domain 2, antigen binding domain 3, antigen binding domain 4, antigen binding domain 5, antigen binding domain 10, antigen binding domain 11 , antigen binding domain 12, antigen binding domain 13, antigen binding domain 14, antigen binding domain 15, antigen binding domain 16, antigen binding domain 17, antigen binding domain 18, antigen binding domain 19, antigen binding domain 20, antigen binding domain 21 , antigen binding domain 22, antigen binding domain 23, antigen binding domain 24, antigen binding domain 25, antigen binding domain 26, antigen binding domain 27, antigen binding domain 28, antigen binding domain 29, antigen binding domain 30, antigen binding domain 31 , antigen binding domain 32, antigen binding domain 33, antigen binding domain 34, antigen binding domain 35, antigen binding domain 36 or antigen binding domain 37.
In embodiments, the antigen binding molecule that binds BCMA is provided, wherein the antigen binding molecule comprises an antigen binding domain is selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and, Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NO:80, and
(iii) a CDR3 of SEQ ID NO:86; and
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) A CDR1 of SEQ ID NO:77,
(ii) A CDR2 of SEQ ID NO:83, and
(iii) A CDR3 of SEQ ID NO:89.
In embodiments, the antigen binding molecule is a chimeric antigen receptor comprising a transmembrane region and an intracellular T-cell receptor signalling domain. Suitably, the hinge region and the transmembrane region is from CD8A and/or the intracellular T-cell receptor signalling domain is from CD247 (CD3-zeta). Suitably, antigen binding molecule further comprises an intracellular domain of a costimulatory protein receptor.
In embodiments, the antigen binding molecule is a bi-specific T-cell engager (BiTE) comprising a T-cell antigen binding domain. Suitably, the T-cell antigen binding domain comprises an antibody fragment that specifically binds CD3.
In embodiments, the antigen binding molecule is an antibody-drug conjugate (ADC) comprising a cytotoxic drug or a therapeutic radioisotope.
In a second aspect, the invention provides a vector encoding the antigen binding molecule of the first aspect of the invention. Suitably, the vector comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NOs 41 to 45, 102 to 107, 132 to 135, 160 to 163, 218 to 226, and 272 to 276.
In a third aspect, the invention provides a pharmaceutical composition comprising the antigen binding molecule of the first aspect of the invention and a pharmaceutically acceptable carrier.
In fourth aspect, the invention provides a chimeric antigen receptor that binds BCMA comprising:
(a) one or more antigen binding domains selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and, Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NO:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89; and,
Antigen binding domain 1 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:6,
(ii) a CDR2 of SEQ ID NO:9, and
(iii) a CDR3 of SEQ ID NO:12; and,
Antigen binding domain 2, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:7,
(ii) a CDR2 of SEQ ID NQ:10, and
(iii) a CDR3 of SEQ ID NO:13; and,
Antigen binding domain 3, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:8,
(ii) a CDR2 of SEQ ID NO:11 , and
(iii) a CDR3 of SEQ ID NO:14; and,
Antigen binding domain 4, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:17,
(ii) a CDR2 of SEQ ID NO:19, and
(iii) a CDR3 of SEQ ID NO:21 ; and,
Antigen binding domain 5, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:18,
(ii) a CDR2 of SEQ ID NO:20, and
(iii) a CDR3 of SEQ ID NO:22; and,
Antigen binding domain 10, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:72,
(ii) a CDR2 of SEQ ID NO:78, and
(iii) a CDR3 of SEQ ID NO:84; and,
Antigen binding domain 11 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:73,
(ii) a CDR2 of SEQ ID NO:79, and
(iii) a CDR3 of SEQ ID NO:85; and,
Antigen binding domain 13, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:75,
(ii) a CDR2 of SEQ ID NO:81 , and
(iii) a CDR3 of SEQ ID NO:87; and,
Antigen binding domain 16, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 12,
(ii) a CDR2 of SEQ ID NO:116, and
(iii) a CDR3 of SEQ ID NQ:120; and,
Antigen binding domain 17, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 13,
(ii) a CDR2 of SEQ ID NO:117, and (iii) a CDR3 of SEQ ID NO:121 ; and,
Antigen binding domain 18, comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 14,
(ii) a CDR2 of SEQ ID NO:118, and
(iii) a CDR3 of SEQ ID NO:122; and,
Antigen binding domain 19, comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 15,
(ii) a CDR2 of SEQ ID NO:119, and
(iii) a CDR3 of SEQ ID NO:123; and,
Antigen binding domain 20, comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:140,
(ii) a CDR2 of SEQ ID NO:144, and
(iii) a CDR3 of SEQ ID NO:148; and,
Antigen binding domain 21 , comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NO:141 ,
(ii) a CDR2 of SEQ ID NO:145, and
(iii) a CDR3 of SEQ ID NO:149; and,
Antigen binding domain 22, comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NO:142,
(ii) a CDR2 of SEQ ID NO:146, and
(iii) a CDR3 of SEQ ID NQ:150; and,
Antigen binding domain 23, comprising a single variable domain on a heavy chain comprising, (i) a CDR1 of SEQ ID NO:143,
(ii) a CDR2 of SEQ ID NO:147, and
(Hi) a CDR3 of SEQ ID NO:151 ; and,
Antigen binding domain 24, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:173,
(ii) a CDR2 of SEQ ID NO:182, and
(iii) a CDR3 of SEQ ID NO:191 ; and,
Antigen binding domain 25, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:174,
(ii) a CDR2 of SEQ ID NO:183, and
(iii) a CDR3 of SEQ ID NO:192; and,
Antigen binding domain 26, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:175,
(ii) a CDR2 of SEQ ID NO:184, and
(iii) a CDR3 of SEQ ID NO:193; and,
Antigen binding domain 27, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:176,
(ii) a CDR2 of SEQ ID NO:185, and
(iii) a CDR3 of SEQ ID NO:194; and,
Antigen binding domain 28, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:177,
(ii) a CDR2 of SEQ ID NO:186, and
(iii) a CDR3 of SEQ ID NO:195; and, Antigen binding domain 29, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:178,
(ii) a CDR2 of SEQ ID NO:187, and
(iii) a CDR3 of SEQ ID NO:196; and,
Antigen binding domain 30, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:179,
(ii) a CDR2 of SEQ ID NO:188, and
(iii) a CDR3 of SEQ ID NO:197; and,
Antigen binding domain 31 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:180,
(ii) a CDR2 of SEQ ID NO:189, and
(iii) a CDR3 of SEQ ID NO:198; and,
Antigen binding domain 32, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:181 ,
(ii) a CDR2 of SEQ ID NQ:190, and
(iii) a CDR3 of SEQ ID NO:199; and,
Antigen binding domain 33, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:232,
(ii) a CDR2 of SEQ ID NO:237, and
(iii) a CDR3 of SEQ ID NO:242; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:247,
(ii) a CDR2 of SEQ ID NO:252, and
(iii) a CDR3 of SEQ ID NO:257; and,
Antigen binding domain 34, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:233,
(ii) a CDR2 of SEQ ID NO:238, and
(iii) a CDR3 of SEQ ID NO:243, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:248,
(ii) a CDR2 of SEQ ID NO:253, and
(iii) a CDR3 of SEQ ID NO:258; and,
Antigen binding domain 35, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:234,
(ii) a CDR2 of SEQ ID NO:239,
(iii) a CDR3 of SEQ ID NO:244, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:249,
(ii) a CDR2 of SEQ ID NO:254, and
(iii) a CDR3 of SEQ ID NO:259; and,
Antigen binding domain 36, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:235,
(ii) a CDR2 of SEQ ID NQ:240,
(iii) a CDR3 of SEQ ID NO:245, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NQ:250,
(ii) a CDR2 of SEQ ID NO:255, and
(iii) a CDR3 of SEQ ID NQ:260; and, Antigen binding domain 37, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:236,
(ii) a CDR2 of SEQ ID NO:241 ,
(iii) a CDR3 of SEQ ID NO:246, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:251 ,
(ii) a CDR2 of SEQ ID NO:256, and
(iii) a CDR3 of SEQ ID NO:261 , and b) A transmembrane region; and c) An intracellular signalling domain.
In embodiments, the chimeric antigen receptor that binds BCMA comprises antigen binding domain 1 , antigen binding domain 2, antigen binding domain 3, antigen binding domain 4, antigen binding domain 5, antigen binding domain 10, antigen binding domain 11 , antigen binding domain 12, antigen binding domain 13, antigen binding domain 14, antigen binding domain 15, antigen binding domain 16, antigen binding domain 17, antigen binding domain 18, antigen binding domain 19, antigen binding domain 20, antigen binding domain 21 , antigen binding domain 22, antigen binding domain 23, antigen binding domain 24, antigen binding domain 25, antigen binding domain 26, antigen binding domain 27, antigen binding domain 28, antigen binding domain 29, antigen binding domain 30, antigen binding domain 31 , antigen binding domain 32, antigen binding domain 33, antigen binding domain 34, antigen binding domain 35, antigen binding domain 36 or antigen binding domain 37.
In embodiments, the chimeric antigen receptor that binds BCMA comprises an antigen binding domain selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iv) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) A CDR1 of SEQ ID NO:77,
(ii) A CDR2 of SEQ ID NO:83, and
(iii) A CDR3 of SEQ ID NO:89.
In embodiments of the CAR of the fourth aspect, the transmembrane region is from CD8A or TNFRSF4, suitably CD8A, and/or the intracellular signalling domain is from CD247 (CD3-zeta). Suitably, the CAR further comprises an intracellular signalling domain of a costimulatory protein receptor.
In embodiments, the CAR comprises a human CD247 (CD3-zeta) cytoplasmic domain (SEQ ID NO:28), a human CD137 co-stimulatory domain (SEQ ID NO:29); a human CD8A hinge/transmembrane domain (SEQ ID NQ:30), the antigen binding molecule of the first aspect; and an CD8A extracellular signal peptide (SEQ ID NO:33).
The CAR may comprise two or more antigen binding domains, wherein at least one of the antigen binding domains is a BCMA antigen binding domain of the first aspect of the present invention. When two or more antigen binding domains are present they may be joined by at least one linker. Suitably, the linker has a sequence selected from the group consisting of: SEQ ID NOS: 31 and 32.
The CAR may be encoded by a nucleic acid sequence comprising two or more CAR sequences, joined by a sequence encoding a self-cleaving linker. Suitably, the self-cleaving linker is a T2A self-cleaving linker with a sequence of: SEQ ID NO 34.
In embodiments, the CAR has a sequence selected from the group consisting of: SEQ ID NOS: 23 to 29, 90 to 95, 124 to 127, 152 to 155, 200 to 208 and 262 to 266.
In a fifth aspect, the invention provides a recombinant T cell comprising the chimeric antigen receptor of the fourth aspect.
In a sixth aspect, the invention provides a bispecific T-cell engager that binds BCMA comprising:
(a) one or more antigen binding domains selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89; and,
Antigen binding domain 1 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:6,
(ii) a CDR2 of SEQ ID NO:9, and
(iii) a CDR3 of SEQ ID NO:12; and,
Antigen binding domain 2, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:7,
(ii) a CDR2 of SEQ ID NQ:10, and
(iii) a CDR3 of SEQ ID NO:13; and,
Antigen binding domain 3, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:8,
(ii) a CDR2 of SEQ ID NO:11 , and
(iii) a CDR3 of SEQ ID NO:14; and,
Antigen binding domain 4, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:17,
(ii) a CDR2 of SEQ ID NO:19, and
(iii) a CDR3 of SEQ ID NO:21 ; and,
Antigen binding domain 5, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:18, (ii) a CDR2 of SEQ ID NO:20, and
(iii) a CDR3 of SEQ ID NO:22; and,
Antigen binding domain 10, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:72,
(ii) a CDR2 of SEQ ID NO:78, and
(iii) a CDR3 of SEQ ID NO:84; and,
Antigen binding domain 11 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:73,
(ii) a CDR2 of SEQ ID NO:79, and
(iii) a CDR3 of SEQ ID NO:85; and,
Antigen binding domain 13, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:75,
(ii) a CDR2 of SEQ ID NO:81 , and
(iii) a CDR3 of SEQ ID NO:87; and,
Antigen binding domain 16, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 12,
(ii) a CDR2 of SEQ ID NO:116, and
(iii) a CDR3 of SEQ ID NQ:120; and,
Antigen binding domain 17, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 13,
(ii) a CDR2 of SEQ ID NO:117, and
(iii) a CDR3 of SEQ ID NO:121 ; and,
Antigen binding domain 18, comprising a single variable domain on a heavy chain comprising, (i) a CDR1 of SEQ ID NO:114,
(ii) a CDR2 of SEQ ID NO:118, and
(Hi) a CDR3 of SEQ ID NO:122; and,
Antigen binding domain 19, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:115,
(ii) a CDR2 of SEQ ID NO:119, and
(iii) a CDR3 of SEQ ID NO:123; and,
Antigen binding domain 20, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:140,
(ii) a CDR2 of SEQ ID NO:144, and
(iii) a CDR3 of SEQ ID NO:148; and,
Antigen binding domain 21 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:141 ,
(ii) a CDR2 of SEQ ID NO:145, and
(iii) a CDR3 of SEQ ID NO:149; and,
Antigen binding domain 22, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:142,
(ii) a CDR2 of SEQ ID NO:146, and
(iii) a CDR3 of SEQ ID NQ:150; and,
Antigen binding domain 23, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:143,
(ii) a CDR2 of SEQ ID NO:147, and
(iii) a CDR3 of SEQ ID NO:151 ; and, Antigen binding domain 24, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:173,
(ii) a CDR2 of SEQ ID NO:182, and
(iii) a CDR3 of SEQ ID NO:191 ; and,
Antigen binding domain 25, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:174,
(ii) a CDR2 of SEQ ID NO:183, and
(iii) a CDR3 of SEQ ID NO:192; and,
Antigen binding domain 26, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:175,
(ii) a CDR2 of SEQ ID NO:184, and
(iii) a CDR3 of SEQ ID NO:193; and,
Antigen binding domain 27, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:176,
(ii) a CDR2 of SEQ ID NO:185, and
(iii) a CDR3 of SEQ ID NO:194; and,
Antigen binding domain 28, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:177,
(ii) a CDR2 of SEQ ID NO:186, and
(iii) a CDR3 of SEQ ID NO:195; and,
Antigen binding domain 29, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:178,
(ii) a CDR2 of SEQ ID NO:187, and (iii) a CDR3 of SEQ ID NO:196; and,
Antigen binding domain 30, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:179,
(ii) a CDR2 of SEQ ID NO:188, and
(iii) a CDR3 of SEQ ID NO:197; and,
Antigen binding domain 31 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:180,
(ii) a CDR2 of SEQ ID NO:189, and
(iii) a CDR3 of SEQ ID NO:198; and,
Antigen binding domain 32, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:181 ,
(ii) a CDR2 of SEQ ID NQ:190, and
(iii) a CDR3 of SEQ ID NO:199; and,
Antigen binding domain 33, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:232,
(ii) a CDR2 of SEQ ID NO:237, and
(iii) a CDR3 of SEQ ID NO:242; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:247,
(ii) a CDR2 of SEQ ID NO:252, and
(iii) a CDR3 of SEQ ID NO:257; and,
Antigen binding domain 34, comprising:
(a) a heavy chain variable region comprising, (i) a CDR1 of SEQ ID NO:233,
(ii) a CDR2 of SEQ ID NO:238, and
(Hi) a CDR3 of SEQ ID NO:243, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:248,
(ii) a CDR2 of SEQ ID NO:253, and
(iii) a CDR3 of SEQ ID NO:258; and,
Antigen binding domain 35, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:234,
(ii) a CDR2 of SEQ ID NO:239,
(iii) a CDR3 of SEQ ID NO:244, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:249,
(ii) a CDR2 of SEQ ID NO:254, and
(iii) a CDR3 of SEQ ID NO:259; and,
Antigen binding domain 36, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:235,
(ii) a CDR2 of SEQ ID NQ:240,
(iii) a CDR3 of SEQ ID NO:245, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NQ:250,
(ii) a CDR2 of SEQ ID NO:255, and
(iii) a CDR3 of SEQ ID NQ:260; and,
Antigen binding domain 37, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:236, (ii) a CDR2 of SEQ ID NO:241 ,
(iii) a CDR3 of SEQ ID NO:246, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:251 ,
(ii) a CDR2 of SEQ ID NO:256, and
(iii) a CDR3 of SEQ ID NO:261 , and b) An antigen binding domain which binds to a T-cell antigen; and c) A linker joining the one or more antigen binding domains of (a) and antigen binding domain which binds to a T-cell antigen of (b).
In embodiments, the bispecific T-cell engager that binds BCMA comprises antigen binding domain 1 , antigen binding domain 2, antigen binding domain 3, antigen binding domain 4, antigen binding domain 5, antigen binding domain 10, antigen binding domain 11 , antigen binding domain 12, antigen binding domain 13, antigen binding domain 14, antigen binding domain 15, antigen binding domain 16, antigen binding domain 17, antigen binding domain 18, antigen binding domain 19, antigen binding domain 20, antigen binding domain 21 , antigen binding domain 22, antigen binding domain 23, antigen binding domain 24, antigen binding domain 25, antigen binding domain 26, antigen binding domain 27, antigen binding domain 28, antigen binding domain 29, antigen binding domain 30, antigen binding domain 31 , antigen binding domain 32, antigen binding domain 33, antigen binding domain 34, antigen binding domain 35, antigen binding domain 36 or antigen binding domain 37.
In embodiments, the bispecific T-cell engager that binds BCMA comprises an antigen binding domain selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and (iii) a CDR3 of SEQ ID NO:89.
In embodiments, the antigen binding domain which binds to a T-cell antigen comprises an antigen binding fragment that specifically binds CD3. Suitably, the antigen binding fragment that specifically binds CD3 comprises:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:35,
(ii) a CDR2 of SEQ ID NO:36, and
(iii) a CDR3 of SEQ ID NO:37; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:38,
(ii) a CDR2 of SEQ ID NO:39, and
(iii) a CDR3 of SEQ ID NQ:40.
In a seventh aspect, the invention provides an antibody-drug conjugate that binds BCMA comprising:
(a) one or more antigen binding domains selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89; and, Antigen binding domain 1 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:6,
(ii) a CDR2 of SEQ ID NO:9, and
(iii) a CDR3 of SEQ ID NO:12; and,
Antigen binding domain 2, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:7,
(ii) a CDR2 of SEQ ID NQ:10, and
(iii) a CDR3 of SEQ ID NO:13; and,
Antigen binding domain 3, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:8,
(ii) a CDR2 of SEQ ID NO:11 , and
(iii) a CDR3 of SEQ ID NO:14; and,
Antigen binding domain 4, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:17,
(ii) a CDR2 of SEQ ID NO:19, and
(iii) a CDR3 of SEQ ID NO:21 ; and,
Antigen binding domain 5, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:18,
(ii) a CDR2 of SEQ ID NQ:20, and
(iii) a CDR3 of SEQ ID NO:22; and,
Antigen binding domain 10, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:72,
(ii) a CDR2 of SEQ ID NO:78, and (iii) a CDR3 of SEQ ID NO:84; and,
Antigen binding domain 11 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:73,
(ii) a CDR2 of SEQ ID NO:79, and
(iii) a CDR3 of SEQ ID NO:85; and,
Antigen binding domain 13, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:75,
(ii) a CDR2 of SEQ ID NO:81 , and
(iii) a CDR3 of SEQ ID NO:87; and,
Antigen binding domain 16, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 12,
(ii) a CDR2 of SEQ ID NO:116, and
(iii) a CDR3 of SEQ ID NQ:120; and,
Antigen binding domain 17, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 13,
(ii) a CDR2 of SEQ ID NO:117, and
(iii) a CDR3 of SEQ ID NO:121 ; and,
Antigen binding domain 18, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 14,
(ii) a CDR2 of SEQ ID NO:118, and
(iii) a CDR3 of SEQ ID NO:122; and,
Antigen binding domain 19, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 15, (ii) a CDR2 of SEQ ID NO:119, and
(iii) a CDR3 of SEQ ID NO:123; and,
Antigen binding domain 20, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:140,
(ii) a CDR2 of SEQ ID NO:144, and
(iii) a CDR3 of SEQ ID NO:148; and,
Antigen binding domain 21 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:141 ,
(ii) a CDR2 of SEQ ID NO:145, and
(iii) a CDR3 of SEQ ID NO:149; and,
Antigen binding domain 22, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:142,
(ii) a CDR2 of SEQ ID NO:146, and
(iii) a CDR3 of SEQ ID NQ:150; and,
Antigen binding domain 23, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:143,
(ii) a CDR2 of SEQ ID NO:147, and
(iii) a CDR3 of SEQ ID NO:151 and,
Antigen binding domain 24, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:173,
(ii) a CDR2 of SEQ ID NO:182, and
(iii) a CDR3 of SEQ ID NO:191 ; and, Antigen binding domain 25, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:174,
(ii) a CDR2 of SEQ ID NO:183, and
(iii) a CDR3 of SEQ ID NO:192; and,
Antigen binding domain 26, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:175,
(ii) a CDR2 of SEQ ID NO:184, and
(iii) a CDR3 of SEQ ID NO:193; and,
Antigen binding domain 27, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:176,
(ii) a CDR2 of SEQ ID NO:185, and
(iii) a CDR3 of SEQ ID NO:194; and,
Antigen binding domain 28, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:177,
(ii) a CDR2 of SEQ ID NO:186, and
(iii) a CDR3 of SEQ ID NO:195; and,
Antigen binding domain 29, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:178,
(ii) a CDR2 of SEQ ID NO:187, and
(iii) a CDR3 of SEQ ID NO:196; and,
Antigen binding domain 30, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:179,
(ii) a CDR2 of SEQ ID NO:188, and
(iii) a CDR3 of SEQ ID NO:197; and,
Antigen binding domain 31 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:180,
(ii) a CDR2 of SEQ ID NO:189, and
(iii) a CDR3 of SEQ ID NO:198; and,
Antigen binding domain 32, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:181 ,
(ii) a CDR2 of SEQ ID NQ:190, and
(iii) a CDR3 of SEQ ID NO:199; and,
Antigen binding domain 33, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:232,
(ii) a CDR2 of SEQ ID NO:237, and
(iii) a CDR3 of SEQ ID NO:242; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:247,
(ii) a CDR2 of SEQ ID NO:252, and
(iii) a CDR3 of SEQ ID NO:257; and,
Antigen binding domain 34, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:233,
(ii) a CDR2 of SEQ ID NO:238, and
(iii) a CDR3 of SEQ ID NO:243, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:248,
(ii) a CDR2 of SEQ ID NO:253, and
(iii) a CDR3 of SEQ ID NO:258; and,
Antigen binding domain 35, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:234,
(ii) a CDR2 of SEQ ID NO:239,
(iii) a CDR3 of SEQ ID NO:244, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:249,
(ii) a CDR2 of SEQ ID NO:254, and
(iii) a CDR3 of SEQ ID NO:259; and,
Antigen binding domain 36, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:235,
(ii) a CDR2 of SEQ ID NQ:240,
(iii) a CDR3 of SEQ ID NO:245, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NQ:250,
(ii) a CDR2 of SEQ ID NO:255, and
(iii) a CDR3 of SEQ ID NQ:260; and,
Antigen binding domain 37, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:236,
(ii) a CDR2 of SEQ ID NO:241 ,
(iii) a CDR3 of SEQ ID NO:246, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:251 ,
(ii) a CDR2 of SEQ ID NO:256, and
(iii) a CDR3 of SEQ ID NO:261 , and b) A therapeutic drug substance; c) A linker joining the one or more antigen binding domains of (a) and the therapeutic drug substance of (b).
In embodiments, the chimeric antigen receptor that binds BCMA comprises antigen binding domain 1 , antigen binding domain 2, antigen binding domain 3, antigen binding domain 4, antigen binding domain 5, antigen binding domain 10, antigen binding domain 11 , antigen binding domain 12, antigen binding domain 13, antigen binding domain 14, antigen binding domain 15, antigen binding domain 16, antigen binding domain 17, antigen binding domain 18, antigen binding domain 19, antigen binding domain 20, antigen binding domain 21 , antigen binding domain 22, antigen binding domain 23, antigen binding domain 24, antigen binding domain 25, antigen binding domain 26, antigen binding domain 27, antigen binding domain 28, antigen binding domain 29, antigen binding domain 30, antigen binding domain 31 , antigen binding domain 32, antigen binding domain 33, antigen binding domain 34, antigen binding domain 35, antigen binding domain 36 or antigen binding domain 37.
In embodiments, the chimeric antigen receptor that binds BCMA comprises an antigen binding domain selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89.
In embodiments, the therapeutic drug substance is a cytotoxic drug.
In embodiments, the one or more antigen binding domains of the chimeric antigen receptor of the fourth aspect of the invention, the one or more antigen binding domains of bispecific T-cell engager (BiTE) of the sixth aspect of the invention, or one or more antigen binding domains of the antibodydrug conjugate (ADC) of the seventh aspect of the invention bind BCMA.
In embodiments, the chimeric antigen receptor of the fourth aspect, the bispecific T-cell engager of the sixth aspect of the invention, or the antibody-drug conjugate of the seventh aspect, wherein the single variable domain on a heavy chain of any one of antigen binding domain 1 to 5 is a VHH. Suitably, the VHH is selected from the group consisting of: SEQ ID NOs 3 to 5, SEQ ID NOs 15 to 16 SEQ ID NOs 66 to 71 , SEQ ID NOs 108 to 111 , SEQ ID NOs 136 to 139, and SEQ ID NOs 164 to 172. Suitably, the ScFv is selected from the group consisting of SEQ ID Nos 227 to 231 .
In embodiments, the antigen binding molecule of the first aspect, the pharmaceutical composition of the third aspect, the chimeric antigen receptor of the fourth aspect, the recombinant T-cell of the fifth aspect, the bispecific T-cell engager of the sixth aspect, the antibody-drug conjugate of the seventh aspect may be for use as a medicament or in medicine.
In embodiments, the antigen binding molecule of the first aspect, the pharmaceutical composition of the third aspect, the chimeric antigen receptor of the fourth aspect, the recombinant T-cell of the fifth aspect, the bispecific T-cell engager of the sixth aspect, the antibody-drug conjugate of the seventh aspect may be for use in killing or inhibiting the growth of cells expressing BCMA.
In embodiments, the antigen binding molecule of the first aspect, the pharmaceutical composition of the third aspect, the chimeric antigen receptor of the fourth aspect, the recombinant T-cell of the fifth aspect, the bispecific T-cell engager of the sixth aspect, the antibody-drug conjugate of the seventh aspect may be for use in the treatment of a disease or disorder selected from the group consisting of: cancers, such as Multiple Myeloma (MM); Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
In an eighth aspect, the invention provides a method of treating a disease or condition associated with the expression of BCMA, said method comprising administering to a patient in need thereof an effective amount of an antigen binding molecule of the first aspect of the invention, a pharmaceutical composition of the second aspect of the invention, a chimeric antigen receptor of the fourth aspect of the invention, a recombinant T-cell of the fifth aspect of the invention, a bi-specific T-cell engager of the sixth aspect of the invention or the antibody-drug conjugate of the seventh aspect of the invention to a patient in need thereof.
In embodiments, the disease or condition is selected from the group consisting of: MM; Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 A shows an embodiment of the general structure of the antigen binding molecule of the present invention wherein the antigen binding domain is a VHH comprising CDRs 1 , 2 and 3, within an example single chimeric antigen receptor scaffold comprising a CD8A signal peptide, a CD8A hinge and TM region, CD137 costimulatory domain and CD247 stimulatory domain.
Figure 1 B shows an embodiment of the general structure of the antigen binding molecule of the present invention wherein the antigen binding domain is a scFv comprising CDRs 1 , 2 and 3 of a heavy chain and comprising CDRs 1 , 2 and 3 of a light chain within an example single chimeric antigen receptor scaffold comprising a CD8A signal peptide, a CD8 hinge, a CD8A TM Domain, CD137 costimulatory domain and CD247 stimulatory domain.
Figure 2 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs. Four days after transduction, cells were exposed for 24h to K562 wild-type cells or BCMA- expressing K562 cells. After 24h GFP expression was assessed via flow cytometry.
Figure 3 shows cells transduced with different BCMA-CAR constructs and expanded for eight days and stained with BCMA-Fc protein. In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies. Median fluorescence intensity (MFI) is shown for CD34+ CAR- T cells. Figure 4 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on RPMI8226 cells cultured with non-transduced T cells.
Figure 5 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (K562_BCMA) at a 1 :1 effector to target cell ratio. 24 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on K562_BCMA cells cultured with non-transduced T cells.
Figure 6 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later supernatant was collected and assessed for IFN-gamma production via LegendPlex™ (Biolegend™) cytokine assay.
Figure 7 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later supernatant was collected and assessed for IL-2 production via LegendPlex™ (Biolegend™) cytokine assay.
Figure 8 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later cells were assessed by flow cytometer for the up-regulation of CD25 and CD69 markers. Figure 9 shows T-cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h and 48h with very low BCMA-expressing cancer cell line (Nalm-6) at a 1 :1 effector to target cell ratio. 24 and 48 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on Nalm-6 cells cultured with non-transduced T cells.
Figure 10 shows T-cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h and 48h with high BCMA-expressing cancer cell line (RPMI-8226) at a 1 :1 effector to target cell ratio. 24 and 48 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on RPMI-8226 cells cultured with non-transduced T cells.
Figure 11 shows cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 48 hours later supernatant was collected and assessed for IFN-gamma production via LegendPlex™ (Biolegend™) cytokine assay.
Figure 12 shows T-cells transduced with different BCMA CAR constructs and expanded for eight days and co-cultured for 24h and 48h with very high BCMA-expressing cancer cell line (U266B1) at a 1 :1 effector to target cell ratio. 24 and 48 hours later BCMA-positive cells were counted by flow cytometer. Killing percentage was calculated based on U266B1 cells cultured with non-transduced T cells.
Figure 13 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs. Four days after transduction, cells were exposed for 24h to BCMA-expressing RPMI-8226 cells. After 24h GFP expression was assessed via flow cytometry.
Figure 14 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs, and expanded for 4 days and stained with BCMA-Fc protein. In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies. Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
Figure 15 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs based on CAR construct BCMA-3. Four days after transduction, cells were exposed for 24h to BCMA-expressing RPMI-8226 cells. After 24h GFP expression was assessed via flow cytometry.
Figure 16 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs based on CAR construct BCMA-3. and expanded for 4 days and stained with BCMA-Fc protein. In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies. Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
Figure 17 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs based on CAR construct BCMA-5. Four days after transduction, cells were exposed for 24h to BCMA-expressing RPMI-8226 cells. After 24h GFP expression was assessed via flow cytometry.
Figure 18 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs based on CAR construct BCMA-5. and expanded for 4 days and stained with BCMA-Fc protein. In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies. Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
Figure 19 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs. Four days after transduction, cells were exposed for 24h to BCMA-expressing RPMI-8226 cells. After 24h GFP expression was assessed via flow cytometry.
Figure 20 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs, and expanded for 4 days and stained with BCMA-Fc protein. In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies. Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
Figure 21 shows Jurkat NFAT_GFP reporter cell line, transduced with the different anti-BCMA CAR constructs. Four days after transduction, cells were exposed for 24h to BCMA-expressing OPM-2 cells. After 24h GFP expression was assessed via flow cytometry.
Figure 22 shows Jurkat NFAT_GFP reporter cell line, transduced with different BCMA-CAR constructs, and expanded for 4 days and stained with BCMA-Fc protein. In a second step, cells were stained with PE-conjugated anti-Fc and APC-conjugated anti-CD34 antibodies. Median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting.
All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Prior to further setting forth the invention, a number of definitions are provided that will assist in the understanding of the invention.
The articles ‘a’, ‘an’ and ‘the’ are used to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.
As used herein, the term ‘comprising’ means any of the recited elements are necessarily included and other elements may optionally be included as well. ‘Consisting essentially of’ means any recited elements are necessarily included, elements which would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. ‘Consisting of’ means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.
Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (up to Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.
The term ‘antibody’ refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric, polymeric and chimeric forms, unless otherwise specified. Specifically encompassed by the term ‘antibody’ are polyclonal antibodies, monoclonal antibodies (mAbs), single domain antibodies, human (FHVH) or heavy-chain antibodies found in camelids (VHH) and antibodylike polypeptides, such as chimeric antibodies and humanized antibodies. The term ‘monoclonal antibody’ refers to an antibody produced by a single clone of cells or cell line and consisting of identical antibodies. As used herein, the term ‘antibody’ or ‘antigen-binding fragments’ or ‘antigen binding molecule’ may more generally encompass any protein or mixed protein/chemical structure that exhibits binding affinity for a particular antigen, or more than one particular antigen. Non-limiting Examples of antigen binding molecules include antibodies, chimeric antigen receptors (CARs), bi-specific T-Cell engagers (e.g. BiTEs), dual affinity re-targeting reagent (DART™), or antibody-drug conjugates (ADCs).
Antigen-binding fragments include those provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments are composed of portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, antigen-binding fragments may comprise at least one variable region (either a heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind a particular antigen. Examples of suitable antigen-binding fragments include, without limitation diabodies, nanobodies and single-chain molecules as well as Fab, F(ab')2, Fc, Fabc, and Fv molecules, single chain (sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in W02007059782, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting essentially of the VH and CH1 domains; a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, a dAb fragment (Ward et al., Nature 341 , 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov.; 21 (11):484-90); camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan.; 5(1): 11 1 -24); an isolated complementarity determining region (CDR), and the like. All antibody isotypes may be used to produce antigen-binding fragments. Additionally, antigen-binding fragments may include non-antibody proteinaceous frameworks that may successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds. Antigen-binding fragments may be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies. The phrase ‘an antibody or antigenbinding fragment’ may be used to denote that a given antigen binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.
As used herein the term ‘antigen binding domain’ refers to a peptide sequence that is intended or able to bind a target of interest. In examples, the antigen binding domain is an antigen-binding fragment as defined above. All types of antigen binding domains are encompassed by the present invention. Examples of some antigen binding domains are scFvs, VHH single domain antibodies or nanobodies, and antigen binding fragments.
The term “VHH” or “VHH” refers to a single-domain antibody (sdAb), also known as a nanobody, and is an antibody fragment consisting of a single monomeric variable antibody domain located on a heavy chain.
An ‘scFv’ or ‘single chain variable fragment’ as used herein, refers to a type of antigen binding domain. Typically, an scFv is a fusion of the variable regions of the heavy (VH) and light chains (VL) of an antibody for a given target connected by a short linker. The VH and VL regions may be in any order around the linker, for example, the scFv may have an opening signal peptide sequence followed by (i) a VH chain, a linker and a VL chain or (ii) the scFv may have (i) a VL chain, a linker and a VH chain, then followed by a hinge region, a TM domain and an intracellular domain. Suitably, the scFv may have a first VH chain, a linker and a VL chain. Antigen binding domains may comprise ‘CDRs’ or ‘complementarity determining regions’ which are predominantly responsible fortarget binding. On a typical antibody, antigen binding molecule or antigen binding domain, multiple CDRs exist and may be selected or varied independently to achieve multiple points of diversity. A ‘recognition sequence’ refers to the nucleic acid sequence encoding one or more antigen binding domains.
‘Specific binding’ or ‘immunospecific binding’ or derivatives thereof when used in the context of antibodies, antigen binding molecules or antibody fragments or domains, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopes of a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a Kd of less than about 1x1 O'8 M, as measured by a surface plasmon resonance assay or a cell binding assay. Phrases such as ‘[antigen] -specific’ antibody (e.g. BCMA-specific antibody) are meant to convey that the recited antibody specifically binds the recited antigen.
As used herein, the term ‘isolated’ means a biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been isolated thus include nucleic acids and proteins purified by standard purification methods. Isolated nucleic acids, peptides and proteins can be part of a composition and still be isolated if such composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An ‘isolated’ antibody or antigen-binding fragment, as used herein, is intended to refer to an antibody or antigen- binding fragment which is substantially free of other antibodies or antigen-binding fragments having different antigenic specificities (for instance, an isolated antibody that specifically binds to BCMA is substantially free of antibodies that specifically bind antigens other than BCMA ). An isolated antibody that specifically binds to an epitope, isoform or variant of BCMA may, however, have cross-reactivity to other related antigens, for instance from other species (such as BCMA species homologs).
As used herein, the term ‘chimeric antigen receptor’ or ‘CAR’ refers to transmembrane receptor that has been engineered to target or bind to a non-native substrate or antigen. In this way the intracellular signalling of the receptor may triggered by binding of a non-native substrate or antigen. Typically, the term CAR refers to a chimeric receptor (i.e. a receptor composed of two or more parts from different sources) that has at least a binding moiety or recognition sequence with a specificity for a target such as an antigen or protein and an intracellular signalling domain that can invoke a signal in the cell in which the CAR is expressed (e.g. a CD247 (CD3 zeta) chain). In embodiments, a ‘chimeric antigen receptor’ or ‘CAR’ is formed of at least three domains: an extracellular antigen binding domain (as defined elsewhere herein), a transmembrane domain and an intracellular domain. A hinge domain between the antigen binding domain and the transmembrane domain is often used to improve recognition of the target. A costimulatory domain may optionally be present in the intracellular domain to modulate the response. To be functional, the domains of the CAR must be ordered correctly. CARs are often used on T-cells (to produce ‘CAR T-cells’) to effect recognition and elicit an appropriate intracellular response which both binds the T-cell to a target cell and triggers the innate immune response of the T-cell, typically causing lysis of the target cell. Such cells have found use in therapy. The term a ‘tandem CAR’ is used herein to refer to a CAR with one or more, suitably two, antigen binding fragments in a single antigen binding domain, optionally joined by a linker. The term a ‘parallel CAR’ is used herein to refer to a CAR sequence (or CARs derived from that sequence) that comprises two or more CAR sequences joined by a linker, suitably a self-cleaving peptide linker, such that following translation, two or more CARs are expressed from the separate CAR sequences.
A ‘transmembrane domain’ or ‘TM domain’ as used herein is any membrane-spanning protein domain. Suitably, the TM domain in a CAR is derived from a known transmembrane protein sequence. However, it can also be artificially designed. A ‘transmembrane sequence’ refers to the nucleic acid sequence encoding a transmembrane domain. As used herein the term ‘hinge domain’ refers to a peptide sequence that connects the antigen binding domain and transmembrane region of a CAR. The hinge domain is located between the antigen binding fragment and the T cell plasma membrane (Moritz D, et al. Gene Ther. 1995;2(8):539-46)
The term ‘signalling domain’ or ‘intracellular domain’ or ‘intracellular signalling domain’ as used herein refers to a moiety that can transmit a signal in a cell, for example an immune cell. The signalling domain typically comprises a domain derived from a receptor that signals by itself in immune cells, such as the T Cell Receptor (TCR) complex or the Fc receptor or DAP10/DAP12 receptors. Additionally, it may contain a costimulatory domain (i.e. a domain derived from a receptor that is required in addition to the TCR to obtain full activation of T cells). The costimulatory domain can be from an activating costimulatory receptor or from an inhibitory costimulatory receptor. An ‘intracellular domain sequence’ refers to the nucleic acid sequence encoding an intracellular signalling domain.
As used herein, the terms ‘bi-specific’, ‘tri-specific’ or ‘multi-specific’ refer to an antibody or antigen binding molecule (i.e. an antibody or antigen binding molecule or antigen binding fragment or domain conjugated to, or a product of conjugation of, a naturally-occurring or synthetic molecule) that comprises one or more further antigen binding domains such that the antibody molecule can have specificity for more than one antigen.
As is known in the art, as used herein, the term ‘bispecific T-cell engager’ or ‘BiTE’ is used synonymously to refer to a single polypeptide chain molecule having two antigen binding domains, one of which binds to a T-cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell (e.g. BCMA ), these two antigen binding domains separated by a linker, specifically a flexible linker, more specifically a flexible protein linker (WO 05/061547; Baeuerle, et al. (2008) Drugs of the Future 33:137-147; Bargou, et al. (2008) Science 321 :974-977). BiTE antibodies have been constructed to various target antigens including CD19, EpCAM, Her2/neu, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or HMW-MAA) (Baeuerle, et al. (2009) Curr. Opin. Mol. Ther. 1 1 :22-30). Key hallmarks of BiTE antibodies that, in their combination, distinguish them from other bispecific antibody constructs, include a high potency of redirected lysis with EC50 values ranging from 0.1 to 50 pmol/L (2-1 ,000 pg/mL) (Baeuerle, et al. (2009) supra); strict target cell-dependent activation of T cells (Brischwein, et al. (2007) J. Immunother. 30: 798-807); and support of serial lysis by activated T cells, i.e., activity at low E: T ratios. BiTE antibodies are typically produced as recombinant, glycosylated proteins secreted by higher eukaryotic cell lines. The terms ‘bi-specific T- Cell engager’ and/or ‘BiTE’ as herein defined encompass, but are not limited to, the specific bi-specific T-Cell engager structure encompassed by the trademark ‘BITE®’ in the name of Amgen Research (Munich) GmbH.
The term ‘DART™’ (Dual Affinity Re-Targeting reagent) diabody refers to an immunoglobulin molecule that comprises at least two polypeptide chains that associate (for example, through a covalent interaction) to form at least two antigen binding domains, which may recognize the same or different antigens. Each of the polypeptide chains of a DART™ diabody comprise an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an antigen binding domain. Rather, the immunoglobulin heavy chain variable region of one (e.g., the first) of the DART™ diabody polypeptide chains interacts with the immunoglobulin light chain variable region of a different (e.g. the second) DART™ polypeptide chain to form an antigen binding domain. Similarly, the immunoglobulin light chain variable region of one (e.g., the first) of the DART™ diabody polypeptide chains interacts with the immunoglobulin heavy chain variable region of a different (e.g., the second) DART™ diabody polypeptide chain to form an antigen binding domain. DART™ diabodies may be monospecific, bispecific, trispecific, etc., thus being able to simultaneously bind one, two, three or more different antigens (which may be of the same or of different antigens). DART™ diabodies may additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., thus being able to simultaneously bind one, two, three, four, five, six or more molecules. These two attributes of DART™ diabodies i.e. degree of specificity and valency, may be combined, for example to produce bispecific antibodies i.e. capable of binding two antigens, which are tetravalent i.e., capable of binding four sets of epitopes, etc. DART™ diabody molecules are disclosed in PCT applications published as WO 2006/113665, WO 2008/157379, and WO 2010/080538. As used herein, the term ‘antibody-drug conjugate’ or ‘ADC’ means a biopharmaceutical drug that comprises an antibody or antigen binding fragment or domain, and a drug substance, typically a cytotoxic drug substance, connected via a linker, suitably a covalent, chemical linker.
As used herein, the term ‘high-throughput screening’ refers to any assay or screening methodology that allows for a higher rate of screening than would be achieved by traditional or previous state of the art techniques. Typically, high-throughput screening enables automation to prepare, screen and/or evaluate libraries of test samples in parallel, reproducibly and rapidly. High-throughput screening can also make use of combinatorial or pooled or mixed sample screening strategies, with associated deconvolution of hits. In the context of CAR high throughput screening, the number of samples for test may be of any size larger than that typically used in prior art non-high-throughput methods. For example, the number of samples may be more than 10. Suitably the number of samples may be more than 11 ,
12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200 or more.
As used herein, the term ‘recognition sequence library’ refers to a set of one or more antigen binding domain sequences or recognition sequences that may be used for cloning into a CAR construct (wherein a CAR construct comprises all components required for a functioning CAR including one or more antigen binding domains, a hinge domain, a transmembrane domain and an intracellular domain) to prepare a CAR library. An ‘scFv library’ is a recognition sequence library formed of scFv recognition sequences from antibodies. A ‘VHH library’ is a recognition sequence library formed of VHH recognition sequences from antibodies. In embodiments, the number of recognition sequences present in the recognition sequence library may be more than 1 or more than 2. Suitably the number of hinge region sequences present in the hinge region sequence library may be more than 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,
13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200 or more.
As used herein, the term ‘CAR scaffold’ refers to the part of the CAR sequence that comprises the components, parts, modules, domains of the CAR excluding the antigen binding domain, that form the sequence of a CAR in a CAR library. The CAR scaffold is formed of the sequences of the individual components, or groups thereof, and combined with the recognition sequence to form a sequence encoding a CAR in any suitable manner, for example, sequentially, convergently, with the recognition sequence being incorporated at any suitable point, i.e. the recognition sequence may be joined to a single unitary sequence of the CAR scaffold, or may be joined to a component part of the CAR scaffold initially and the full CAR sequence of the recognition sequence and scaffold completed subsequently. The component parts of the CAR scaffold may derive from single sequences leading to one or a small number of CAR scaffolds for addition to a recognition sequence, or the component parts of the CAR scaffold may derive from libraries of one or more of the individual components leading to a CAR scaffold library comprising a plurality of sequences. A CAR scaffold library may be formed by combination, suitably in a combinatorial, or directed, manner of the individual components or component libraries.
As used herein, the term ‘CAR library’ refers to a set of sequences encoding a functional CAR structure, including at least one recognition domain, such as an scFv or VHH, a hinge domain, a transmembrane domain and intracellular domain(s). In embodiments, the number of CARs present in the CAR library may be more than 10. Suitably the number of CARs present in the CAR library may be more than 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200 or more. The term ‘CAR library’ may also be used interchangeably to refer to the plasmid or vector, or otherwise modified sequence of the sequence(s) encoding for a functional CAR structure.
As used herein, the term ‘CAR-cell library’ refers to a collection or set of cells expressing CARs on their surface. Each cell in the CAR-cell library may express a single CAR (i.e. express only one CAR encoded by a single sequence), or a single cell in the CAR-cell library may express two or more CARs, each encoded by the same or different sequences. Suitably, each cell in the CAR-cell library expresses a single CAR or all CARs present in the library. In embodiments, the number of CARs present in the CARcell library may be more than 10. Suitably the number of CARs present in the CAR-cell library may be more than 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200 or more.
As used herein, the term ‘next generation sequencing’ of ‘NGS’ refers to a catch-all term used to describe a number of different modern sequencing technologies. These technologies allow for sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing. Examples are Solex™ sequencing by Illumina™, Roche 454™ sequencing, Ion Torrent™ sequencing and nanopore based sequencing methods (e.g. Oxford Nanopore Technologies™ Grid ION™).
The phrase ‘nucleic acid molecule’ synonymously referred to as ‘nucleotides’ or ‘nucleic acids’ or ‘polynucleotide’ refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Nucleic acid molecules include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, ‘polynucleotide’ refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. ‘Modified’ bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, ‘polynucleotide’ embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. ‘Polynucleotide’ also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
There are various means by which a nucleic acid sequence may be inserted into a genome, including but not limited to plasmid or vector transfection, transposition and genome editing. All are contemplated for use in the present invention.
As used herein a ‘vector’ is a replicon, such as plasmid, phage, cosmid, or virus in which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment. A ‘transposon’ or ‘transposable elements’ are DNA sequences that can change their position within a genome. ‘Genome editing’ refers to the ability to edit the genome to insert the required sequence, for example using CRISPR-Cas9 genome editing technology.
A ‘clone’ is a population of cells derived from a single cell or common ancestor by mitosis.
A ‘cell line’ is a clone of a primary cell that is capable of stable growth in vitro for many generations. In some examples provided herein, cells are transformed by transfecting the cells with DNA.
The terms ‘express’ and ‘produce’ are used synonymously herein and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post-translational modifications.
The term ‘subject’ refers to human and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, goats, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In most particular embodiments of the described methods, the subject is a human.
The terms ‘treating’ or ‘treatment’ refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations.
Sequence homology
When referring to BCMA, it will be appreciated that reference thereto encompasses fragments thereof, as well as related polypeptides, which include, but are not limited to, allelic variants, splice variants, derivative variants, substitution variants, deletion variants, and/or insertion variants including the addition of an N-terminal methionine, fusion polypeptides, and interspecies homologs. In certain embodiments, a BMCA polypeptide includes terminal residues, such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues.
Amino acid sequence of an example isoform of BCMA to which the antibodies of the present invention bind is provided as SEQ ID NO: 1 .
The DNA sequence of BCMA (SEQ ID NO: 1) is provided (SEQ ID NO: 2). Splice variants and other isoforms of BCMA are known and are encompassed by the present disclosure.
For all aspects and embodiments of the present invention, the amino acid sequence of BCMA encompasses a polypeptide that has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity to BCMA (for example SEQ ID NO: 1). Likewise, the BCMA polynucleotide sequence can comprise a polynucleotide that has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity to BCMA (for example, SEQ ID NO: 2). Sequence identity can also be to a fragment or portion of the full-length polynucleotide or polypeptide. Hence, a sequence may have only 50% overall sequence identity with a sequence of the invention but in a particular region, domain or subunit could share 80%, 90%, or as much as 99% sequence identity with sequence of the invention.
According to the present invention, homology to a nucleic acid sequence is not limited simply to sequence identity. Many nucleic acid sequences can demonstrate biologically significant homology to each other despite having apparently low sequence identity. In the present invention homologous nucleic acid sequences are considered to be those that will hybridise to each other under conditions of low stringency (Sambrook J. et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY).
The present application shows a systematic high-throughput screen output of a CAR-T cell library. This allowed identification of multiple novel antigen binding molecules or antigen binding fragments or domains (e.g. VHHs or scFvs) and their associated heavy chain CDRs and light chain CDRs, that bind to BCMA and outperform other known antibody or antigen binding fragments in several experimental models.
Accordingly, in at least one aspect, the invention provides an antibody or antigen binding fragments or antigen binding molecules having specificity for BCMA (Antigen binding molecule Nos. 1 to 5, 10 to 37), wherein the antibody or antigen binding fragments or antigen binding molecules have an antigen binding domain comprising the amino acid sequences (SEQ ID NOS: 3 to 22, 66 to 89, 108 to 123, 136 to 151 , 164 to 199 and 227 to 261) as shown in Table 1 below:
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Table 1 : Exemplified BCMA-binding domains and fragments of the present invention
The antigen-binding molecules, or antigen binding domains, of the present invention were identified from a functional high-throughput screen of CAR-T cells, wherein the CAR-T cells express a unique chimeric antigen receptor (CARs), each CAR being or comprising an antigen binding molecule comprising an antigen binding domain as a VHH derived from a diverse VHH library or a scFv from a diverse scFv library. The antigen binding domains as identified in Tables 1 and 2 are those exhibiting surprisingly good functional response (i.e. were positive hits) in various assays against cells expressing BCMA and are therefore deemed to provide superior CAR-T cells overthat known in the art and provide novel and effective antigen binding molecules or antigen binding domains of BCMA.
The high-throughput screening method employed to identify antigen binding molecules or antigen binding domains, and/or bi-specific, tri-specific or multi-specific antigen binding molecules and/or antibody-drug conjugates and/or CAR-T cells derived therefrom is described in the Applicant’s copending International application PCT/GB2022/050158 (published as WO2022/157500), the content of which is incorporated by reference.
In embodiments, the antibody or antigen binding molecule can be an isolated monospecific or bispecific antibody having specificity for BCMA (suitably human BMCA) and can be a full-length antibody or an antibody fragment. The antibody can be polyclonal, monoclonal, recombinant, chimeric, or humanised. Furthermore, the antibody can be of any isotype including without limitation IgA, IgD, IgE, IgG, or IgM. Thus, for example, the antibody can be any IgA such as lgA1 or lgA2, or any IgG such as lgG1 , lgG2, lgG3, lgG4, or synthetic IgG. The antibody can also be any antibody fragment having specificity for BCMA, such as F(ab)2, Fv, scFv, F(ab' )2, F(ab), VL, VH, VHH, dsFv, Fv, scFv-Fc, (scFv)2, a diabody, and a bivalent antibody. The antibody can be any modified or synthetic antibody, including, but not limited to, non-depleting IgG antibodies, T-bodies, or other Fe or Fab variants of antibodies.
In some embodiments, the invention provides an antigen binding molecule or antigen binding domain with avidity for BCMA of about 10 pM or less, 5 pM or less, 2 pM or less, 1 pM or less, 500 nM or less, 400 nM or less, 300 nM or less, or 200 nM or less, or 100 nM or less, or 75 nM or less, or 50 nM or less, or 25 nM or less, or 10 nM or less, or 5 nM or less. Avidity can be measured using art-known techniques, such as ELISA or BIACORE.
The antigen binding molecule or antigen binding domain of the invention can be produced by any suitable technique, for example, using any suitable eukaryotic or non-eukaryotic expression system. In certain embodiments, the antigen binding molecule is produced using a mammalian expression system. Alternatively, the antigen binding molecule or antigen binding domain of the invention can be produced using a suitable non-eukaryotic expression system such as a bacterial expression system. Bacterial expression systems can be used to produce fragments such as a F(ab)2, Fv, scFv, F(ab' )2, F(ab), VL, VH, VHH, dsFv, Fv, scFv-Fc, (scFv)2, and diabodies.
The antigen binding molecule can be a product of conjugation to a synthetic molecule, or the antigen binding fragment of the invention can be conjugated to a naturally-occurring or synthetic molecule. Conjugation of, or to produce, the antigen binding molecule or antigen binding domain of the invention to the naturally-occurring or synthetic molecule may be by any suitable method, for example recombinant engineering. The naturally-occurring or synthetic molecule can be any molecule such as a drug targeting a tumour or tumour cells. The naturally-occurring or synthetic molecule can also be a peptide/protein or an antibody or an antigen binding fragment of a different target affinity such that the resultant molecule has specificity for more than one antigen, wherein the resulting fusion protein can be produced by conventional recombinant protein expression systems and methods.
In this respect, a further aspect of the invention relates to chimeric antigen receptors (CARs). CARs, are engineered receptors, which confer specificity for a desired antigen onto an immune effector cell, such as a T-cell. CARs may be expressed on the extracellular surface of a cell. Expression may be by any suitable means via retroviral vector expression. The most common form of these molecules are fusions of an antibody or antigen binding molecule or antigen binding fragment or domain, such as an scFv or VHH, to the transmembrane and intracellular domains of a native T-cell activation receptor, such as CD3, typically CD3-zeta. Such molecules result in the transmission of a signal, suitably an activation signal, in response to recognition by the scFv or VHH of its target. ‘First-generation’ CARs typically have the intracellular domain from the CD3-zeta chain, which is the primary transmitter of signals from endogenous TCRs. ‘Second-generation’ CARs add intracellular signalling domains from various costimulatory protein receptors (e.g., CD28, CD137, 41 BB, ICOS) to the intracellular portion of the CAR to provide additional signals to the T cell (see Figure 1). Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells (Maher, et al. (2002) Nat. Biotechnol. 20:70-75; Kowolik, et al. (2006) Cancer Res. 66: 10995-11004). ‘Third-generation’ CARs combine multiple signalling domains, such as CD3z-CD28-41 BB or CD3z-CD28-OX40, to further augment potency (Zhao, et al. (2009) J. Immunol. 183:5563-5574; Pule, et al. (2005) Mol. Ther. 12:933- 941 ; Zhong, et al. (2010) Mol. Ther. 18:413-420). Accordingly, in an embodiment of this invention, a CAR is provided that is or comprises an anti-BCMA antigen-binding molecule or antibody or antigen binding fragment or domain, such as a VHH fragment, as defined in SEQ ID NOs 3 to 5, 15 to 16, 66 to 71 , 108 to 111 , 136 to 139, and 164 to 172 or a scFv fragment, as defined in SEQ ID Nos 227 to 231 .
CARs of this invention can be prepared using standard recombinant protein techniques using sequences of CD3, e.g. CD247 (CD3-zeta) and optionally other costimulatory molecules known in the art. For example, the human CD247 (CD3-zeta) sequence is available under GENBANK accession number NP_932170, the human CD28 sequence is available under GENBANK accession number NP 006130, the human CD8A sequence is available under GENBANK accession number AAH25715, and the human CD137 sequence is available under GENBANK accession number NP_001552.
In embodiments, the CARs of the present invention include, but are not necessarily limited to: a human CD247 (CD3-zeta) cytoplasmic domain (SEQ ID NO:28), a human CD137 co-stimulatory domain (SEQ ID NO:29); a human CD8A hinge/transmembrane domain (SEQ ID NQ:30); one or more antigen binding fragments or domains of the present invention (SEQ ID NOS: 3 to 5, 15 and 16, 66 to 71 , 108 to 111 , 136 to 139, 164 to 172 and 227 to 231)); and a CD8A extracellular signal peptide (SEQ ID NO: 33). In particular embodiments, the CARs of the present invention are represented by the SEQ ID NOS: 23 to 27, 90 to 95, 124 to 127, 152 to 155, 200 to 208 and 262 to 266. In specific embodiments, the CARs of the present invention have a single BCMA VHH antigen binding domain, with a sequence as defined in SEQ ID NOS: 3 to 5, 15, 16, 66 to 71 , 108 to 111 , 136 to 139, and 164 to 172.
In specific embodiments, the CARs of the present invention have a scFv BCMA antigen binding domain, with a sequence as defined in SEQ ID NOS: 227 to 231 .In specific embodiments, the CARs as defined in SEQ ID NOS: 23 to 27, 90 to 95, 124 to 127, 152 to 155, 200 to 208 and 262 to 266 are encoded by DNA sequences represented by the SEQ ID NOS: 41 to 45, 102 to 107, 132 to 135, 160 to 163, 218 to 226, and 272 to 276.
In some embodiments, the antigen binding molecule can be the product of conjugation to a naturally- occurring or synthetic molecule, or the antigen binding fragment or domain can be conjugated to a naturally-occurring or synthetic molecule that can confer specificity for one or more antigens in addition to BCMA. For example, the antigen binding molecule of the invention can be engineered (e.g. as a bivalent diabody or a conjugated Fab dimer or trimer) to have specificity for BCMA and another tumour antigen, e.g., an antigen associated with a disease or disorder as disclosed herein. Alternatively, the antigen binding molecule can be engineered to have specificity for BCMA and an antigen that promotes activation or targeting of other cells, such as cytotoxic effector cells or T cells. Accordingly, the invention also includes bispecific, tri-specific and multi-specific molecules such as BiTEs (bi-specific T-cell engagers) and DART™s (dual affinity retargeting reagents).
As is known in the art, a bi-specific T-cell engager (BiTE) refers to a single polypeptide chain molecule having two antigen binding domains, one of which binds to a T-cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell (WO 05/061547; Baeuerle, et al. (2008) Drugs of the Future 33:137-147; Bargou, et al. (2008) Science 321 :974-977). BiTE antibodies have been constructed to various target antigens including CD19, EpCAM, Her2/neu, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or HMW-MAA) (Baeuerle, et al. (2009) Curr. Opin. Mol. Ther. 1 1 :22-30). Accordingly, in another embodiment of this invention, an anti-BCMA antigen binding molecule or antigen binding domain (e.g. a scFv or VHH) is a component of a bi-specific T-cell engager (BiTE). In particular embodiments, the bi-specific T-cell engager (BiTE) of this invention is composed of an anti- BCMA antigen binding molecule or antigen binding fragment or domain and an anti-CD3 antibody fragment fused together by a linker, e.g., a flexible protein linker. See, for example, US 5,929,212. The term ‘BiTE’ as used herein may encompass but is not limited to the bispecific T-cell engager named BiTE® available from Amgen®.
In embodiments, the antibody fragment that specifically binds CD3 comprises:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:35,
(ii) a CDR2 of SEQ ID NO:36, and
(iii) a CDR3 of SEQ ID NO:37; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:38,
(ii) a CDR2 of SEQ ID NO:39, and
(iii) a CDR3 of SEQ ID NQ:40.
In other embodiments, the invention provides the antigen binding molecule or the antigen binding fragment or domain of the invention coupled to a naturally-occurring or synthetic molecule as defined herein to provide a DART™. DART™ refers to an immunoglobulin molecule that includes at least two polypeptide chains that associate (especially through a covalent interaction) to form at least two antigen binding sites, which may recognize the same or different antigens. Each of the polypeptide chains of a DART™ include a VHH heavy chain region, or an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope binding site. Rather, the immunoglobulin heavy chain variable region of one (e.g., the first) of the DART™ polypeptide chains interacts with the immunoglobulin light chain variable region of a different (e.g. the second) DART™ polypeptide chain to form an epitope binding site. Similarly, the immunoglobulin light chain variable region of one (e.g. the first) of the DART™ polypeptide chains interacts with the immunoglobulin heavy chain variable region of a different (e.g., the second) DART™ polypeptide chain to form an epitope binding site. DART™s may be monospecific, bi-specific, tri-specific, etc., thus being able to simultaneously bind one, two, three or more different antigens (which may be of the same or of different antigens). DART™s may additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., thus being able to simultaneously bind one, two, three, four, five, six or more molecules. These two attributes of DART™s (i.e., degree of specificity and valency may be combined, for example to produce bispecific antibodies (i.e., capable of binding two epitopes) that are tetravalent (i.e. capable of binding four sets of antigens/epitopes), etc. The construction of DART™ molecules is disclosed in WO 2006/113665, WO 2008/157379, and WO 2010/080538. Accordingly, in another embodiment of this invention, the anti-BCMA antigen binding molecule is a DART™ or the antigen binding fragment or domain is included in a DART™.
In other embodiments, the antigen binding molecule or antigen binding fragment or domain of the present invention is conjugated, linked or joined to other naturally occurring or synthetic molecules including therapeutic agents (or ‘payloads’) such as cytotoxic, cytostatic, or anti-angiogenic agents and radioisotopes, labels or nanoparticles. Such molecules are terms antibody-drug conjugates or ADCs. Examples of ADCs include: Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Trastuzumab emtansine and Trastuzumab emtansine.
The invention provides a method of inhibiting or killing cells that express BCMA (BCMA cells) by contacting the cells with an antigen binding molecule, antigen binding fragment or domain or antibody, antigen-binding molecule or antigen binding domain conjugated to a naturally-occurring or synthetic molecule (e.g. a CAR, a bispecific T-cell engager such as a BiTE, DART™, or ADC), all of which are aspects of the invention. The method can be used to inhibit BCMA cells in vitro or in a subject (i.e., in vivo). The contacted BCMA cells can be in, for example, a cell culture or animal model of a disorder associated with aberrant expression or levels of BCMA. The method is useful, for example, to measure and/or rank (relative to another antibody or antigen binding molecule) the antigen binding molecule's inhibitory activity for a BCMA cell type. Inhibiting BCMA cells can include blocking or reducing the activity or growth of BCMA. Inhibiting can also include the killing of BCMA. Cytotoxicity of an antigen binding molecule, antibody, antigen binding fragment, antigen binding domain or antigen binding molecule of antigen binding domain conjugated to a naturally-occurring or synthetic molecule, such as a CAR, BiTE, DART™, or ADC ofthe invention can be assessed using any conventional assay including, e.g. a lactate dehydrogenase cytotoxicity assay such as the CYTOTOX 96™ non-radioactive cytotoxicity assay commercially available from PROMEGA™. Similarly, the invention provides an antigen binding molecule, antigen binding fragment or domain or antibody conjugated to, or as a product of conjugation of, a naturally-occurring or synthetic molecule (e.g. a bispecific T-cell engager such as a CAR, BiTE, DART™, or ADC) of the invention for use in inhibiting and killing cells expressing BCMA.
The invention also provides a method of treating a subject that has, is suspected to have, or is at risk for a disorder associated with aberrant levels of BCMA cells. As used in the context of the present invention, the term ‘aberrant’ is intended to include increased or decreased BCMA expression on a cell as compared to expression of BCMA in normal or healthy cells, or an increase in cells expressing BCMA. Generally, the method of treatment includes administering a therapeutically effective amount of an isolated antigen binding molecule, antibody, antigen binding fragment or domain, or fusion protein of the invention to the subject. The antibody can be any anti-BCMA antigen binding molecule or antibody, antigen binding fragment or domain (e.g. chimeric, humanised, synthetic, F(ab)2 , Fv, scFv, F(ab')2, F(ab), VL, VH, VHH, dsFv, Fv, or (scFv)2) or an antigen binding molecule or domain conjugated to a naturally-occurring or synthetic molecule (e.g., a bispecific T-cell engager such as a CAR, BiTE, DART™, or ADC) as described herein. Similarly, the invention provides an antibody, antigen binding fragment or antibody conjugated to, or a product of conjugation of, a naturally-occurring or synthetic molecule (e.g., a bispecific T-cell engager such as a CAR, BiTE, DART™, or ADC) of the invention for use in medicine, and for the treatment of disease and disorders associated with aberrant or elevated BCMA expression. Disorders that can be treated include, for example, cancers, such as Multiple myeloma (MM); Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
The invention also provides a method of treating a subject that has, is suspected to have, or is at risk for a disorder associated with elevated levels of BCMA on a cell, or by elevated levels of BCMA cells by adoptive transfer of the recombinant host cells, e.g. T-cells described herein, which express an antigen binding molecule, antibody or antigen binding fragment or domain, optionally conjugated to, or as a product of conjugation to, a naturally-occurring or synthetic molecule of the invention as a CAR that selectively binds BCMA. Similarly, the invention provides recombinant cells expressing an antigen binding molecule or antigen binding fragment or domain that is conjugated to, or a product of conjugation of, a naturally-occurring or synthetic molecule (e.g. CAR) of the invention for use in medicine.
Recombinant technology can be used to introduce CAR-encoding genetic material into any suitable T- cells, e.g. effector memory T-cells from the subject to be treated. The recombinant T-cells are transferred, typically by infusion, to the patient. The transferred T-cells of the invention can then mount an immune response against BCMA expressing cells (BCMA cells) in the patient.
The adoptive transfer method can be used, for example, to treat subjects that have or are suspected to have cancers, such as Multiple myeloma (MM); Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
In embodiments, and suitable for use in treatment, the invention also provides a pharmaceutical composition containing an antigen binding molecule or antigen binding fragment or domain or antibody conjugated to, or a product of conjugation of, a naturally-occurring or synthetic molecule (e.g., a CAR, a bispecific T-cell engager such as a BiTE, DART™, or ADC) as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared from any of the antigen binding molecules, antibody, antigen binding fragment or domain (e.g. chimeric, humanised, synthetic, F(ab)2 , Fv, scFv, F(ab’)2 , F(ab), VL, VH, VHH, dsFv, Fv, or (scFv)2) or antibody conjugated to a naturally-occurring or synthetic molecule (e.g., a CAR, a bispecific T-cell engager such as a BiTE, DART™, or ADC) as described herein.
The composition of the invention can include a carrier for the antigen binding molecule or antigen binding fragment, or antigen binding molecule conjugated to, or a product of, conjugation of, a naturally- occurring or synthetic molecule, desirably a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier. The term ‘pharmaceutically acceptable carrier’, as used herein, means one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances, which are suitable for administration into a human or veterinary patient (e.g. a physiologically acceptable carrier or a pharmacologically acceptable carrier). The term ‘carrier’ denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The pharmaceutically acceptable carrier can be comingled with one or more of the active components, e.g., a hybrid molecule, and with each other, when more than one pharmaceutically acceptable carrier is present in the composition in a manner so as not to substantially impair the desired pharmaceutical efficacy. ‘Pharmaceutically acceptable’ materials typically are capable of administration to a patient without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset.
The pharmaceutical composition can contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt. The pharmaceutical compositions also optionally can contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
The pharmaceutical composition can be presented in unit dosage form and can be prepared by any suitable method, many of which are well-known in the pharmaceutical arts. Such methods include the step of bringing the antigen binding molecule of the invention into association with a carrier that constitutes one or more accessory ingredients. In general, the composition is prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
High throughput screening to identify CARs/antibodies
The present invention benefits from the high-throughput screening methodology as described in the copending International application no. PCT/GB2022/050158, the content of which is incorporated herein by reference.
The high-throughput screening method allows resources to be focussed primarily on the diversity of the CAR library and identification of the CARs that are able to demonstrate promising activity in a functional assay relevant to a clinical context.
The invention is described in greater detail by the following non-limiting examples.
EXAMPLES
Example 1 :
A synthetic human single domain heavy chain (VHH) phage library was panned against recombinant human BCMA protein (R&D Biosystems™). To this end, each library was grown to log phase, and then rescued with M13KO7 helper phage (Antibody Design Lab™, PH010L) before being amplified overnight at 32°C in a shaker. Each phage library was subsequently precipitated with PEG/NaCI, re-suspended in PBS and stored at -80°C. Protein G coated magnetic beads were coated with BCMA-Fc or Fc recombinant protein in PBS and subsequently blocked in PBS + BSA. Phage particles were incubated for 30 minutes with negative magnetic particles (Fc protein). Subsequently, magnetic particles were pelleted and the supernatant was incubated for 1 h with BCMA coated magnetic beads under rotation. After 1 h incubation, unbound and non-specifically bound phages were washed away by rinsing the beads with PBST. Bound phages were eluted by 100 mM triethylamine (TEA), and the eluate was neutralized by 1 M Tris-HCI (pH 7.4). Each eluate was then used to infect exponentially growing E. coli TGI cells. The panning was repeated for an additional two to four cycles.
Example 2:
After the last panning step, VHH sequences were PCR amplified from the eluted phages or the isolated plasmids using Q5 DNA polymerase (NEB) and VHH-specific forward and reverse primers. Forward and reverse primers contained overhang sequences, including protein linker sequences and Esp3l restriction sites. Resulting amplicons were cleaned by PCR clean-up and PCR amplicons were directly used as inserts for golden gate assembly. A backbone, a CAR scaffold library, consisting of pooled plasmids containing different CAR scaffolds with variation in VHH domain, hinge domain, transmembrane domain and intracellular signalling domains, was used. Golden gate assembly was performed with NEB Golden Gate kit and incubated, cycling between 16C and 42C for 40x cycles. The resulting CAR-library was electroporated into electrocompetent bacteria and grown overnight at 30°C, followed by plasmid maxi-prep isolation. The CAR sequences from the resulting plasmid library were briefly PCR amplified and sequenced on an Oxford Nanopore Technology™ MinlON™, using the amplicon sequencing kit.
Example 3:
In orderto assess if the CAR library was functionally expressed and recognizing BCMA, the CAR library was used to produce lentiviral vector particles and was then transduced into human T cells or Jurkat cell line. The resulting CAR-T cell library was expanded for four more days after transduction. Transduction efficiency was assessed by flow cytometry through staining with APC-conjugated anti- CD34 and transduced cells were enriched through CD34 microbeads (Miltenyi™). Enriched cells were then assessed for BCMA binding. BCMA CAR-T cell library cells were stained with BCMA-Fc fusion protein (recombinant Fc, R&D systems™). In a second step, cells were stained with APC-conjugated anti-Fc and Pacific Blue-conjugated anti-CD34 antibody to detect transduced cells. The expression of CARs on the cell surface was assessed via flow cytometry. BCMA-positive cells were sorted by flow cytometry using a SH800S cell sorter (Sony Biotechnology). Genomic DNA was isolated and PCR amplicons were sequenced using Illumina NextSeqI OOO.
Example 4:
A CAR-T cell library was produced by activation of PBMCs with TransAct™ (Miltenyi™) in the presence of human IL-2 (100 lU/ml) and transduced at day two with the lentiviral BCMA CAR library. The next day, cells were washed and further expanded until day 8 of the process. Transduction was assessed by flow cytometry and transduced cells were enriched through CD34 microbeads (Miltenyi™). Purity was assessed by flow cytometry. The CAR-T-cell library was co-cultured with BCMA-positive (BCMA- expressing) cells (K562 cells genetically engineered to express BCMA or RPMI8226 cell line) or K562 wild-type cells. After 6h or24h activation, CAR-T cells were prepared for single cell sequencing analysis. CAR-T cell libraries from different donors were prepared for 10x genomics single cell gene expression analysis (1 Ox genomics 3’ sequencing kit V3 or Single Cell 5’ Kit v2) and sequenced on a NovaSeq™ 6000 (Illumina™). Cell ranger™ software (10x Genomics™) was used for downstream processing and alignment of reads. In order to deconvolute the single cell sequencing data and identify the CAR sequence and cell barcode identity, CAR sequences were amplified using long PCR with a forward read 1 primer and CAR-specific reverse primer. After 10 cycles of PCR, the product was cleaned using SPRI bead clean-up and a second, nested PCR was performed. PCR products were barcoded and an Oxford Nanopore™ library was prepared using the Oxford Nanopore ligation sequencing kit. Libraries were then sequenced on a MinlON® flow cell and CAR sequences and 10x cell barcodes were determined.
Example 5:
Single CAR constructs were selected based on best activation propensity from the single cell gene expression analysis, and CAR sequences were synthesised by Integrated DNA technologies™ (IDT™). Primary T cells expressing the different CAR constructs were generated and used for functional assays.
Comparative antibodies 6 to 9 are prior art BCMA CARs (SEQ ID NOS: 56 to 59, encoded by DNA sequences represented by the SEQ ID NOS: 61 to 64). Comparative antibody 6 is used as an “antibody targeting B-cell maturation antigen and methods of use (US201801 18842A1). Comparative antibody 6 is used as an “antibody targeting B-cell maturation antigen and methods of use (US20180118842A1). Comparative antibody 7 is obtained from US20170281766A1 . Comparative antibody 8 has been demonstrated in a “method for treating tumor using immune effector cell (W02020020210A1)”. Comparative antibody 9 has been demonstrated use in cancer chemotherapy in WO2018028647A1 .
Comparative antibodies 6 to 8 comprise ScFv antigen binding fragments (SEQ ID NOS: 46-48 respectively, encoded by the DNA SEQ ID NOS: 51 -53). Comparative antibody 9 comprises a dual VHH antigen binding fragment (SEQ ID NO: 49, encoded by the DNA SEQ ID NO: 54).
The CAR labelled CD19 (SEQ ID NO: 60, encoded by DNA SEQ ID NO: 65) relates to a CAR with a CD19 antigen binding domain and acts as a negative control. CD19 CAR has been shown efficacy in “Biomarkers predictive of therapeutic responsiveness to chimeric antigen receptor therapy and uses thereof’ (W02016057705A1). CD19 is used as a negative control in these BCMA assays.
The activation level of the BCMA CAR T cells was assessed by quantifying the percentage change in GFP expression in Jurkat NFAT_reporter cell line upon exposure to the BCMA-expressing cells for 24 hours. The activation was calculated by comparing the GFP fluorescence before the exposure to the BCMA-expressing cells against the GFP fluorescence following 24 h exposure to the BCMA-expressing cells.
As is evident from Figure 2, each of the CARs (SEQ ID NOS 23 to 27) showed similar activation level against BCMA compared to the prior art CARs. Similarly, the activation level of the BCMA CAR T cells transduced with BCMA CAR 10-15 (SEQ ID NOs 90 to 95) (Figure 13) or 24 to 32 (SEQ ID NOs 200 to 208) (Figure 19) are comparable to that of the comparative antibody 9, BCMA CAR T cells transduced with CAR 16 to 19 constructs (SEQ ID NOs 124 to 127) are comparable to that of the BCMA CAR-3 transduced T cells (Figure 15), and BCMA CAR T cells transduced with CAR 20 to 23 (SEQ ID NOs 152 to 155) are comparable to that of the BCMA CAR-5 transduced T cells (Figure 17). Where ‘%GFP+’ is stated in the figures, this denotes percentage activation in a NFAT_GFP reporter cell line. ‘%AGFP’ (percentage delta GFP) denotes the difference between the percentage activation for Jurkat cells in the presence of target cells and Jurkat cells in the absence of target cells (i.e. removes background GFP activation due to tonic signalling), i.e. %GFP+ cells before target cell exposure is subtracted from percentage %GFP+ cells after target cell exposure. Figure 2 shows both (specific plus non-specific activation); whereas Figure 13 is the value of specific activation minus tonic activation.
This demonstrates the surprisingly beneficial effect of the antibody/antigen binding fragments of the present invention, and the utility of such antibody/antigen binding fragments in CARs, other bi-specific, tri-specific and multi-specific molecules (such as BiTEs and DART™s), and ADCs.
The affinity/avidity of the BCMA CAR T cells against BCMA was assessed by quantifying the median fluorescence intensity from PE (phycoerythrin) conjugated anti-Fc that detects BCMA bound to BCMA CAR-T cells transduced with CAR constructs. In the first step, the BCMA CAR-T cells transduced with different CAR constructs were stained with BCMA-Fc. In the second step, the cells were stained with PE-conjugated anti-Fc. To gate only on CAR-positive, transduced cells, select active/enriched T cells, the cells were stained with APC conjugated anti-CD34 antibodies. The median fluorescence intensity (MFI) is shown for CD34+ CAR-T cells.
In Figure 3, BCMA CAR-T cells transduced with CAR-1 through CAR-5 constructs exhibit a degree of BCMA binding comparable to comparative antibodies 6 to 9. BCMA CAR-T cells transduced with CAR- 10 to CAR-15 constructs demonstrate a comparable or higher degree of BCMA binding (Figure 14), while those transduced with CAR-16 through CAR-19 constructs show a significantly higher degree of BCMA binding relative to the comparative antibodies 6 to 9 (Figure 16). BCMA CAR-T cells transduced with CAR-20 to CAR-23 (Figure 18) and CAR-24 to CAR-32 (Figure 20) demonstrate a comparable or higher degree of BCMA binding.
Example 6:
Additionally, CAR-T cell activation was assessed via upregulation of activation markers, such as CD69 and CD25 on CD3+, CD34+ CAR-T cells after a 24h co-culture with RPMI8226 cells. As a non-activated control, CAR-T cells were cultured in the absence of target cells for 24h. After 24h cells were assessed for activation markers via flow cytometry (Figure 8). In addition, the supernatant of the cell co-cultures was used for IFNg ELISA assay. IFNg was quantified using human IFN-gamma and IL-2 Legendplex (Biolegend) (Figure 6, Figure 7, Figure 11).
Figure 8 shows cells transduced with BCMA CAR-1 to BCMA CAR-5 constructs and expanded for eight days and co-cultured for 24h with the BCMA-expressing cancer cell line (RPMI8226) at a 1 :1 effector to target cell ratio. 24 hours later cells were assessed by flow cytometer for the up-regulation of CD25 and CD69 markers. It demonstrates that BCMA CAR-1 to BCMA CAR-5 demonstrate comparable CD25 and CD69 upregulation compared to that of the prior art CARs 6, 7 and 9.
Figure 6 demonstrates that BCMA CAR T cells transduced with BCMA CAR-1 to CAR-5 constructs produce comparable IFN-gamma level to that of the comparative antibodies 6,7 and 9.
Figure 7 illustrates that BCMA CAR T cells transduced with BCMA CAR-1 or CAR-3 produce comparable IL-2 level to that of the comparative antibody 9, and higher than that of the comparative antibody 8.
Figure 11 illustrates that BCMA CAR T cells transduced with BCMA CAR-10 to BCMA CAR-15 constructs produce higher or comparable IFN-gamma to that of the comparative antibody 9.
Example 7:
To further evaluate the activity of different BCMA CARs, BCMA-CAR T-cells were evaluated for cellular toxicity and activity against cancer cells.
The ability of the different CARs to elicit functional responses was explored by incubating the CAR T- cells for 24 hours and/or 48 hours at 1 :1 ratio with BCMA-expressing cancer cell line (RPMI8226, K562_BCMA, Nalm-6, or U266B1). After 24h co-culture, cells were stained with anti-CD3 Pacific Blue, which label T cells and anti-BCMA APC cells, which label cancer cells, in order to separate T cells and cancer cells. Cells were acquired and counted using flow cytometry and cell killing percentage in the presence of BCMA-CAR T cells was calculated relative to cancer cells cultured in the presence of nontransduced T cells (Figure 4, Figure 5, Figure 9, Figure 10, Figure 12).
BCMA CAR T cells transduced with BCMA CAR-1 to CAR-5 constructs demonstrate a higher cell killing level relative to that of the comparative antibody 8, and similar level to that of the comparative antibodies 6,7 and 9 (Figure 4). In figure 4, the killing percentage of BCMA CAR T cells for with each CAR construct was calculated relative to the killing percentage observed in RPMI8226 cells cultured with nontransduced T cells.
BCMA CAR T cells transduced with BCMA CAR-1 to CAR-5 constructs demonstrate a higher cell killing level relative to that of the comparative antibody 8 and comparative antibody CD19 CAR, and similar level to that of the comparative antibodies 6,7 and 9 (Figure 5). In figure 5, the killing percentage of BCMA CAR T cells with each CAR construct was calculated relative to the killing percentage observed in K562_BCMA cells cultured with non-transduced T cells.
BCMA CAR T cells transduced with BCMA CAR-10 to BCMA-15 constructs demonstrate a comparable cell killing level relative to that of the comparative antibody 9 (Figure 9). In figure 9, the killing percentage of BCMA CAR T cells with each CAR construct was calculated relative to the killing percentage observed in Nalm-6 cells cultured with non-transduced T cells.
BCMA CAR T cells transduced with BCMA CAR-10 to BCMA-15 constructs demonstrate a comparable cell killing level relative to that of the comparative antibody 9 (Figure 10). In figure 10, the killing percentage of BCMA CAR T cells with each CAR construct was calculated relative to the killing percentage observed in RPMI8226 cells cultured with non-transduced T cells.
BCMA CAR T cells transduced with BCMA CAR-10 to CAR-15 constructs demonstrate a higher level relative to that of the comparative antibody 9 (Figure 12). BCMA CAR-T cells transduced with BCMA CAR 13 demonstrate comparable cell killing level compared to that of the comparative antibody 9. The killing percentage was calculated relative to that of U266B1 cells cultured with non-transduced T cells.
CD19 CAR of Figures 1 to 8 is synonymous (mans the same as) “negative control” in Figures 9 to 14, 19 and 20.
As is evident from the above, each of the BCMA CARs 1 to 5 (corresponding to CAR SEQ ID NOs 23 to 27 comprising the VHHs SEQ ID NOs 3, 4, 5, 15, 16) and each of the CARs 10-15 (corresponding to SEQ ID NOs 90 to 95 comprising the VHHs SEQ ID Nos 66 to 71), and the associated CDRs, as detailed in Table 1 , showed enhanced activity against BCMA-expressing cells than the prior art CARs with regards to anti-cancer killing activity.
This demonstrates the surprisingly beneficial effect of the antigen binding molecules/fragments of the present invention, and the utility of such antigen binding molecules fragments as CARs, other bispecific, tri-specific and multi-specific molecules (such as BiTEs and DART™s), and ADCs.
Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the invention. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention. Sequence listing:
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Claims

1 . An antigen binding molecule that binds BCMA; wherein the antigen binding molecule comprises an antigen binding domain selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89; and,
Antigen binding domain 1 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:6,
(ii) a CDR2 of SEQ ID NO:9, and
(iii) a CDR3 of SEQ ID NO:12; and,
Antigen binding domain 2, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:7,
(ii) a CDR2 of SEQ ID NQ:10, and
(iii) a CDR3 of SEQ ID NO:13; and, Antigen binding domain 3, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:8,
(ii) a CDR2 of SEQ ID NO:11 , and
(iii) a CDR3 of SEQ ID NO:14; and,
Antigen binding domain 4, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:17,
(ii) a CDR2 of SEQ ID NO:19, and
(iii) a CDR3 of SEQ ID NO:21 ; and,
Antigen binding domain 5, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:18,
(ii) a CDR2 of SEQ ID NQ:20, and
(iii) a CDR3 of SEQ ID NO:22; and,
Antigen binding domain 10, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:72,
(ii) a CDR2 of SEQ ID NO:78, and
(iii) a CDR3 of SEQ ID NO:84; and,
Antigen binding domain 11 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:73,
(ii) a CDR2 of SEQ ID NO:79, and
(iii) a CDR3 of SEQ ID NO:85; and,
Antigen binding domain 13, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:75,
(ii) a CDR2 of SEQ ID NO:81 , and
(iii) a CDR3 of SEQ ID NO:87; and,
Antigen binding domain 16, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:112,
(ii) a CDR2 of SEQ ID NO:116, and
(iii) a CDR3 of SEQ ID NQ:120; and,
Antigen binding domain 17, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:113,
(ii) a CDR2 of SEQ ID NO:117, and
(iii) a CDR3 of SEQ ID NO:121 ; and,
Antigen binding domain 18, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:114,
(ii) a CDR2 of SEQ ID NO:118, and
(iii) a CDR3 of SEQ ID NO:122; and,
Antigen binding domain 19, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:115,
(ii) a CDR2 of SEQ ID NO:119, and
(iii) a CDR3 of SEQ ID NO:123; and,
Antigen binding domain 20, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:140,
(ii) a CDR2 of SEQ ID NO:144, and
(iii) a CDR3 of SEQ ID NO:148; and,
Antigen binding domain 21 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:141 , (ii) a CDR2 of SEQ ID NO:145, and
(iii) a CDR3 of SEQ ID NO:149; and,
Antigen binding domain 22, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:142,
(ii) a CDR2 of SEQ ID NO:146, and
(iii) a CDR3 of SEQ ID NQ:150; and,
Antigen binding domain 23, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:143,
(ii) a CDR2 of SEQ ID NO:147, and
(iii) a CDR3 of SEQ ID NO:151 ; and,
Antigen binding domain 24, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:173,
(ii) a CDR2 of SEQ ID NO:182, and
(iii) a CDR3 of SEQ ID NO:191 ; and,
Antigen binding domain 25, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:174,
(ii) a CDR2 of SEQ ID NO:183, and
(iii) a CDR3 of SEQ ID NO:192; and,
Antigen binding domain 26, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:175,
(ii) a CDR2 of SEQ ID NO:184, and
(iii) a CDR3 of SEQ ID NO:193; and, Antigen binding domain 27, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:176,
(ii) a CDR2 of SEQ ID NO:185, and
(iii) a CDR3 of SEQ ID NO:194; and,
Antigen binding domain 28, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:177,
(ii) a CDR2 of SEQ ID NO:186, and
(iii) a CDR3 of SEQ ID NO:195; and,
Antigen binding domain 29, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:178,
(ii) a CDR2 of SEQ ID NO:187, and
(iii) a CDR3 of SEQ ID NO:196; and,
Antigen binding domain 30, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:179,
(ii) a CDR2 of SEQ ID NO:188, and
(iii) a CDR3 of SEQ ID NO:197; and,
Antigen binding domain 31 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:180,
(ii) a CDR2 of SEQ ID NO:189, and
(iii) a CDR3 of SEQ ID NO:198; and,
Antigen binding domain 32, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:181 ,
(ii) a CDR2 of SEQ ID NQ:190, and (iii) a CDR3 of SEQ ID NO:199; and,
Antigen binding domain 33, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:232,
(ii) a CDR2 of SEQ ID NO:237, and
(iii) a CDR3 of SEQ ID NO:242; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:247,
(ii) a CDR2 of SEQ ID NO:252, and
(iii) a CDR3 of SEQ ID NO:257; and,
Antigen binding domain 34, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:233,
(ii) a CDR2 of SEQ ID NO:238, and
(iii) a CDR3 of SEQ ID NO:243, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:248,
(ii) a CDR2 of SEQ ID NO:253, and
(iii) a CDR3 of SEQ ID NO:258; and,
Antigen binding domain 35, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:234,
(ii) a CDR2 of SEQ ID NO:239,
(iii) a CDR3 of SEQ ID NO:244, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:249,
(ii) a CDR2 of SEQ ID NO:254, and
(iii) a CDR3 of SEQ ID NO:259; and,
Antigen binding domain 36, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:235,
(ii) a CDR2 of SEQ ID NO:240,
(iii) a CDR3 of SEQ ID NO:245, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NQ:250,
(ii) a CDR2 of SEQ ID NO:255, and
(iii) a CDR3 of SEQ ID NQ:260; and,
Antigen binding domain 37, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:236,
(ii) a CDR2 of SEQ ID NO:241 ,
(iii) a CDR3 of SEQ ID NO:246, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:251 ,
(ii) a CDR2 of SEQ ID NO:256, and
(iii) a CDR3 of SEQ ID NO:261 .
2. The antigen binding molecule of claim 1 , wherein the antigen binding molecule comprises an antigen binding domain selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and (iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89.
3. The antigen binding molecule of claim 1 or 2, wherein the antigen binding domain binds to BCMA.
4. The antigen binding molecule of claim 1 to 3, wherein, the antigen binding domains 1 to 5 and 10 to 32 is a VHH or wherein, the antigen binding domains 33 to 37 is a ScFv.
5. The antigen binding molecule of claim 4, wherein:
(a) the VHH is selected from the group consisting of: SEQ ID NOs 3 to 5, SEQ ID NOs 15 to 16, SEQ ID NOs 66 to 71 , SEQ ID NOs 108 to 111 , SEQ ID NOs 136 to 139, and SEQ ID NOs 164 to 172, or
(b) the ScFv is selected from the group consisting of SEQ ID Nos 227 to 231 .
6. The antigen binding molecule of any one of claims 1 to 5, wherein the antigen binding molecule is a chimeric antigen receptor comprising a transmembrane region and an intracellular T-cell receptor signalling domain.
7. The antigen binding molecule of claim 6, wherein the hinge region and the transmembrane region is from CD8A and/or the intracellular T-cell receptor signalling domain is from CD247 (CD3-zeta).
8. The antigen binding molecule of claim 6 or 7, further comprising an intracellular domain of a costimulatory protein receptor.
9. The antigen binding molecule of any one of claims 1 to 5, wherein the antigen binding molecule is a bi-specific T-cell engager (BiTE) comprising a T-cell antigen binding domain.
10. The antigen binding molecule of claim 9, wherein the T-cell antigen binding domain comprises an antibody fragment that specifically binds CD3.
11 . The antigen binding molecule of any one of claims 1 to 5, wherein the antigen binding molecule is an antibody-drug conjugate (ADC) comprising a cytotoxic drug or a therapeutic radioisotope.
12. A vector encoding the antigen binding molecule of any one of claims 1 to 10.
13. The vector of claim 12, wherein the vector comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NOs 41 to 45, SEQ ID NOs 102 to 107, SEQ ID NOs 132 to 135, SEQ ID NOs 160 to 163, SEQ ID NOs 218 to 226, and SEQ ID NOs 272 to 276.
14. A pharmaceutical composition comprising the antigen binding molecule of any one of claims 1 to 11 and a pharmaceutically acceptable carrier.
15. A chimeric antigen receptor that binds BCMA comprising:
(a) one or more antigen binding domains selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89; and,
Antigen binding domain 1 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:6, (ii) a CDR2 of SEQ ID NO:9, and
(iii) a CDR3 of SEQ ID NO:12; and,
Antigen binding domain 2, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:7,
(ii) a CDR2 of SEQ ID NQ:10, and
(iii) a CDR3 of SEQ ID NO:13; and,
Antigen binding domain 3, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:8,
(ii) a CDR2 of SEQ ID NO:11 , and
(iii) a CDR3 of SEQ ID NO:14; and,
Antigen binding domain 4, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:17,
(ii) a CDR2 of SEQ ID NO:19, and
(iii) a CDR3 of SEQ ID NO:21 ; and,
Antigen binding domain 5, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:18,
(ii) a CDR2 of SEQ ID NQ:20, and
(iii) a CDR3 of SEQ ID NO:22; and,
Antigen binding domain 10, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:72,
(ii) a CDR2 of SEQ ID NO:78, and
(iii) a CDR3 of SEQ ID NO:84; and, Antigen binding domain 11 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:73,
(ii) a CDR2 of SEQ ID NO:79, and
(iii) a CDR3 of SEQ ID NO:85; and,
Antigen binding domain 13, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:75,
(ii) a CDR2 of SEQ ID NO:81 , and
(iii) a CDR3 of SEQ ID NO:87; and,
Antigen binding domain 16, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:112,
(ii) a CDR2 of SEQ ID NO:116, and
(iii) a CDR3 of SEQ ID NQ:120; and,
Antigen binding domain 17, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:113,
(ii) a CDR2 of SEQ ID NO:117, and
(iii) a CDR3 of SEQ ID NO:121 ; and,
Antigen binding domain 18, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:114,
(ii) a CDR2 of SEQ ID NO:118, and
(iii) a CDR3 of SEQ ID NO:122; and,
Antigen binding domain 19, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:115,
(ii) a CDR2 of SEQ ID NO:119, and
(iii) a CDR3 of SEQ ID NO:123; and, Antigen binding domain 20, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:140,
(ii) a CDR2 of SEQ ID NO:144, and
(iii) a CDR3 of SEQ ID NO:148; and,
Antigen binding domain 21 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:141 ,
(ii) a CDR2 of SEQ ID NO:145, and
(iii) a CDR3 of SEQ ID NO:149; and,
Antigen binding domain 22, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:142,
(ii) a CDR2 of SEQ ID NO:146, and
(iii) a CDR3 of SEQ ID NQ:150; and,
Antigen binding domain 23, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:143,
(ii) a CDR2 of SEQ ID NO:147, and
(iii) a CDR3 of SEQ ID NO:151 ; and,
Antigen binding domain 24, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:173,
(ii) a CDR2 of SEQ ID NO:182, and
(iii) a CDR3 of SEQ ID NO:191 ; and,
Antigen binding domain 25, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:174, (ii) a CDR2 of SEQ ID NO:183, and
(iii) a CDR3 of SEQ ID NO:192; and,
Antigen binding domain 26, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:175,
(ii) a CDR2 of SEQ ID NO:184, and
(iii) a CDR3 of SEQ ID NO:193; and,
Antigen binding domain 27, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:176,
(ii) a CDR2 of SEQ ID NO:185, and
(iii) a CDR3 of SEQ ID NO:194; and,
Antigen binding domain 28, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:177,
(ii) a CDR2 of SEQ ID NO:186, and
(iii) a CDR3 of SEQ ID NO:195; and,
Antigen binding domain 29, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:178,
(ii) a CDR2 of SEQ ID NO:187, and
(iii) a CDR3 of SEQ ID NO:196; and,
Antigen binding domain 30, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:179,
(ii) a CDR2 of SEQ ID NO:188, and
(iii) a CDR3 of SEQ ID NO:197; and, Antigen binding domain 31 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:180,
(ii) a CDR2 of SEQ ID NO:189, and
(iii) a CDR3 of SEQ ID NO:198; and,
Antigen binding domain 32, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:181 ,
(ii) a CDR2 of SEQ ID NQ:190, and
(iii) a CDR3 of SEQ ID NO:199; and,
Antigen binding domain 33, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:232,
(ii) a CDR2 of SEQ ID NO:237, and
(iii) a CDR3 of SEQ ID NO:242; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:247,
(ii) a CDR2 of SEQ ID NO:252, and
(iii) a CDR3 of SEQ ID NO:257; and,
Antigen binding domain 34, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:233,
(ii) a CDR2 of SEQ ID NO:238, and
(iii) a CDR3 of SEQ ID NO:243, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:248,
(ii) a CDR2 of SEQ ID NO:253, and
(iii) a CDR3 of SEQ ID NO:258; and, Antigen binding domain 35, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:234,
(ii) a CDR2 of SEQ ID NO:239,
(iii) a CDR3 of SEQ ID NO:244, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:249,
(ii) a CDR2 of SEQ ID NO:254, and
(iii) a CDR3 of SEQ ID NO:259; and,
Antigen binding domain 36, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:235,
(ii) a CDR2 of SEQ ID NQ:240,
(iii) a CDR3 of SEQ ID NO:245, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NQ:250,
(ii) a CDR2 of SEQ ID NO:255, and
(iii) a CDR3 of SEQ ID NQ:260; and,
Antigen binding domain 37, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:236,
(ii) a CDR2 of SEQ ID NO:241 ,
(iii) a CDR3 of SEQ ID NO:246, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:251 ,
(ii) a CDR2 of SEQ ID NO:256, and
(iii) a CDR3 of SEQ ID NO:261 , and b) A transmembrane region; and c) An intracellular signalling domain.
16. The chimeric antigen receptor of claim 15, wherein the one or more antigen binding domain is selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89.
17. The chimeric antigen receptor of claim 15 to 16, wherein the transmembrane region is from CD8A and/or the intracellular signalling domain is from CD247 (CD3-zeta).
18. The chimeric antigen receptor of claim 15 or 17, further comprising an intracellular signalling domain of a costimulatory protein receptor.
19. The chimeric antigen receptor of any one of claims 15 to 18, wherein the CAR comprises a human CD247 (CD3-zeta) cytoplasmic domain (SEQ ID NO:28), a human CD137 costimulatory domain (SEQ ID NO:29); a human CD8A hinge/transmembrane domain (SEQ ID NQ:30); and a CD8A extracellular signal peptide (SEQ ID NO:33).
20. The chimeric antigen receptor of any one of claims 15 to 19, wherein the CAR has a sequence selected from the group consisting of: SEQ ID NOS: 23 to 27, 90 to 95, 124 to 127, 152 to 155, 200 to 208 and 262 to 266.
21 . A recombinant T cell comprising the chimeric antigen receptor of any one of claims 15 to 20.
22. A bispecific T-cell engager that binds BCMA comprising:
(a) one or more antigen binding domains selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89; and,
Antigen binding domain 1 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:6,
(ii) a CDR2 of SEQ ID NO:9, and
(iii) a CDR3 of SEQ ID NO:12; and,
Antigen binding domain 2, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:7,
(ii) a CDR2 of SEQ ID NQ:10, and
(iii) a CDR3 of SEQ ID NO:13; and, Antigen binding domain 3, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:8,
(ii) a CDR2 of SEQ ID NO:11 , and
(iii) a CDR3 of SEQ ID NO:14; and,
Antigen binding domain 4, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:17,
(ii) a CDR2 of SEQ ID NO:19, and
(iii) a CDR3 of SEQ ID NO:21 ; and,
Antigen binding domain 5, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:18,
(ii) a CDR2 of SEQ ID NQ:20, and
(iii) a CDR3 of SEQ ID NO:22; and,
Antigen binding domain 10, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:72,
(ii) a CDR2 of SEQ ID NO:78, and
(iii) a CDR3 of SEQ ID NO:84; and,
Antigen binding domain 11 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:73,
(ii) a CDR2 of SEQ ID NO:79, and
(iii) a CDR3 of SEQ ID NO:85; and,
Antigen binding domain 13, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:75,
(ii) a CDR2 of SEQ ID NO:81 , and
(iii) a CDR3 of SEQ ID NO:87; and,
Antigen binding domain 16, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:112,
(ii) a CDR2 of SEQ ID NO:116, and
(iii) a CDR3 of SEQ ID NO:120; and,
Antigen binding domain 17, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:113,
(ii) a CDR2 of SEQ ID NO:117, and
(iii) a CDR3 of SEQ ID NO:121 ; and,
Antigen binding domain 18, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:114,
(ii) a CDR2 of SEQ ID NO:118, and
(iii) a CDR3 of SEQ ID NO:122; and,
Antigen binding domain 19, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:115,
(ii) a CDR2 of SEQ ID NO:119, and
(iii) a CDR3 of SEQ ID NO:123; and,
Antigen binding domain 20, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:140,
(ii) a CDR2 of SEQ ID NO:144, and
(iii) a CDR3 of SEQ ID NO:148; and,
Antigen binding domain 21 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:141 ,
(ii) a CDR2 of SEQ ID NO:145, and (iii) a CDR3 of SEQ ID NO:149; and,
Antigen binding domain 22, comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NO:142,
(ii) a CDR2 of SEQ ID NO:146, and
(iii) a CDR3 of SEQ ID NQ:150; and,
Antigen binding domain 23, comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NO:143,
(ii) a CDR2 of SEQ ID NO:147, and
(iii) a CDR3 of SEQ ID NO:151 ; and,
Antigen binding domain 24, comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NO:173,
(ii) a CDR2 of SEQ ID NO:182, and
(iii) a CDR3 of SEQ ID NO:191 ; and,
Antigen binding domain 25, comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NO:174,
(ii) a CDR2 of SEQ ID NO:183, and
(iii) a CDR3 of SEQ ID NO:192; and,
Antigen binding domain 26, comprising a single variable domain on heavy chain comprising,
(i) a CDR1 of SEQ ID NO:175,
(ii) a CDR2 of SEQ ID NO:184, and
(iii) a CDR3 of SEQ ID NO:193; and,
Antigen binding domain 27, comprising a single variable domain on a heavy chain comprising, (i) a CDR1 of SEQ ID NO:176,
(ii) a CDR2 of SEQ ID NO:185, and
(Hi) a CDR3 of SEQ ID NO:194; and,
Antigen binding domain 28, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:177,
(ii) a CDR2 of SEQ ID NO:186, and
(iii) a CDR3 of SEQ ID NO:195; and,
Antigen binding domain 29, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:178,
(ii) a CDR2 of SEQ ID NO:187, and
(iii) a CDR3 of SEQ ID NO:196; and,
Antigen binding domain 30, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:179,
(ii) a CDR2 of SEQ ID NO:188, and
(iii) a CDR3 of SEQ ID NO:197; and,
Antigen binding domain 31 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:180,
(ii) a CDR2 of SEQ ID NO:189, and
(iii) a CDR3 of SEQ ID NO:198; and,
Antigen binding domain 32, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:181 ,
(ii) a CDR2 of SEQ ID NQ:190, and
(iii) a CDR3 of SEQ ID NO:199; and, Antigen binding domain 33, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:232,
(ii) a CDR2 of SEQ ID NO:237, and
(iii) a CDR3 of SEQ ID NO:242; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:247,
(ii) a CDR2 of SEQ ID NO:252, and
(iii) a CDR3 of SEQ ID NO:257; and,
Antigen binding domain 34, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:233,
(ii) a CDR2 of SEQ ID NO:238, and
(iii) a CDR3 of SEQ ID NO:243, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:248,
(ii) a CDR2 of SEQ ID NO:253, and
(iii) a CDR3 of SEQ ID NO:258; and,
Antigen binding domain 35, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:234,
(ii) a CDR2 of SEQ ID NO:239,
(iii) a CDR3 of SEQ ID NO:244, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:249,
(ii) a CDR2 of SEQ ID NO:254, and
(iii) a CDR3 of SEQ ID NO:259; and, Antigen binding domain 36, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:235,
(ii) a CDR2 of SEQ ID NO:240,
(iii) a CDR3 of SEQ ID NO:245, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NQ:250,
(ii) a CDR2 of SEQ ID NO:255, and
(iii) a CDR3 of SEQ ID NQ:260; and,
Antigen binding domain 37, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:236,
(ii) a CDR2 of SEQ ID NO:241 ,
(iii) a CDR3 of SEQ ID NO:246, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:251 ,
(ii) a CDR2 of SEQ ID NO:256, and
(iii) a CDR3 of SEQ ID NO:261 , and b) An antigen binding domain which binds to a T-cell antigen; and c) A linker joining the one or more antigen binding domains of (a) and antigen binding domain which binds to a T-cell antigen of (b).
23. The bi-specific T-cell engager of claim 22, wherein the one or more antigen binding domains is selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and (iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89.
24. The bi-specific T-cell engager of claim 22 to 23, wherein the antigen binding domain which binds to a T-cell antigen comprises an antigen binding fragment that specifically binds CD3.
25. The bi-specific T-cell engager of claim 22 to 24, wherein the antigen binding fragment that specifically binds CD3 comprises:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:35,
(ii) a CDR2 of SEQ ID NO:36, and
(iii) a CDR3 of SEQ ID NO:37; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:38,
(ii) a CDR2 of SEQ ID NO:39, and
(iii) a CDR3 of SEQ ID NQ:40.
26. An antibody-drug conjugate that binds BCMA comprising:
(a) one or more antigen binding domains selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and, Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NO:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89; and,
Antigen binding domain 1 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:6,
(ii) a CDR2 of SEQ ID NO:9, and
(iii) a CDR3 of SEQ ID NO:12; and,
Antigen binding domain 2, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:7,
(ii) a CDR2 of SEQ ID NQ:10, and
(iii) a CDR3 of SEQ ID NO:13; and,
Antigen binding domain 3, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:8,
(ii) a CDR2 of SEQ ID NO:11 , and
(iii) a CDR3 of SEQ ID NO:14; and,
Antigen binding domain 4, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:17,
(ii) a CDR2 of SEQ ID NO:19, and
(iii) a CDR3 of SEQ ID NO:21 ; and,
Antigen binding domain 5, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:18,
(ii) a CDR2 of SEQ ID NO:20, and
(iii) a CDR3 of SEQ ID NO:22; and,
Antigen binding domain 10, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:72,
(ii) a CDR2 of SEQ ID NO:78, and
(iii) a CDR3 of SEQ ID NO:84; and,
Antigen binding domain 11 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:73,
(ii) a CDR2 of SEQ ID NO:79, and
(iii) a CDR3 of SEQ ID NO:85; and,
Antigen binding domain 13, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:75,
(ii) a CDR2 of SEQ ID NO:81 , and
(iii) a CDR3 of SEQ ID NO:87; and,
Antigen binding domain 16, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 12,
(ii) a CDR2 of SEQ ID NO:116, and
(iii) a CDR3 of SEQ ID NQ:120; and,
Antigen binding domain 17, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:1 13,
(ii) a CDR2 of SEQ ID NO:117, and
(iii) a CDR3 of SEQ ID NO:121 ; and,
Antigen binding domain 18, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:114,
(ii) a CDR2 of SEQ ID NO:118, and
(iii) a CDR3 of SEQ ID NO:122; and,
Antigen binding domain 19, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:115,
(ii) a CDR2 of SEQ ID NO:119, and
(iii) a CDR3 of SEQ ID NO:123; and,
Antigen binding domain 20, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:140,
(ii) a CDR2 of SEQ ID NO:144, and
(iii) a CDR3 of SEQ ID NO:148; and,
Antigen binding domain 21 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:141 ,
(ii) a CDR2 of SEQ ID NO:145, and
(iii) a CDR3 of SEQ ID NO:149; and,
Antigen binding domain 22, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:142,
(ii) a CDR2 of SEQ ID NO:146, and
(iii) a CDR3 of SEQ ID NQ:150; and,
Antigen binding domain 23, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:143, (ii) a CDR2 of SEQ ID NO:147, and
(iii) a CDR3 of SEQ ID NO:151 ; and,
Antigen binding domain 24, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:173,
(ii) a CDR2 of SEQ ID NO:182, and
(iii) a CDR3 of SEQ ID NO:191 ; and,
Antigen binding domain 25, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:174,
(ii) a CDR2 of SEQ ID NO:183, and
(iii) a CDR3 of SEQ ID NO:192; and,
Antigen binding domain 26, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:175,
(ii) a CDR2 of SEQ ID NO:184, and
(iii) a CDR3 of SEQ ID NO:193; and,
Antigen binding domain 27, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:176,
(ii) a CDR2 of SEQ ID NO:185, and
(iii) a CDR3 of SEQ ID NO:194; and,
Antigen binding domain 28, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:177,
(ii) a CDR2 of SEQ ID NO:186, and
(iii) a CDR3 of SEQ ID NO:195; and, Antigen binding domain 29, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:178,
(ii) a CDR2 of SEQ ID NO:187, and
(iii) a CDR3 of SEQ ID NO:196; and,
Antigen binding domain 30, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:179,
(ii) a CDR2 of SEQ ID NO:188, and
(iii) a CDR3 of SEQ ID NO:197; and,
Antigen binding domain 31 , comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NQ:180,
(ii) a CDR2 of SEQ ID NO:189, and
(iii) a CDR3 of SEQ ID NO:198; and,
Antigen binding domain 32, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:181 ,
(ii) a CDR2 of SEQ ID NQ:190, and
(iii) a CDR3 of SEQ ID NO:199; and,
Antigen binding domain 33, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:232,
(ii) a CDR2 of SEQ ID NO:237, and
(iii) a CDR3 of SEQ ID NO:242; and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:247,
(ii) a CDR2 of SEQ ID NO:252, and
(iii) a CDR3 of SEQ ID NO:257; and, Antigen binding domain 34, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:233,
(ii) a CDR2 of SEQ ID NO:238, and
(iii) a CDR3 of SEQ ID NO:243, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:248,
(ii) a CDR2 of SEQ ID NO:253, and
(iii) a CDR3 of SEQ ID NO:258; and,
Antigen binding domain 35, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:234,
(ii) a CDR2 of SEQ ID NO:239, and
(iii) a CDR3 of SEQ ID NO:244, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:249,
(ii) a CDR2 of SEQ ID NO:254, and
(iii) a CDR3 of SEQ ID NO:259; and,
Antigen binding domain 36, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:235,
(ii) a CDR2 of SEQ ID NQ:240, and
(iii) a CDR3 of SEQ ID NO:245, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NQ:250,
(ii) a CDR2 of SEQ ID NO:255, and
(iii) a CDR3 of SEQ ID NQ:260; and, Antigen binding domain 37, comprising:
(a) a heavy chain variable region comprising,
(i) a CDR1 of SEQ ID NO:236,
(ii) a CDR2 of SEQ ID NO:241 , and
(iii) a CDR3 of SEQ ID NO:246, and
(b) a light chain variable region comprising,
(i) a CDR1 of SEQ ID NO:251 ,
(ii) a CDR2 of SEQ ID NO:256, and
(iii) a CDR3 of SEQ ID NO:261 , and b) A therapeutic drug substance; c) A linker joining the one or more antigen binding domains of (a) and the therapeutic drug substance of (b).
27. An antibody-drug conjugate of claim 26, wherein the one or more antigen binding domains is selected from the group consisting of:
Antigen binding domain 14, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:76,
(ii) a CDR2 of SEQ ID NO:82, and
(iii) a CDR3 of SEQ ID NO:88; and,
Antigen binding domain 12, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:74,
(ii) a CDR2 of SEQ ID NQ:80, and
(iii) a CDR3 of SEQ ID NO:86; and,
Antigen binding domain 15, comprising a single variable domain on a heavy chain comprising,
(i) a CDR1 of SEQ ID NO:77,
(ii) a CDR2 of SEQ ID NO:83, and
(iii) a CDR3 of SEQ ID NO:89.
28. The antibody-drug conjugate of claim 26 or 27, wherein the therapeutic drug substance is a cytotoxic drug.
29. The chimeric antigen receptor of claims 15 to 20, the bispecific T-cell engager of claims 22 to 25, or the antibody-drug conjugate of claims 26 to 28 wherein the one or more antigen binding domains bind BCMA.
30. The chimeric antigen receptor of claims 15 to 20 and 29, the bispecific T-cell engager of claims 22 to 25 and 29, or the antibody-drug conjugate of claims 26 to 28, wherein, the single variable domain on a heavy chain of any one of antigen binding domain 1 to 32 is a VHH or a ScFv.
31. The chimeric antigen receptor, the bispecific T-cell engager, or the antibody-drug conjugate of claim 27, wherein:
(a) the VHH is selected from the group consisting of: SEQ ID NOs 3 to 5, SEQ ID NOs 15 to 16, SEQ ID NOs 66 to 71 , SEQ ID NOs 108 to 111 , SEQ ID NOs 136 to 139, and SEQ ID NOs 164 to 172, or
(b) the ScFv is selected from the group consisting of SEQ ID Nos 227 to 231 .
32. The antigen binding molecule of claims 1 to 11 , the pharmaceutical composition of claim 14, the chimeric antigen receptor of claims 15 to 20 and 29, the recombinant T-cell of claim 21 , the bispecific T-cell engager of claims 22 to 25 and 29, the antibody-drug conjugate of claims 26 to 29, for use in medicine.
33. The antigen binding molecule of claims 1 to 11 , the pharmaceutical composition of claim 14, the chimeric antigen receptor of claims 15 to 20 and 29, the recombinant T-cell of claim 21 , the bispecific T-cell engager of claims to 25 and 29, the antibody-drug conjugate of claims 25 to 29 for use in killing or inhibiting the growth of cells expressing BCMA.
34. The antigen binding molecule of claims 1 to 11 , the pharmaceutical composition of claim 14, the chimeric antigen receptor of claims 15 to 20 and 29, the recombinant T-cell of claim 21 , the bispecific T-cell engager of claims 22 to 25 and 29, the antibody-drug conjugate of claims 26 to 29 for use in the treatment of a disease or disorder selected from the group consisting of: MM; Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
35. A method of treating a disease or condition associated with the expression of BCMA, said method comprising administering to a patient in need thereof an effective amount of an antigen binding molecule, of any one of claims 1 to 11 , a pharmaceutical composition of claim 14, a chimeric antigen receptor of any one of claims 15 to 20, a recombinant T-cell of claim 21 , a bispecific T-cell engager of any one of claims 22 to 25 or the antibody-drug conjugate of any one of claims 26 to 29 to a patient in need thereof.
36. The method of treatment of Claim 35, wherein the disease or condition is selected from the group consisting of: MM; Hodgkin’s and non-Hodgkin’s lymphoma; various leukaemias; glioblastoma; Waldentrsom macroglobulinemia; multiple sclerosis; neuroborreloiosis; systemic lupus erythematosus (SLE); rheumatoid arthritis (RA) and Sjogren’s syndrome.
PCT/GB2024/052828 2023-11-07 2024-11-07 Bcma binding molecules Pending WO2025099434A1 (en)

Applications Claiming Priority (2)

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