WO2023183758A2 - Molécules multispécifiques ciblant cd3 et magea4 et leurs utilisations - Google Patents

Molécules multispécifiques ciblant cd3 et magea4 et leurs utilisations Download PDF

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WO2023183758A2
WO2023183758A2 PCT/US2023/064649 US2023064649W WO2023183758A2 WO 2023183758 A2 WO2023183758 A2 WO 2023183758A2 US 2023064649 W US2023064649 W US 2023064649W WO 2023183758 A2 WO2023183758 A2 WO 2023183758A2
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amino acid
seq
acid sequence
antigen
binding complex
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WO2023183758A3 (fr
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Jennifer Finney
Lauric Haber
Chia-Yang Lin
Ryan MCKAY
Thomas Meagher
Eric Smith
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Regeneron Pharmaceuticals, Inc.
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Publication of WO2023183758A2 publication Critical patent/WO2023183758A2/fr
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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    • 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
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to alternative formats for multivalent antigen-binding proteins, and methods of use thereof.
  • the multivalent antigen-binding proteins including bispecific and multispecific molecules, may comprise a first polypeptide chain with an N-terminal and/or a C- terminal antigen-binding domain that specifically binds a T-cell antigen (e.g., CD3), and a second polypeptide chain comprising at least one antigen-binding domain that binds a target antigen (e.g., MAGE-A4).
  • MAGE-A4 is a cancer-testis antigen (CTA) on the X chromosome.
  • CTA cancer-testis antigen
  • the function of MAGE-A4 is unknown, but it may be involved in cell cycle progression/regulation, transcriptional control, cell survival and/or apoptosis.
  • overexpression of MAGE-A4 has been shown to promote growth of spontaneously transformed oral keratinocytes and inhibit growth arrest of cells in G1 .
  • MAGE-A4 is abundantly expressed by many tumors of different histological types, such as head and neck squamous cell carcinoma, lung carcinoma, such as non-small cell lung carcinoma, esophageal squamous cell carcinoma, colon carcinoma, bladder cancer, mucosal and cutaneous melanomas, ovarian carcinoma, e.g., serous carcinoma, and uterine carcinoma but, in normal healthy adult tissues, MAGE-A4 expression is restricted to the testes.
  • lung carcinoma such as non-small cell lung carcinoma, esophageal squamous cell carcinoma, colon carcinoma, bladder cancer, mucosal and cutaneous melanomas, ovarian carcinoma, e.g., serous carcinoma, and uterine carcinoma
  • ovarian carcinoma e.g., serous carcinoma, and uterine carcinoma
  • MAGE-A4 antigens have rendered MAGE-A4 a good candidate for cancer immunotherapy.
  • Bispecific and multispecific antibodies and antigen-binding molecules are molecules that can bind to two or more antigens.
  • FcFc* structure a traditional bispecific antibody with Fab antigen-binding domains on either arm of the antibody and an Fc region with a modified CH3 domain that changes Protein A binding affinity to permit isolation of the heterodimer from the homodimeric impurities.
  • bispecific or multispecific antigen-binding molecules including newer formats for such molecules, that improve desired functionalities.
  • the present invention provides multispecific antigen-binding molecules that bind both a T-cell antigen (TCA) (e.g., CD3) and a target antigen (TA) (e.g., MAGE-A4).
  • TCA T-cell antigen
  • TA target antigen
  • the present invention provides a multispecific molecular binding complex comprising a first binding segment (S1) and a second binding segment (S2), wherein S1 comprises a first polypeptide unit comprising, from N-terminus to C-terminus, a first antigen-binding domain (ABD1), a first multimerizing domain (M1), and a second antigenbinding domain (ABD2); wherein S2 comprises a second polypeptide unit comprising, from N-terminus to C-terminus, a third antigen-binding domain (ABD3), a second multimerizing domain (M2), and a fourth antigenbinding domain (ABD4); wherein S1 and S2 associate with one another via interactions between M1 and M2, wherein ABD1 specifically binds a first CD3 polypeptide sequence, ABD2 specifically binds a second CD3 polypeptide sequence, ABD3 specifically binds a first HI_A-bound Melanoma- Associated Antigen A4 (M
  • the first polypeptide unit may comprise one polypeptide, or more than one polypeptide (e.g., two, three).
  • the second polypeptide unit may comprise one polypeptide, or more than one polypeptide (e.g., two, three).
  • the first HLA-bound MAGE-A4 peptide and the second HLA bound MAGE-A4 peptide comprise the same sequence.
  • the first HLA-bound MAGE-A4 peptide comprises amino acids 286- 294, or a portion thereof, of SEQ ID NO: 187.
  • the first HLA-bound MAGE-A4 peptide comprises amino acids 230- 239, or a portion thereof, of SEQ ID NO: 187.
  • the first HLA-bound MAGE-A4 peptide and the second HLA bound MAGE-A4 peptide comprise different sequences.
  • the first HLA-bound MAGE-A4 peptide comprises amino acids 286- 294, or a portion thereof, of SEQ ID NO: 187.
  • the second HLA bound MAGE-A4 peptide comprises amino acids 230-239, or a portion thereof, of SEQ ID NO: 187.
  • the first CD3 polypeptide sequence and the second CD3 polypeptide sequence are the same. In some embodiments, the first CD3 polypeptide sequence and the second CD3 polypeptide sequence are different.
  • ABD1 , ABD2, ABD3, and/or ABD4 each comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR).
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • one or more of ABD1 , ABD2, ABD3, and ABD4 are a Fab or scFv.
  • the Fab comprises a heavy chain variable region (HCVR) and a heavy chain CH1 domain paired with a light chain variable region (LCVR) and a CL domain.
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • the first polypeptide unit comprises at least one first light chain polypeptide comprising an LCVR and a CL domain.
  • the second polypeptide unit comprises at least one second light chain polypeptide comprising an LCVR and a CL domain.
  • the scFv comprises a heavy chain variable region (HCVR) comprising a cysteine mutation at residue 44, and a light chain variable region comprising a cysteine mutation at residue 100 (Kabat numbering).
  • HCVR heavy chain variable region
  • Kabat numbering a light chain variable region comprising a cysteine mutation at residue 100
  • the scFv comprises an HCVR and an LCVR joined together via a polypeptide linker of from 5 to 30 amino acids.
  • the polypeptide linker is a (G4S) 4 (SEQ ID NO: 268) linker.
  • the scFv(s) are connected to the C-terminus of the first and/or second multimerizing domain via a polypeptide linker of from 5 to 25 amino acids.
  • the polypeptide linker is a (G4S)3 (SEQ ID NO: 269) linker.
  • the Fab(s) are connected to the C-terminus of the first and/or second multimerizing domain via a polypeptide linker of from 5 to 25 amino acids.
  • the polypeptide linker is a (G4S)s (SEQ ID NO: 269) linker.
  • ABD1 comprises a Fab.
  • ABD1 comprises an HCVR comprising three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 231.
  • CDRs heavy chain complementarity determining regions
  • HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 233
  • HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 235
  • HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 237.
  • ABD1 comprises an HCVR comprising an amino acid sequence of SEQ ID NO: 231 , or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 231.
  • ABD1 comprises an LCVR comprising three light chain complementarity determining regions (CDRs) (LCDR1 , LCDR2 and LCDR3) contained within an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 255.
  • LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 257
  • LCDR2 comprises the amino acid sequence set forth in AAS
  • LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 261.
  • ABD1 comprises an LCVR comprising an amino acid sequence an amino acid sequence of SEQ ID NO: 255, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 255.
  • ABD2 is an scFv.
  • ABD2 comprises an HCVR comprising three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 247.
  • CDRs heavy chain complementarity determining regions
  • HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 249
  • HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 251
  • HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 253.
  • ABD2 comprises an HCVR comprising an amino acid sequence an amino acid sequence of SEQ ID NO: 247, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 247.
  • ABD2 comprises an LCVR comprising three light chain complementarity determining regions (CDRs) (LCDR1 , LCDR2 and LCDR3) contained within an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 239.
  • LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 241
  • LCDR2 comprises the amino acid sequence set forth in AAS
  • LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 245.
  • ABD2 comprises an LCVR comprising an amino acid sequence an amino acid sequence of SEQ ID NO: 239, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 239.
  • ABD3 is a Fab.
  • ABD3 comprises an HCVR comprising three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 207.
  • CDRs heavy chain complementarity determining regions
  • HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 209
  • HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 211
  • HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 213.
  • ABD3 comprises an HCVR comprising an amino acid sequence an amino acid sequence of SEQ ID NO: 207, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 207.
  • ABD3 comprises an LCVR comprising three light chain complementarity determining regions (CDRs) (LCDR1 , LCDR2 and LCDR3) contained within an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 255.
  • LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 257
  • LCDR2 comprises the amino acid sequence set forth in AAS
  • LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 261.
  • ABD3 comprises an LCVR comprising an amino acid sequence an amino acid sequence of SEQ ID NO: 255, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 255.
  • ABD4 is an scFv.
  • ABD4 comprises an HCVR comprising three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 223.
  • CDRs heavy chain complementarity determining regions
  • HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 225
  • HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 227
  • HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 229.
  • ABD4 comprises an HCVR comprising an amino acid sequence an amino acid sequence of SEQ ID NO: 223, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 223.
  • ABD4 comprises an LCVR comprising three light chain complementarity determining regions (CDRs) (LCDR1 , LCDR2 and LCDR3) contained within an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 215.
  • LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 217
  • LCDR2 comprises the amino acid sequence set forth in AAS
  • LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 221.
  • ABD4 comprises an LCVR comprising an amino acid sequence an amino acid sequence of SEQ ID NO: 215, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 215.
  • the first and second multimerizing domains are immunoglobulin Fc domains.
  • the first and second multimerizing domains associate with one another via disulfide bonding.
  • the first multimerizing domain and the second multimerizing domain are human IgG 1 or human lgG4 Fc domains.
  • the first multimerizing domain or the second multimerizing domain comprises an amino acid substitution that reduces affinity for Protein A binding compared to a wildtype Fc domain of the same isotype.
  • the amino acid substitution comprises an H435R modification, or H435R and Y436F modifications (EU numbering).
  • the first multimerizing domain comprises the H435R and Y436F modifications.
  • the first polypeptide, the second polypeptide, or both the first and the second polypeptides comprise a modified hinge domain that reduces binding affinity for an Fey receptor relative to a wild-type hinge domain of the same isotype.
  • S1 comprises a first polypeptide comprising an amino acid sequence of SEQ ID NO: 265, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 265; and/or a first light chain polypeptide comprising an amino acid sequence of SEQ ID NO: 267, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 267.
  • S2 comprises a second polypeptide comprising an amino acid sequence of SEQ ID NO: 263, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 263; and/or a second light chain polypeptide comprising an amino acid sequence of SEQ ID NO: 267, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 267.
  • the present disclosure provides a multispecific molecular binding complex comprising a first binding segment (S1) and a second binding segment (S2), wherein S1 comprises i) a first polypeptide comprising, from N-terminus to C-terminus, a first heavy chain variable region (HCVR), a first heavy chain CH1 domain, a first immunoglobulin Fc domain, and a first scFv that specifically binds a first CD3 polypeptide sequence; and ii) a first light chain polypeptide comprising a first light chain variable region (LCVR) and a first CL domain, wherein the first light chain polypeptide pairs with the first HCVR and the first heavy chain CH1 domain to form a first Fab that specifically binds a second CD3 polypeptide sequence; wherein S2 comprises i) a second polypeptide comprising, from N-terminus to C-terminus, a third HCVR, a second heavy chain CH1 domain, a second immunoglobulin
  • the present disclosure provides an isolated nucleic acid molecule encoding the multispecific molecular binding complex of any of the above embodiments.
  • the nucleic acid molecule comprises a nucleotide sequence encoding the first polypeptide of the multispecific molecular binding complex comprising SEQ ID NO: 264, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 264.
  • the nucleic acid molecule comprises a nucleotide sequence encoding a first light chain polypeptide of the multispecific molecular binding complex comprising SEQ ID NO: 266, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 266.
  • the nucleic acid molecule comprises a nucleotide sequence encoding a second polypeptide of the multispecific molecular binding complex comprising SEQ ID NO: 262, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 262.
  • the nucleic acid molecule comprises a nucleotide sequence encoding a second light chain polypeptide of the multispecific molecular binding complex comprising SEQ ID NO: 266, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 266.
  • the present disclosure provides an isolated nucleic acid molecule encoding the first polypeptide of the multispecific molecular binding complex of any of the above embodiments.
  • the isolated nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 264, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 264.
  • the present disclosure provides an isolated nucleic acid molecule encoding the at least one first light chain polypeptide of the multispecific molecular binding complex of any one of the above embodiments.
  • the isolated nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 266, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 266.
  • the present disclosure provides an isolated nucleic acid molecule encoding the second polypeptide of the multispecific molecular binding complex of any one of the above embodiments.
  • the isolated nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 262, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 262.
  • the present disclosure provides an isolated nucleic acid molecule encoding the at least one second light chain polypeptide of the multispecific molecular binding complex of any one of above embodiments.
  • the isolated nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 266, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 95.5%, or 99.9% sequence identity to the nucleotide sequence of SEQ ID NO: 266.
  • the polynucleotide is operably linked to a regulatory sequence at least one regulator element for expression of said polypeptide.
  • the at least one regulator element is a promoter.
  • the isolated nucleic acid molecule is a DNA molecule or a derivative thereof.
  • the isolated nucleic acid molecule is an RNA molecule or a derivative thereof.
  • the present disclosure provides a vector comprising the nucleic acid molecule of any one of the above embodiments.
  • the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, an adenovirus vector, or a retroviral vector.
  • the present disclosure provides a cell comprising the nucleic acid described herein or the vector described herein.
  • the present disclosure provides a pharmaceutical composition comprising the multispecific molecular binding complex described herein, the nucleic acid molecule described herein, or the vector described herein, and a pharmaceutically acceptable carrier or diluent.
  • the present disclosure provides a method of treating a cancer, comprising administering the multispecific molecular binding complex described herein, the nucleic acid molecule described herein, the vector described herein, or the pharmaceutical composition described herein to a subject in need thereof, wherein one or more cells of the cancer presents the first and/or second HLA bound MAGE-A4 peptide on the cell surface.
  • the cancer is multiple myeloma, synovial sarcoma, esophageal cancer, head and neck cancer, lung cancer, bladder cancer, ovarian cancer, uterine cancer, stomach cancer, cervical cancer, breast cancer, or melanoma.
  • the cancer is melanoma.
  • the cancer is lung cancer.
  • the cancer is bladder cancer.
  • the bladder cancer is urinary bladder squamous cell carcinoma.
  • the cancer is synovial sarcoma.
  • the molecule is administered in combination with one or more second therapeutic agents.
  • the one or more second therapeutic agents comprise a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds a target antigen (TA) and a second antigen-binding domain that binds a T-cell antigen.
  • the target antigen is a tumor-cell antigen.
  • the one or more second therapeutic agents comprise a bispecific anti-TA x anti-CD28 antibody.
  • the one or more second therapeutic agents comprise a bispecific anti-EGFR x anti-CD28 antibody.
  • the one or more second therapeutic agents comprise an antibody that binds a check-point inhibitor on a T cell. In some embodiments, the one or more second therapeutic agents comprise an anti-PD-1 antibody.
  • the molecule is administered in combination with a bispecific anti- EGFR x anti-CD28 antibody and an anti-PD-1 antibody.
  • the present disclosure provides a method for making the multispecific molecular binding complex described herein, comprising expressing the nucleic acid described herein or the vector described herein in a cell.
  • any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure. [0066] Other embodiments will become apparent from a review of the ensuing detailed description.
  • Figs. 1 A and 1 B illustrate known bispecific antibody and antigen-binding molecule formats.
  • Figs. 1C, 1 E, 1F, 1G, 1 H, 11, 1J,1 K, 1 L, 1M, 1N, 10, 1 P, 1Q, 1 R and 1S illustrate bispecific or multispecific antigen-binding molecule formats in accordance with embodiments of the present invention.
  • a first polypeptide chain comprises both an N-terminal and a C- terminal antigen-binding domain (e.g., a Fab or scFv) that specifically binds a T-cell antigen (TCA) (e.g., CD3), and a second polypeptide chain comprising at least one antigen-binding domain (e.g., a Fab or scFv) that binds a target antigen (TA) (e.g., a tumor cell antigen).
  • TCA T-cell antigen
  • TA target antigen
  • FIG. 1 D illustrates a format in which the two antigen-binding domains that specifically bind a T-cell antigen (e.g., CD3) are located on different polypeptide chains (at the N-terminus on one polypeptide chain, and at the C- terminus on the second polypeptide chain).
  • T-cell antigen e.g., CD3
  • FIG. 1 D illustrates a format in which the two antigen-binding domains that specifically bind a T-cell antigen (e.g., CD3) are located on different polypeptide chains (at the N-terminus on one polypeptide chain, and at the C- terminus on the second polypeptide chain).
  • Fig. 1T illustrates exemplary 2+2 bispecific antibody (bsAb) formats.
  • bsAb bispecific antibody
  • Various valencies and geometries are displayed. Orientation of the targeting arms and/or valency may allow specific T-cell activation and/or cytotoxic activity against cells expressing, e.g., peptide antigens.
  • Fig. 2A shows a schematic representation of a multispecific molecular binding complex of the present disclosure.
  • Fig. 2B shows a schematic representation of a 2+2 bispecific antibody design.
  • the bsAb may comprise, for example, a first polypeptide comprising an N-terminus effector arm and/or a C- terminus effector arm which may comprise an antigen-binding fragment(s), e.g., a fragment antigenbinding region (Fab region) and/or a single chain fragment variable (scFv).
  • a second polypeptide of the bsAb may comprise an N-terminus and/or a C-terminus peptide in groove (PIG) target arm which may comprise a Fab and/or an scFv.
  • a multimerization domain(s) comprising an FC domain of an lgG4 is also depicted.
  • Fig. 2C shows a schematic representation of bsAb9930 designed to target two different melanoma-associated antigen 4 (MAGE-A4) peptides.
  • Fig. 2D shows a cartoon of an exemplary mechanism of action for bsAb9930 in combination with a CD28-based bispecific antibody (epidermal growth factor receptor [EGFR] x cluster of differentiation 28 [CD28]) and an anti-programmed cell death protein 1 (PD1) antibody targeting A375 cells.
  • Figs. 3A-3C demonstrate bsAb9930 shows maximal efficacy in preventing A375 tumor growth when combined with PD-1 blockade and tumor localized CD28 costimulation.
  • Fig. 3A shows coadministration of bsAb9930 Alti-flex with tumor localized CD28 costimulation and anti-PD-1 blockade completely regresses A-375 tumors.
  • FIG. 3B demonstrates coadministration of tumor localized CD28 costimulation and bsAb9930 Alti-flex selectively enhances the proportion of activated CD4+ and CD8+ T cells in the tumor relative to the spleen at 48 hours following initial dosing.
  • Fig. 3C demonstrates that bsAb9930 shows efficacy in preventing SCaBER urinary bladder tumor growth when combined with PD-1 blockade and tumor localized CD28 costimulation.
  • Figs. 4A-4B demonstrate conventional tumor-associated antigen (TAA)xCD3 bsAbs can be inefficient at redirecting T cells to kill tumor cell lines expressing endogenous MHC-peptide tumor neoantigens.
  • Fig. 4A shows selected HLA-A2: MAGE-A4 (286-294) antibody paired to a CD3 arm to generate a bispecific antibody. The bispecific antibody’s binding to Jurkat and A375 cells overexpressing the peptide of interest was assessed by flow cytometry.
  • Fig. 4A-4B demonstrate conventional tumor-associated antigen (TAA)xCD3 bsAbs can be inefficient at redirecting T cells to kill tumor cell lines expressing endogenous MHC-peptide tumor neoantigens.
  • Fig. 4A shows selected HLA-A2: MAGE-A4 (286-294) antibody paired to a CD3 arm to generate a bispecific antibody. The bispecific antibody’s binding to Jurkat and A375 cells overexpressing the peptid
  • HLA-A2 MAGE-A4 (286-294) x CD3 bispecific antibody against A375 cells expressing endogenous level of MAGE-A4 peptide, or A375 cells engineered to overexpress HLA- A2/MAGE-A4 (286-294) peptide assessed by flow cytometry.
  • Figs. 5A-5D show 2+2 bsAbs are the most potent at redirecting T cells to kill tumor cells expressing endogenous levels of peptide and that the potency relies on both the geometry and valency of these molecules, and is driven by two CD3 arms.
  • Figs. 5A and 5B show binding to Jurkat cells (Fig. 5A) and to a cell line overexpressing the peptide of interest (Fig. 5B) from multiple AltibodiesTM technology formats evaluated by flow cytometry, demonstrating the functionality of all binding arms.
  • Fig. 5C demonstrates the in-vitro cytotoxic potency of a panel of alternative formats against the A375 cell line, expressing endogenous level of the peptide of interest, assessed by flow cytometry.
  • Fig. 5D shows the contribution of 2+2 bsAb’s binding arms assessed in a flow cytometrybased cytotoxicity assay, evaluating molecules engineered to remove one binding moiety at the time.
  • Figs. 6A-6B show an exemplary 2+2 bsAb format allows for targeting two different peptides, and its potency is enhanced when combined with a co-stimulatory bispecific and anti- PD1.
  • Fig. 6A shows the cytotoxic potency of an exemplary 2+2 bsAb targeting a single MAGE-A4 peptide or bsAb9930 against A375 cells expressing endogenous levels of MAGE-A4 peptides as assessed in-vitro by flow cytometry.
  • Fig. 6A shows the cytotoxic potency of an exemplary 2+2 bsAb targeting a single MAGE-A4 peptide or bsAb9930 against A375 cells expressing endogenous levels of MAGE-A4 peptides as assessed in-vitro by flow cytometry.
  • 6B shows the specific cytotoxic potency of bsAb9930 as single agent or in combination with a CD28-based bispecific antibody (EGFRxCD28) and anti-PD1 as assessed in a flow cytometry-based cytotoxicity assay targeting A375 cells.
  • EGFRxCD28 CD28-based bispecific antibody
  • Figs. 7A-7B show bsAb9930 does not exhibit in-vitro cytotoxic activity against an HLA- A2+/MAGE-A4- cell line, and does not activate T cells in the absence of target MHC-peptide tumor antigen.
  • Fig. 7A shows the specific cytotoxic potency of bsAb9930 demonstrated in a flow cytometry-based cytotoxicity assay targeting a cell line that expresses HLA-A2 but does not express MAGE-A4 (J82 cells).
  • Fig. 7A-7B show bsAb9930 does not exhibit in-vitro cytotoxic activity against an HLA- A2+/MAGE-A4- cell line, and does not activate T cells in the absence of target MHC-peptide tumor antigen.
  • Fig. 7A shows the specific cytotoxic potency of bsAb9930 demonstrated in a flow cytometry-based cytotoxicity assay targeting a cell line that expresses HLA-A2 but does not express MAGE-
  • FIG. 7B shows a flow cytometry-based T cell activation assay, monitoring CD25 over-expression on CD2+ T cells upon antibody treatment (20 pg/ml) demonstrates that T cells incubated with bsAb9930 do not upregulate CD25 in the absence of target cells.
  • Bispecific antibodies (which can be referred to herein as bsAbs or bsABs) engineered to recruit T cells as a means to kill tumor cells are a promising class of therapeutics in the field of oncology.
  • a challenge for this class of agents is the requirement for cell surface expressed tumor specific targets, as a large number of tumor-specific antigens are only expressed intracellularly.
  • One strategy to access these targets is to take advantage of the natural processing and presentation of intracellular antigens in the context of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • the present disclosure provides the generation and characterization of a 2+2 format bispecific antibody (2+2 bsAb) engineered to target MHC-peptide tumor neoantigens (see, e.g., Fig. 1T and Figs. 2B-2C).
  • the orientation of the targeting arms of the 2+2 bsAbs and the valency of these reagents allow specific T-cell activation and potent cytotoxic activity against cell lines expressing endogenous level of peptide antigens, both in-vitro and in-vivo.
  • This bispecific format is an important tool to expand the range of tumors able to be specifically targeted with T-cell redirecting biologies.
  • results discussed in the Examples support 2+2 bsAb’s clinical potential in redirecting T cells to tumor expressing challenging intracellular tumor antigens.
  • results demonstrate a conventional CD3 bispecific antibody targeting an MHC-peptide tumor neoantigen redirected T cells to kill tumor cells when the peptide antigen was over expressed on the surface of the cells.
  • ALTI BODIESTM technology Leveraging the ALTI BODIESTM technology, a suite of bispecific alternative formats was generated which exhibited a range of valencies and geometries (Fig. 1T).
  • an exemplary 2+2 bsAb of the disclosure was selected as being potent (Fig. 2C), specifically redirecting T cells to kill tumor cells expressing endogenous levels of peptide antigen.
  • the in-vitro potency of this format was mainly driven by the two CD3 binding moieties, and T cell activation measured by the upregulation of the CD25 marker was not observed in the absence of tumor cells expressing the peptide antigen.
  • Selected 2+2 bsAb, bsAb9930, targeting 2 different peptides demonstrated robust in-vivo activity, exhibiting maximal efficacy when combined with an EGFR x CD28 costimulatory bispecific and PD1 blockade.
  • An exemplary mechanism of action for bsAb9930 in combination with a CD28-based bispecific antibody and an anti-PD1 antibody, in particular, targeting A375 cells is shown in Fig. 2D.
  • T cell refers to immune cells expressing CD3, including CD4+ cells (helper T cells), CD8+ cells (cytotoxic T cells), regulatory T cells (Tregs), and tumor infiltrating lymphocytes.
  • T-cell antigen refers to a cell-surface expressed protein present on a T cell, and includes “co-stimulatory molecules.”
  • a “co-stimulatory molecule” refers to a protein expressed by a T cell that binds a cognate ligand or receptor (e.g., on an antigen-presenting cell) to provide a stimulatory signal, which, in combination with the primary signal provided by engagement of the T cell’s TCR with a peptide/MHC, stimulates the activity of the T cell. Stimulation of a T cell can include activation, proliferation and/or survival of the T cell.
  • cell surface-expressed or “cell-surface molecule” means one or more protein(s) that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody or an antigen-binding domain of the multispecific antigen-binding molecules discussed herein.
  • CD3 refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, the expression “CD3” means human CD3 unless specified as being from a non-human species, e.g., "mouse CD3,” “monkey CD3,” etc.
  • an antibody that binds CD3 or an “anti-CD3 antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers).
  • the antigen-binding domains of the present invention may bind soluble CD3 and/or cell surface expressed CD3.
  • Soluble CD3 includes natural CD3 proteins as well as recombinant CD3 protein variants such as, e.g., monomeric and dimeric CD3 constructs, that lack a transmembrane domain or are otherwise unassociated with a cell membrane.
  • the expression "cell surface-expressed CD3” means one or more CD3 protein(s) that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody.
  • Cell surface-expressed CD3 includes CD3 proteins contained within the context of a functional T cell receptor in the membrane of a cell.
  • cell surface-expressed CD3 includes CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers).
  • the expression, “cell surface-expressed CD3” also includes a CD3 chain (e.g., CD3- epsilon, CD3-delta or CD3-gamma) that is expressed by itself, without other CD3 chain types, on the surface of a cell.
  • a “cell surface-expressed CD3” can comprise or consist of a CD3 protein expressed on the surface of a cell which normally expresses CD3 protein.
  • “cell surface-expressed CD3” can comprise or consist of CD3 protein expressed on the surface of a cell that normally does not express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.
  • MAGE-A4 refers to Melanoma-Associated Antigen A4.
  • MAGE- A4 is an intracellular protein expressed by a variety of different tumor cells.
  • MAGE-A4 refers to the human MAGE-A4 protein unless specified as being from a non-human species (e.g., "mouse MAGE-A4,” “monkey MAGE-A4,” etc.).
  • the human MAGE-A4 protein has the amino acid sequence shown in SEQ ID NO: 187. Reference to particular regions of a MAGE-A4 polypeptide (e.g., MAGE-A4 286-294 or MAGE-A4230-239) are with respect to SEQ ID NO: 187.
  • MAGE-A4 286-294 MAGE-A4 (286-294),” and “MAGEA4286-294” may be used interchangeably.
  • MAGE-A4 230-239 MAGE-A4 (230-239)
  • MAGEA4230-239 may be used interchangeably.
  • the polypeptide sequence of MAGE-A4 (286-294) KVLEHVVRV
  • the polypeptide sequence of MAGE-A4 (230-239) (GVYDGREHTV) is given as SEQ ID NO: 271.
  • an antibody that binds MAGE-A4" or an "anti- MAG E-A4 antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize MAGE-A4.
  • an antibody that binds MAGE-A4 interacts with amino acids 286-294 of MAGE-A4 or amino acids 230-239 of MAGE-A4.
  • antigen-binding domain refers to that portion of a multispecific molecule or a corresponding antibody that binds specifically to a predetermined antigen (e.g., CD3 or a tumor associated antigen).
  • the antigen binding domain can comprise an HCVR and an LCVR on the same polypeptide or on more than one polypeptide.
  • References to a “corresponding antibody” refer to the antibody from which the CDRs or variable regions (HCVR and LCVR) used in a multispecific molecule are derived.
  • the Fig. 1C structured molecules discussed in the examples include Fabs and scFvs with variable regions derived from specific anti-CD3 antibodies and antiMAG E-A4 antibodies. These antibodies are the “corresponding antibodies” to the respective multispecific molecules.
  • multispecific antigen-binding molecule includes molecules that bind two or more (e.g., three or four) different epitopes or antigens. In some cases, the multispecific antigen-binding molecules are bispecific. In some cases, the multispecific antigen-binding molecules are trispecific. In some cases, the multispecific antigen-binding molecules are tetraspecific.
  • antibody means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD3 or a target antigen (TA)).
  • CDR complementarity determining region
  • the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • antibody also includes immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1 , CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CL1).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • Each V H and V is composed of three CDRs and four FRs, arranged from amino- terminus to carboxy-terminus in the following order: FR1 , CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FRs of the anti-TA antibody or anti-CD3 antibody may be identical to the human germline sequences, or may be naturally or artificially modified.
  • An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigenbinding fragment," as used herein.
  • SMIPs small modular immunopharmaceuticals
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain V H -V H , V H -V L or V L -V L dimers.
  • the antigenbinding fragment of an antibody may contain a monomeric V or V domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) H-CH1 ; (ii) VH-CH2; (iii) VH-CH3; (iv) VH- CH1-C H 2; (v) V H -CH1-CH2-CH3; (vi) VH-C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V -CH1 -CH2; (xii) V -CH1-CH2-CH3; (xiii) V L -CH2-CH3; and (xiv) V -CL.
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
  • the antibodies are human antibodies.
  • the term "human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term "human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the antibodies discussed herein may, in some embodiments, be recombinant human antibodies.
  • the term "recombinant human antibody” is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the antibodies referenced herein may be isolated antibodies.
  • An "isolated antibody,” as used herein, means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody.”
  • An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. An isolated antibody may be substantially free of other cellular material and/or chemicals.
  • the antibodies referenced herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope.
  • a single antigen may have more than one epitope.
  • different antibodies may bind to different areas on an antigen and may have different biological effects.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • a "multimerization domain” or “multimerizing domain” is any macromolecule that has the ability to associate (covalently or non-covalently) with a second macromolecule of the same or similar structure or constitution.
  • a multimerization domain may be a polypeptide comprising an immunoglobulin CH3 domain.
  • a non-limiting example of a multimerization domain is an Fc portion of an immunoglobulin, e.g., an Fc domain of an IgG selected from the isotypes lgG1, lgG2, I gG3, and lgG4, as well as any allotype within each isotype group.
  • the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif. In some embodiments, the multimerizing domain is an immunoglobulin Fc domain and the multispecific antigen-binding molecules of the present invention are formed by association of two such Fc domains via interchain disulfide bonding as in a conventional antibody.
  • nucleic acid or “polynucleotide” refers to nucleotides and/or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PCR polymerase chain reaction
  • Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids can be either single stranded or double stranded.
  • recombinant is intended to include all molecules that are prepared, expressed, created or isolated by recombinant means, such as multispecific molecules (e.g. bispecific molecules) expressed using a recombinant expression vector transfected into a host cell, multispecific molecules (e.g., bispecific molecules) isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or multispecific molecules prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin and/or MHC gene sequences to other DNA sequences.
  • Such recombinant multispecific molecules can include antigen-binding domains having variable and constant regions derived from human germline immunoglobulin sequences.
  • subject or "patient” as used herein includes all members of the animal kingdom including non-human primates and humans. In one embodiment, patients are humans with a disease or disorder, e.g., an infection or a cancer.
  • a disease or disorder e.g., an infection or a cancer.
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below.
  • a nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
  • the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331 , herein incorporated by reference.
  • Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamateaspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference.
  • a "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1.
  • FASTA e.g., FASTA2 and FASTA3
  • FASTA2 and FASTA3 provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra).
  • Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.
  • HLA refers to the human leukocyte antigen (HLA) system or complex, which is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. These cell-surface proteins are responsible for the regulation of the immune system in humans. HLAs corresponding to MHC class I (A, B, and C) present peptides from inside the cell.
  • HLA human leukocyte antigen
  • MHC major histocompatibility complex
  • HLA-A refers to the group of human leukocyte antigens (HLA) that are coded for by the HLA-A locus.
  • HLA-A is one of three major types of human MHC class I cell surface receptors.
  • the receptor is a heterodimer, and is composed of a heavy a chain and smaller p chain.
  • the a chain is encoded by a variant HLA-A gene, and the p chain (P2-microglobulin) is an invariant P2 microglobulin molecule.
  • HLA-A2 is one particular class I major histocompatibility complex (MHC) allele group at the HLA-A locus; the a chain is encoded by the HLA-A*02 gene and the p chain is encoded by the p2-microglobulin or B2M locus.
  • MHC major histocompatibility complex
  • vector and “expression vector” include, but are not limited to, a viral vector, a plasmid, an RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids.
  • the vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and are commercially available.
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
  • RNA viruses such as picornavirus and alphavirus
  • double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
  • herpesvirus e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus
  • poxvirus e.g., vaccinia, fowlpox and canarypox
  • Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses examples include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, and lentivirus. Multispecific Antigen-Binding Molecules
  • the multispecific antigen-binding molecules (e.g. , bispecific or trispecific or tetraspecific) of the present invention comprise (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first antigen-binding domain that specifically binds a T cell antigen (e.g., CD3), (ii) a first multimerizing domain, and (iii) a second antigen-binding domain that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third antigen-binding domain that specifically binds a target antigen (e.g., MAGE-A4), and (ii) a second multimerizing domain, wherein the first and the second multimerizing domains associate with one another (e.g., via interchain disulfide bonding) to form the molecule.
  • a first polypeptide comprising, from
  • the multispecific antigen-binding molecules (e.g., bispecific or trispecific or tetraspecific) of the present invention comprise (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first antigen-binding domain that specifically binds a T cell antigen (e.g., CD3), (ii) a first multimerizing domain, and (iii) a second antigen-binding domain that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N- terminus to C-terminus (i) a third antigen-binding domain that specifically binds a target antigen (e.g., MAGE-A4), such as MAGE-A4 286-294, (ii) a second multimerizing domain, and (iii) a fourth antigen-binding domain that specifically binds a target antigen (e.g., MAGE
  • MAGE-A4 such
  • the antigen-binding domains referenced above and herein can be Fab domains, comprising a heavy chain variable region (HCVR) and a heavy chain CH1 domain paired with a light chain variable region (LCVR) and a CL domain.
  • the antigen-binding domains referenced above and herein can also be single chain variable fragment (scFv) domains, comprising an HCVR and LCVR connected together by a short peptide linker of, e.g., from about 10 to about 25 amino acids.
  • the linker between the HCVR and LCVR of each scFv is (G4S)4 (SEQ ID NO: 268).
  • the antigen-binding domains of the multispecific molecules of the present invention can be all Fab domains, all scFv domains, or a combination of Fab domains and scFv domains.
  • one or more of the antigen-binding domains is a Fab domain.
  • one or more of the antigen-binding domains is a scFv domain.
  • the first antigen-binding domain and the third antigen-binding domain are Fab domains.
  • the second antigen-binding domain is an scFv domain.
  • the fourth antigen-binding domain is an scFv domain.
  • the first and third antigenbinding domains are Fab domains, and the second and fourth antigen-binding domains are scFv domains.
  • the first, second and third antigen-binding domains are Fab domains.
  • the first, second, third and fourth antigen-binding domains are Fab domains.
  • the scFv domains are connected to the C-terminus of the respective multimerizing domain via a linker peptide.
  • the linker is between 1-10 amino acids long. In some embodiments, the linker is between 1-20 amino acids long. In this regard, the linker may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In some embodiments, the linker may be 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. Ranges including the numbers discussed herein are also encompassed within this disclosure, e.g., a linker 10-30 amino acids long. In some embodiments, the linkers are flexible linkers.
  • Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
  • Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (GS) n , where n is an integer of at least one (e.g., from 1-20), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • the linker between each scFv domain and the C-terminus of the respective multimerizing domain is (G4S) 3 (SEQ ID NO: 269).
  • the scFv can be a stabilized scFv, in which one or more modifications is made to the HCVR and/or LCVR sequence in order to produce and maintain a proper conformation of the scFv.
  • the scFv includes cysteine mutations at residue 44 of the HCVR and residue 100 of the LCVR (Kabat numbering) to produce inter-disulfide bonding between the variable regions (see, Zhao et al., Int. J. Mol.
  • the scFv includes mutations at residue 39 of the HCVR and residue 38 of the LCVR (Kabat numbering) to modify the glutamine residues to glutamic acid or lysine residues to inhibit conformational isomerization (see, Igawa et al., Protein Engineering, Design and Selection, 23(8):667-677, 2010).
  • the LCVR (and optionally the CL) of any of the antigen-binding domains can be a cognate LCVR that corresponds to the HCVR, or the LCVR can be a universal LCVR (and optionally CL) common to multiple antigen-binding domains.
  • the light chain of the Fab domains is a common light chain.
  • the light chain of the Fab domains is a cognate light chain corresponding to the target antigen binding domain, and the light chain is common to both Fab domains.
  • the LCVR of the scFv domains is a cognate LCVR.
  • the light chain of the Fab domains is a common light chain and the LCVR of the scFv domains is a cognate LCVR.
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a first scFv that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a second Fab that specifically binds a target antigen (e.g., MAGE- A4, such as MAGE-A4 286-294), (ii) a second immunoglobulin Fc domain, and (iii) a second scFv that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4230-239), wherein the first and the second immuno
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4286-294), (ii) a second immunoglobulin Fc domain, and (iii) a fourth Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4 230-239), wherein the first and the second immunoglobulin domains
  • a target antigen
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a first scFv that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a second Fab that specifically binds a first target antigen (e.g., MAGE-A4, such as MAGE-A4 286-294 or 230-239), (ii) a second immunoglobulin Fc domain, and (iii) a second scFv that specifically binds a second target antigen different from the first
  • a first polypeptide comprising, from N-terminus to C-terminus
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third Fab that specifically binds a first target antigen (e.g., MAGE-A4, such as MAGE-A4286-294), (ii) a second immunoglobulin Fc domain, and (iii) a fourth Fab that specifically binds a second target antigen different from the first target antigen, wherein the first and the second immunoglobulin domains associate with one another via disulfide bonding to form
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a first scFv that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a second Fab that specifically binds a target antigen (e.g., MAGE- A4, such as MAGE-A4 286-294 or 230-239), (ii) a second immunoglobulin Fc domain, and (iii) a second scFv that specifically binds a T cell antigen (e.g.
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4286-294 or 230-239), (ii) a second immunoglobulin Fc domain, and (iii) a fourth Fab that specifically binds a T cell antigen (e.g., CD3), wherein the first and the second immunoglobulin domains associate with one another via disulf
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a second Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4286-294 or 230-239), and (ii) a second immunoglobulin Fc domain, wherein the first and the second immunoglobulin domains associate with one another via disulfide bonding to form the molecule.
  • a target antigen e.g., MAGE-A4, such as MAGE-A4286-294 or
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a T cell antigen (e.g., CD3); and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4286-294 or 230-239), and (ii) a second immunoglobulin Fc domain, wherein the first and the second immunoglobulin domains associate with one another via disulfide bonding to form the molecule.
  • a target antigen e.g., MAGE-A4, such as MAGE-A4286-294 or
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a first T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a first scFv that specifically binds a second T cell antigen; and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a second Fab that specifically binds a target antigen (e.g., MAGE- A4, such as MAGE-A4 286-294), (ii) a second immunoglobulin Fc domain, and (iii) a second scFv that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4230-239), wherein the first and the second immunoglobulin domains associate
  • a target antigen
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a first T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a second T cell antigen; and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4286-294), (ii) a second immunoglobulin Fc domain, and (iii) a fourth Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4 230-239), wherein the first and the second immunoglobulin domains associate with one another via dis
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a first T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a first scFv that specifically binds a second T cell antigen; and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a second Fab that specifically binds a first target antigen (e.g., MAGE-A4, such as MAGE-A4 286-294 or 230-239), (ii) a second immunoglobulin Fc domain, and (iii) a second scFv that specifically binds a second target antigen different from the first target antigen, wherein
  • a first target antigen e.g., MAGE-A4, such as MAGE-A4 28
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a first T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a second T cell antigen; and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third Fab that specifically binds a first target antigen (e.g., MAGE-A4, such as MAGE-A4286-294 or 230-239), (ii) a second immunoglobulin Fc domain, and (iii) a fourth Fab that specifically binds a second target antigen different from the first target antigen, wherein the first and the second immunoglobulin domains associate with one another via disulfide bonding to form the
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a first T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a first scFv that specifically binds a second T cell antigen; and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a second Fab that specifically binds a target antigen (e.g., MAGE- A4, such as MAGE-A4 286-294 or 230-239), (ii) a second immunoglobulin Fc domain, and (iii) a second scFv that specifically binds a T cell antigen (optionally may bind the first T cell antigen, the second T cell antigen, or a third T cell antigen), where
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a first T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a second T cell antigen; and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4286-294 or 230-239), (ii) a second immunoglobulin Fc domain, and (iii) a fourth Fab that specifically binds a T cell antigen (optionally may bind the first T cell antigen, the second T cell antigen, or a third T cell antigen), wherein the first and the second
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a first T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a second T cell antigen; and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a second Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4286-294 or 230-239), and (ii) a second immunoglobulin Fc domain, wherein the first and the second immunoglobulin domains associate with one another via disulfide bonding to form the molecule.
  • a target antigen e.g., MAGE-A4, such as MAGE-A4286-294 or 230-239
  • the multispecific antigen-binding molecules of the present invention comprise: (a) a first polypeptide comprising, from N-terminus to C-terminus (i) a first Fab that specifically binds a first T cell antigen (e.g., CD3), (ii) a first immunoglobulin Fc domain, and (iii) a second Fab that specifically binds a second T cell antigen; and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third Fab that specifically binds a target antigen (e.g., MAGE-A4, such as MAGE-A4286-294 or 230-239), and (ii) a second immunoglobulin Fc domain, wherein the first and the second immunoglobulin domains associate with one another via disulfide bonding to form the molecule.
  • a target antigen e.g., MAGE-A4, such as MAGE-A4286-294 or 230-239
  • the multispecific antigen-binding molecules e.g., bispecific or trispecific or tetraspecific
  • the multispecific antigen-binding molecules may comprise one or more light chain polypeptides that pair with the corresponding heavy chain portions in the two polypeptide chains to form the Fab domain(s).
  • the multispecific antigen-binding molecules of the present invention comprise any of the various exemplary 2+2 bispecific antibody formats illustrated in Fig. 1T.
  • the multispecific antigen-binding molecules of the present invention comprise a 2+2 bispecific antibody format illustrated in Fig. 2B.
  • the present disclosure encompasses any of the various compositions and methods disclosed in International Patent Application W02021030680, the content of which is incorporated herein by reference in its entirety for all purposes. Any such compositions and/or methods may be used in the practice of the invention disclosed herein.
  • the fourth antigen-binding domain can specifically bind a target antigen (e.g., CD3) or a T cell antigen (e.g., MAGE-A4).
  • the third antigen-binding domain and the fourth antigen-binding domain specifically bind distinct target antigens (different epitopes on the same protein, or different proteins).
  • the distinct target antigens are expressed on the surface of the same target cell (e.g., tumor cell).
  • the third antigen-binding domain and the fourth antigen-binding domain specifically bind the same target antigen (the same epitope on the same protein).
  • the first and second antigen-binding domains, and the fourth antigen-binding domain can bind the same or distinct T-cell antigens, as illustrated in the figures.
  • the first, second and fourth antigen-binding domains specifically bind distinct T-cell antigens (different epitopes on the same protein, or different proteins).
  • the first, second and fourth antigen-binding domains specifically bind the same T-cell antigen (the same epitope on the same protein).
  • the distinct T-cell antigens are a costimulatory molecule (e.g., CD28) and a check-point inhibitor (e.g., PD-1) on the surface of a T cell.
  • the multispecific molecules of the invention can provide a costimulatory signal to the T cell as well as prevent checkpoint inhibition.
  • references to “same” target antigen or T-cell antigen does not necessarily mean that the antigen-binding domains are binding to the same surface molecule, but rather that the antigen-binding domains have the same specificity (e.g., they each bind CD3 or a TA).
  • references to a “distinct” target antigen or T-cell antigen mean that it is different from another target antigen (e.g., MAGE-A4 vs. EGFR) or another T-cell antigen (e.g., CD28 vs. PD-1), or is another epitope on the same protein.
  • the target antigen can be a tumor- associated antigen or an infection-associated antigen (e.g., a viral antigen, a bacterial antigen, a fungal antigen, or an antigen expressed by a parasite).
  • the target antigen is a tumor-associated antigen.
  • the target antigen is an infection-associated antigen.
  • the target antigen is a viral antigen.
  • the target antigen is a bacterial antigen.
  • the target antigen is a fungal antigen.
  • the target antigen is an antigen expressed by a parasite.
  • the target antigen is a peptide in the context of the groove (PiG) of a major histocompatibility complex (MHC) protein.
  • the PiG is a peptide consisting of about 5 to about 40 amino acid residues, from about 6 to about 30 amino acid residues, from about 8 to about 20 amino acid residues, or about 9, 10, or 11 amino acid residues.
  • the PiG is a tumor-associated antigen.
  • the target antigen is a peptide in the context of the groove of any class, subtype or allele of human leukocyte antigen, including any of HLA-A, HLA-B, HLA-C, HLA- DR, HLA-DQ or HLA-DP.
  • the target antigen is a peptide in the context of the groove of the human leukocyte antigen HLA-A2.
  • the target antigen is a peptide/MHC complex. In some cases, the peptide in the peptide/MHC complex is a fragment of a tumor-associated antigen.
  • the antigen is a tumor-associated antigen or an antigen expressed by a tumor cell.
  • the tumor-associated antigen is MAGE-A4.
  • the MAGE-A4 is an HLA-bound MAGE-A4 peptide.
  • the HLA- bound MAGE-A4 peptide comprises amino acids 286-294, or a portion thereof, of SEQ ID NO: 187.
  • the HLA bound MAGE-A4 peptide comprises amino acids 230-239, or a portion thereof, of SEQ ID NO: 187.
  • the MAGE-A4-binding domain binds to human MAGE-A4.
  • the MAGE-A4-binding domain binds weakly to human MAGE-A4. In some embodiments, the MAGE- A4-binding domain binds or associates weakly with human and cynomolgus (monkey) MAGE-A4, yet the binding interaction is not detectable by in vitro assays known in the art. In some embodiments, the MAGE-A4-binding domain binds with weak affinity to human MAGE-A4. In some embodiments, the MAGE-A4-binding domain binds with moderate affinity to human MAGE-A4. In some embodiments, the MAGE-A4-binding domain binds with high affinity to human MAGE-A4.
  • the present disclosure provides antigen-binding domains derived from corresponding antibodies that bind human MAGE-A4 (e.g., at 25°C) with a K D of less than about 5 nM as measured by surface plasmon resonance.
  • the corresponding antibodies bind MAGE-A4 with a KD of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM as measured by surface plasmon resonance.
  • the MAGE-A4-binding domain exhibits an ECso value of less than less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, or less than 500 pM, as measured in an in vitro flow cytometry binding assay.
  • the MAGE-A4-binding domain exhibits an ECso value of about or greater than about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 500 nM or 1 pM, as measured in an in vitro flow cytometry binding assay.
  • MAGE-A4-binding domains e.g., MAGE-A4-binding domains derived from corresponding antibodies of fragments thereof, bind MAGE-A4 with a dissociative halflife (t 1 /2) of greater than about 10 minutes or greater than about 125 minutes as measured by surface plasmon resonance at 25°C.
  • the corresponding antibodies bind MAGE-A4 with a t 1 / 2 of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes, as measured by surface plasmon resonance at 25°C.
  • the multispecific antigen-binding molecules comprises an antigenbinding domain that specifically targets a second target antigen which is different from the first target antigen (e.g., MAGE-A4).
  • the second target antigen may be an tumor-associated antigen selected from the group consisting of AFP, ALK, BAGE proteins, BIRC5 (survivin), BIRC7, [3- catenin, bcr-abl, BRCA1 , BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, CEA, cyclin-B1, CYP1B1 , EGFR, EGFRvlll, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1 , FOLR1 , GAGE proteins (e.g., GAGE-1 , -2), GD2, GD2, GD2, GD
  • the T cell antigen can be an antigen expressed at the surface of a T cell, a T cell receptor complex antigen, a co-stimulatory molecule or a check point inhibitor on a T cell, CD3, CD27, CD28, 4-1 BB or PD-1.
  • the T cell antigen is a T cell receptor complex antigen.
  • the T cell antigen is CD3.
  • the multispecific antigen-binding molecules comprises an antigenbinding domain that specifically targets a second T cell antigen which is different from the first T cell antigen (e.g., CD3).
  • the second T cell antigen is a co-stimulatory molecule or a check-point inhibitor on a T cell.
  • the second T cell antigen is selected from the group consisting of CD27, CD28, 4-1 BB and PD-1.
  • the second T cell antigen is selected from the group consisting of CD27, CD28, 4-1 BB and PD-1.
  • the second T cell antigen is selected from the group consisting of CD28, IGOS, HVEM, CD27, 4-1 BB, 0X40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1 , and TIM2.
  • the CD3-binding domain binds to human CD3 and induces human T cell activation. In certain embodiments, the CD3-binding domain binds weakly to human CD3 and induces human T cell activation. In some embodiments, the CD3-binding domain binds weakly to human CD3 and induces tumor-associated antigenexpressing cell killing. In some embodiments, the CD3-binding domain binds or associates weakly with human and cynomolgus (monkey) CD3, yet the binding interaction is not detectable by in vitro assays known in the art. In some embodiments, the CD3-binding domain binds with weak affinity to human CD3.
  • the CD3-binding domain binds with moderate affinity to human CD3. In some embodiments, the CD3-binding domain binds with high affinity to human CD3. In some embodiments, the CD3-binding domain binds to human CD3 (e.g., at 25°C) with a KD of less than about 15 nM as measured by surface plasmon resonance (e.g., mAb-capture or antigen-capture format) or a substantially similar assay.
  • surface plasmon resonance e.g., mAb-capture or antigen-capture format
  • the CD3-binding domain binds human CD3 with an K D value of greater than about 15 nM, greater than about 20 nM, greater than about 30 nM, greater than about 40 nM, greater than about 50 nM, greater than about 60 nM, greater than about 100 nM, greater than about 200 nM, or greater than about 300 nM, as measured in a surface plasmon resonance binding assay (e.g., mAb-capture or antigen-capture format) or a substantially similar assay.
  • a surface plasmon resonance binding assay e.g., mAb-capture or antigen-capture format
  • the antibodies or antigen-binding fragments of the present invention bind CD3 with a K D of less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 180 pM, less than about 160 pM, less than about 140 pM, less than about 120 pM, less than about 100 pM, less than about 80 pM, less than about 60 pM, less than about 40 pM, less than about 20 pM, or less than about 10 pM, as measured by surface plasmon resonance, e.g., using an assay format (e.g., mAb-capture or antigen-capture format), or a substantially similar assay.
  • an assay format e.g., mAb-capture or antigen-capture format
  • the CD3-binding domain exhibits an ECso value of less than less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, or less than 500 pM, as measured in an in vitro flow cytometry binding assay.
  • the CD3-binding domain exhibits an ECso value of about or greater than about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 500 nM or 1 pM, as measured in an in vitro flow cytometry binding assay.
  • the multispecific antigen-binding molecule of the present disclosure comprises a MAGE-A4-binding arm as set forth in Table 1a. In some embodiments, the multispecific antigen-binding molecule of the present disclosure comprises a CD3 binding arm as set forth in Table 1b. In some embodiments, the multispecific antigen-binding molecule of the present disclosure comprises one or more light chain polypeptides as set forth in Table 1c. In some embodiments, the multispecific antigen-binding molecule of the present disclosure comprises the full-length polypeptides as set forth in Table 1c.
  • the MAGE-A4-binding domain can comprise any of the HCVR/LCVR or CDR (e.g., the six CDRs contained within a pair of HCVR/LCVR sequences) amino acid sequences set forth in the below Table 2a or Table 2b.
  • Table 2a sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of exemplary anti-MAGE-A4 antibodies of the present disclosure.
  • the mAb31345 and mAb31345* sequences of Table 2a are identical except for one extra C-terminal amino acid in the “called” LCVR sequence of mAb31345* (i.e., the full-length antibodies are identical but one additional amino acid was assigned to the LCVR of mAb31345* when annotating the LCVR region).
  • the corresponding nucleic acid sequence identifiers are set forth in Table 2b.
  • Table 2b Nucleic Acid Sequence Identifiers of Exemplary Anti-MAGE-A4 Antibodies 158] Methods for generating and/or evaluating anti-MAGE-A4 antibodies are described in International Patent Applications WO2020257288A2 and WO2021016585, the content of which is incorporated herein by reference in its entirety for all purposes. Any such compositions and/or methods may be used in the practice of the invention disclosed herein.
  • the CD3-binding domain can comprise any of the HCVR/LCVR or CDR (e.g., the six CDRs contained within a pair of HCVR/LCVR sequences) amino acid sequences of the anti-CD3 antibodies disclosed in WO 2014/047231 or WO 2017/053856, including the antibodies identified as 7195P, 7221G, 7221G5 and 7221G20.
  • CDR e.g., the six CDRs contained within a pair of HCVR/LCVR sequences
  • an anti- CD3 antibody identified as a “strong binder” has an affinity for human CD3 in the single digit nanomolar range (e.g., from 1-9 nM) as measured in a surface plasmon resonance assay (e.g., at 25°C in an antigen-capture format with measurements conducted on a T200 BIACORE instrument).
  • an anti-CD3 antibody identified as a “moderate binder” has an affinity for human CD3 in the double digit nanomolar range (e.g., from 10-99 nM, optionally from 10-50 nM or 10-25 nM) as measured in a surface plasmon resonance assay.
  • an anti- CD3 antibody identified as a “weak binder” has an affinity for human CD3 in the three digit nanomolar range (e.g., from 100-999 nM, optionally from 100-500 nM or from 500 nM to 1 pM) as measured in a surface plasmon resonance assay.
  • an anti-CD3 antibody identified as a “very weak binder” has an affinity for human CD3 that is greater than 10 pM or is undetectable as measured in a surface plasmon resonance assay.
  • the CD3-binding domain can comprise any of the HCVR/LCVR or CDR (e.g., the six CDRs contained within a pair of HCVR/LCVR sequences) amino acid sequences set forth in the following tables (the “G” versions are taken from WO 2017/053856)
  • the CD3-binding domains e.g., in the Fab arm of a molecule having the structure of Fig. 1C or 1 F
  • the cognate light chain of the target antigen binding domain is common to both the target antigen-binding domain and the CD3-binding domain (e.g., in the N- terminal Fab domains of the structure of Fig. 1C or 1 F).
  • Each of the antibodies set forth in Table 3 comprises a common light chain variable region comprising the amino acid sequence set forth in Table 5.
  • Each of the “G” designated antibodies may also be referred to herein with a “7221” prefix, e.g., 7221G, 7221G5, 7221G20, etc.
  • the amino acid residue at position 44 of the heavy chain variable region may be replaced with a cysteine residue, for example, as shown in SEQ ID NO: 169 (the modified heavy chain corresponding to 7195P) or SEQ ID NO: 170 (the modified heavy chain corresponding to 7221 G).
  • the multispecific antigen-binding molecules (e.g., bispecific or trispecific or tetraspecific) of the present invention may comprise two polypeptide chains, each of which includes a multimerizing domain that facilitates association of the two polypeptide chains (e.g., via interchain disulfide bonding) to form a single multispecific antigen-binding molecule.
  • the multispecific antigen-binding molecules (e.g., bispecific or trispecific or tetraspecific) of the present invention may comprise one or more light chain polypeptides that pair with the corresponding heavy chain portions in the two polypeptide chains to form the Fab domain(s).
  • the first and second multimerizing domains can be immunoglobulin Fc domains (e.g. of human IgG isotype). In some cases, the first and second multimerizing domains associate with one another via disulfide bonding. In some embodiments, the first multimerizing domain and the second multimerizing domain are human I gG 1 or human lgG4 Fc domains. In some cases, the first and second multimerizing domains comprise a hinge domain, a CH2 domain and a CH3 domain of human I gG1 or human lgG4.
  • the first multimerizing domain or the second multimerizing domain comprises an amino acid substitution that reduces affinity for Protein A binding compared to a wildtype Fc domain of the same isotype (e.g., human lgG1 or human lgG4).
  • the amino acid substitution comprises an H435R modification, or H435R and Y436F modifications (EU numbering).
  • the first multimerizing domain comprises the H435R and Y436F modifications.
  • the second multimerizing domain comprises the H435R and Y436F modifications.
  • the first polypeptide, the second polypeptide, or both the first and the second polypeptides comprise a modified hinge domain that reduces binding affinity for an Fey receptor relative to a wild-type hinge domain of the same isotype (e.g., human lgG1 or human lgG4).
  • the constant region may be chimeric, combining sequences derived from more than one immunoglobulin isotype.
  • a chimeric Fc domain can comprise part or all of a CH2 sequence derived from a human lgG1 , human lgG2 or human lgG4 CH2 region, and part or all of a CH3 sequence derived from a human I gG 1 , human lgG2 or human lgG4.
  • a chimeric Fc domain can also contain a chimeric hinge region.
  • a chimeric hinge may comprise an "upper hinge" sequence, derived from a human lgG1, a human lgG2 or a human lgG4 hinge region, combined with a "lower hinge” sequence, derived from a human I gG 1 , a human lgG2 or a human lgG4 hinge region.
  • a particular example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [lgG4 CH1 ] - [lgG4 upper hinge] - [lgG2 lower hinge] - [lgG4 CH2] - [lgG4 CH3],
  • Another example of a chimeric Fc domain that can be included in any of the antigenbinding molecules set forth herein comprises, from N- to C-terminus: [IgG 1 CH1] - [I gG 1 upper hinge] - [lgG2 lower hinge] - [lgG4 CH2] - [I gG 1 CH3]
  • positions 233-236 within the hinge domain may be G, G, G and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, with positions numbered by EU numbering.
  • the heavy chain constant region comprises from N-terminal to C-terminal the hinge domain, a CH2 domain and a CH3 domain.
  • the heavy chain constant region comprises from N-terminal to C- terminal a CH1 domain, the hinge domain, a CH2 domain and a CH3 domain.
  • the CH1 region, if present, remainder of the hinge region, if any, CH2 region and CH3 region are the same human isotype.
  • the CH1 region, if present, remainder of the hinge region, if any, CH2 region and CH3 region are human lgG1.
  • the CH1 region, if present, remainder of the hinge region, if any, CH2 region and CH3 region are human lgG2.
  • the CH1 region if present, remainder of the hinge region, if any, CH2 region and CH3 region are human lgG4.
  • the constant region has a CH3 domain modified to reduce binding to protein A.
  • the association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a multispecific antigen-binding molecule.
  • the multimerizing domain may be any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution.
  • a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain.
  • a nonlimiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes lgG1 , lgG2, lgG3, and lgG4, as well as any allotype within each isotype group.
  • the first and second multimerizing domains may be of the same IgG isotype such as, e.g., lgG1/lgG1, lgG2/lgG2, lgG4/lgG4.
  • the first and second multimerizing domains may be of different IgG isotypes such as, e.g., IgG 1/lgG2, lgG1/lgG4, lgG2/lgG4, etc.
  • the multimerizing domain is an Fc fragment or an amino acid sequence of from 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerizing domain is a cysteine residue, or a short cysteine-containing peptide.
  • Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.
  • the multimerizing domains may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wildtype, naturally occurring version of the Fc domain.
  • the invention includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn.
  • the bispecific antigen-binding molecule comprises a modification in a CH2 or a CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0).
  • Nonlimiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434.
  • a modification at position 250 e.g., E or Q
  • 250 and 428 e.g., L or F
  • 252 e.g., L/Y/F/W or T
  • 254 e.g., S or T
  • the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P).
  • a 428L e.g., M428L
  • 434S e.g., N434S
  • 428L, 2591 e.g., V259I
  • 308F e.g., V308F
  • 433K
  • the present invention also includes multispecific antigen-binding molecules comprising a first Ig CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference.
  • the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering).
  • the second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU).
  • Antigen-binding domains specific for particular antigens can be prepared by any antibody generating technology known in the art. Once obtained, different antigen-binding domains, specific for two or more different antigens (e.g., CD3 and MAGE-A4), can be appropriately arranged relative to one another to produce the structures of the multispecific antigen-binding molecules of the present invention using routine methods.
  • one or more of the individual components (e.g., heavy and light chains or parts thereof) of the multispecific antigen-binding molecules of the invention are derived from chimeric, humanized or fully human antibodies. Methods for making such antibodies are well known in the art.
  • one or more of the heavy and/or light chains of the multispecific antigen-binding molecules of the present invention can be prepared using VELOCIMMUNETM technology.
  • VELOCIMMUNETM technology or any other human antibody generating technology
  • high affinity chimeric antibodies to a particular antigen e.g., CD3 or MAGE-A4
  • the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc.
  • the mouse constant regions are replaced with a desired human constant region to generate fully human heavy and/or light chains that can be incorporated into the multispecific antigen-binding molecules of the present invention.
  • Genetically engineered animals may be used to make human multispecific antigen-binding molecules.
  • a genetically modified mouse can be used which is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus.
  • Such genetically modified mice can be used to produce fully human multispecific antigen-binding molecules comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable region gene segments. (See, e.g., US 2011/0195454).
  • Fully human refers to an antibody, or antigen-binding fragment or immunoglobulin domain thereof, comprising an amino acid sequence encoded by a DNA derived from a human sequence over the entire length of each polypeptide of the antibody or antigen-binding fragment or immunoglobulin domain thereof.
  • the fully human sequence is derived from a protein endogenous to a human.
  • the fully human protein or protein sequence comprises a chimeric sequence wherein each component sequence is derived from human sequence. While not being bound by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the creation of immunogenic epitopes in the junctions of component sequences, e.g. compared to any wild-type human immunoglobulin regions or domains.
  • the methods and techniques discussed above are used to generate antibodies to a T-cell antigen and a target antigen, and the antigen-binding domains of these antibodies (e.g., the HCVR, LCVR, or CDRs) are used to produce the multispecific antigenbinding molecules as discussed herein or having, e.g., the structures illustrated in Figs. 1C and 1 E- 1S.
  • binding in the context of the binding of an antibody (e.g.. a corresponding antibody), immunoglobulin, antigen-binding domain or multispecific antigen-binding molecule to, e.g., a predetermined antigen, such as a cell surface protein or fragment thereof, typically refers to an interaction or association between a minimum of two entities or molecular structures, such as an antigen-binding domain I antigen interaction.
  • binding affinity typically corresponds to a K D value of about 10' 7 M or less, such as about 10' 8 M or less, such as about 10 -9 M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody, Ig, antibody-binding domain or multispecific antigen-binding molecule as the analyte (or anti-ligand).
  • SPR surface plasmon resonance
  • Flow cytometry assays are also routinely used.
  • the antibody e.g., a corresponding antibody
  • antigen-binding domain or multispecific antigen-binding molecule of the invention binds to the predetermined antigen or cell surface molecule having an affinity corresponding to a K D value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein).
  • a non-specific antigen e.g., BSA, casein
  • the affinity of an antibody e.g., a corresponding antibody
  • antigen-binding domain or multispecific antigen-binding molecule corresponding to a KD value that is equal to or less than tenfold lower than a non-specific antigen may be considered non-detectable binding, however such an antibody may be paired with a second antigen binding arm for the production of a bispecific antibody of the invention.
  • K D refers to the dissociation equilibrium constant of a particular antibody (or antigen-binding domain)-antigen interaction, or the dissociation equilibrium constant of an antibody (or antigen-binding domain) or antibody-binding fragment binding to an antigen.
  • a higher binding affinity (or KD) of a particular molecule (e.g. antibody or antigen-binding domain) to its interactive partner molecule (e.g. antigen X) compared to the binding affinity of the molecule (e.g. antibody or antigen-binding domain) to another interactive partner molecule (e.g. antigen Y) may be expressed as a binding ratio determined by dividing the larger K D value (lower, or weaker, affinity) by the smaller K D (higher, or stronger, affinity), for example expressed as 5-fold or 10-fold greater binding affinity, as the case may be.
  • kd (sec -1 or 1/s) refers to the dissociation rate constant of a particular antibody (or antigen-binding domain)-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding domain. Said value is also referred to as the k o tr value.
  • k a (M-1 x sec-1 or 1/M) refers to the association rate constant of a particular antibody (or antigen-binding domain)-antigen interaction, or the association rate constant of an antibody or antibody-binding domain.
  • the term "KA” (M-1 or 1/M) refers to the association equilibrium constant of a particular antibody (or antigen-binding domain)-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding domain.
  • the association equilibrium constant is obtained by dividing the k a by the k d .
  • the term “EC50” or “EC50” refers to the half maximal effective concentration, which includes the concentration of an antibody (or antigen-binding domain or multispecific molecule) which induces a response halfway between the baseline and maximum after a specified exposure time.
  • the EC50 essentially represents the concentration of an antibody (or antigen-binding domain or multispecific molecule) where 50% of its maximal effect is observed.
  • the EC50 value equals the concentration of a multispecific molecule of the invention that gives half- maximal binding to cells expressing CD3 or target antigen (e.g., MAGE-A4), as determined by e.g. a flow cytometry binding assay.
  • a multispecific molecule of the invention that gives half- maximal binding to cells expressing CD3 or target antigen (e.g., MAGE-A4), as determined by e.g. a flow cytometry binding assay.
  • decreased binding can be defined as an increased EC50 molecule concentration which enables binding to the half-maximal amount of target cells.
  • the EC50 value represents the concentration of a molecule of the invention that elicits half-maximal depletion of target cells by T cell cytotoxic activity.
  • increased cytotoxic activity e.g. T cell-mediated tumor cell killing
  • EC50, or half maximal effective concentration value pH-Dependent Binding
  • the present invention includes antigen-binding domains and multispecific antigen-binding molecules with pH-dependent binding characteristics.
  • a molecule of the present invention may exhibit reduced binding to a T-cell antigen or a target antigen at acidic pH as compared to neutral pH.
  • molecules of the invention may exhibit enhanced binding to a T-cell antigen or a target antigen at acidic pH as compared to neutral pH.
  • acidic pH includes pH values less than about 6.2, e.g., about 6.0, 5.95, 5,9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1 , 5.05, 5.0, or less.
  • neutral pH means a pH of about 7.0 to about 7.4.
  • neutral pH includes pH values of about 7.0, 7.05, 7.1 , 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.
  • "reduced binding ... at acidic pH as compared to neutral pH” is expressed in terms of a ratio of the KD value of the molecule (or antigen-binding domain) binding to its antigen at acidic pH to the KD value of the molecule (or antigen-binding domain) binding to its antigen at neutral pH (or vice versa).
  • a molecule or antigen-binding domain may be regarded as exhibiting "reduced binding to a T-cell antigen or a target antigen at acidic pH as compared to neutral pH” for purposes of the present invention if the molecule or antigen-binding domain exhibits an acidic/neutral K D ratio of about 3.0 or greater.
  • the acidic/neutral KD ratio for a molecule or antigen-binding domain of the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.
  • Multispecific molecules with pH-dependent binding characteristics may be obtained, e.g., by screening a population of corresponding antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH as compared to neutral pH. Additionally, modifications of the antigen-binding domain at the amino acid level may yield molecules with pH-dependent characteristics. For example, by substituting one or more amino acids of an antigen-binding domain (e.g., within a CDR) with a histidine residue, a molecule with reduced antigen-binding at acidic pH relative to neutral pH may be obtained.
  • the present invention can include multispecific antigen-binding molecules and antigenbinding domains thereof that are capable of simultaneously binding to a human T-cell antigen (e.g., CD3) and a human target antigen or antigens (e.g., MAGE-A4).
  • a human T-cell antigen e.g., CD3
  • a human target antigen or antigens e.g., MAGE-A4
  • the present invention can include multispecific antigen-binding molecules that bind a human T-cell antigen (e.g., CD3) and induce T cell activation in the presence of target cells.
  • a human T-cell antigen e.g., CD3
  • the present invention includes multispecific antigen-binding molecules that bind a human T-cell antigen (e.g., CD3) and induce T cell cytotoxic activity in the presence of cells expressing the target antigen or target antigens (e.g., MAGE-A4).
  • the present invention can include multispecific antigen-binding molecules that bind a human T-cell antigen (e.g., CD3) and induce T cell activation without increasing cytokine production relative to a conventionally structured bispecific anti-CD3 x anti-TA antibody (e.g., Fig. 1A).
  • a human T-cell antigen e.g., CD3
  • a conventionally structured bispecific anti-CD3 x anti-TA antibody e.g., Fig. 1A
  • the present invention can include multispecific antigen-binding molecules that are capable of depleting or reducing cell populations in which the cells express the target antigen or target antigens.
  • the multispecific antigen-binding molecules of the present invention are capable of inducing T-cell mediated cytotoxicity more potently than molecules having conventional bispecific antibody formats (e.g., Figs. 1A and 1 B).
  • the present invention can include multispecific antigen-binding molecules that bind a human T-cell antigen (e.g., CD3) and two distinct target antigens (e.g., a molecule having the structure of Fig. 1 F), and induce cytotoxic activity and/or T-cell activation in the presence of cells expressing the two target antigens.
  • a human T-cell antigen e.g., CD3
  • two distinct target antigens e.g., a molecule having the structure of Fig. 1 F
  • cancers express a variety of intracellular antigens that are processed inside the cell by the proteosome and associated peptides are presented at the surface of the cell in the context of HLA molecules. Targeting peptides from different proteins may be used to increase the specificity of the multispecific molecules of the present invention.
  • cancers characterized by PiG antigens or low density cancer antigens escape conventional cancer therapies because they are often present in low target copy numbers within tumors.
  • solid tumors characterized by PiGs or low density cancer antigens can be more resistant to therapy and more difficult to treat because they are not cell surface antigens, but are present in grooves within the cancer related peptide.
  • a multispecific molecule of the present invention targeting two distinct antigens can effectively target PiGs and/or low density cancer antigens to increase/enhance efficacy of therapy in cancers, especially those cancers characterized by solid tumors.
  • the multispecific antigen-binding molecules of the present invention are capable of inducing T-cell mediated cytotoxicity in cell populations when the density of the target antigen ranges from about 100 copies per cell to about 1 million copies per cell or more.
  • the target antigen is present at a copy number/cell of about 100, about 200, about 300, about 400, about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 15000, about 20000, about 25000, about 30000, about 35000, about 40000, about 45000, about 50000, about 75000, about 100000 (/.e., 100K), about 200K, about 300K, about 400K, about 500K, about 600K, about 700K, about 800K, about 900K, about 1 million, about 2 million, about 3 million, about 4 million, about 5 million, or about 10 million.
  • the inventors postulate that the improved cytotoxic potency of the molecular format of the present invention is a function of the presence of two T-cell antigen (e.g., CD3) binding domains on a single chain of the molecule.
  • T-cell antigen e.g., CD3
  • the geometry of the molecular structures of the present invention selectively induces lytic synapse formation at low concentrations without inducing stimulatory synapse formation, the latter of which is responsible for cytokine production from cytotoxic T lymphocytes.
  • the epitope on the T-cell antigen (e.g., CD3) and/or the target antigen (e.g., MAGE-A4) to which the antigen-binding molecules of the present invention bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a protein.
  • the epitope may consist of a plurality of noncontiguous amino acids (or amino acid sequences) of the protein.
  • the molecules of the invention may interact with, e.g., amino acids contained within a single CD3 chain (e.g., CD3-epsilon, CD3- delta or CD3-gamma), or may interact with amino acids on two or more different CD3 chains.
  • the term "epitope,” as used herein, refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antigen-binding domain known as a paratope.
  • a single antigen may have more than one epitope.
  • different antigen-binding domains may bind to different areas on an antigen and may have different biological effects.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • Various techniques known to persons of ordinary skill in the art can be used to determine whether an antigen-binding domain of a molecule "interacts with one or more amino acids" within a polypeptide or protein.
  • Exemplary techniques include, e.g., routine cross-blocking assay such as that described Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutational analysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis.
  • methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496).
  • Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding domain of a molecule interacts is hydrogen/deuterium exchange detected by mass spectrometry.
  • the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the molecule to the deuterium-labeled protein. Next, the protein/molecule complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the molecule (which remain deuterium-labeled).
  • the target protein After dissociation of the molecule, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium- labeled residues which correspond to the specific amino acids with which the molecule interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267 (2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of the antigen/molecule complex may also be used for epitope mapping purposes.
  • the present invention includes multispecific antigen-binding molecules that are bioequivalent to any of the exemplary multispecific antigen-binding molecules set forth herein.
  • Two antigen-binding proteins are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose.
  • antigen-binding proteins will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
  • two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.
  • two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
  • two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
  • Bioequivalence may be demonstrated by in vivo and in vitro methods.
  • Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antigen-binding protein or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antigen-binding protein (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.
  • Bioequivalent variants of the exemplary multispecific antigen-binding molecules set forth herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity.
  • cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.
  • bioequivalent antigen-binding proteins may include variants of the exemplary multispecific antigen-binding molecules set forth herein comprising amino acid changes which modify the glycosylation characteristics of the molecules, e.g., mutations which eliminate or remove glycosylation.
  • antigen-binding molecules which bind to human T cell antigen (e.g., CD3) but not to the same antigen from other species.
  • antigen-binding molecules which bind to human target antigens (e.g., MAGE-A4) but not to the same target antigens from other species.
  • the present invention also includes antigen-binding molecules that bind to human antigens and corresponding antigens from one or more non-human species.
  • antigen-binding molecules which bind to human CD3 and/or a human tumor antigen (e.g., MAGE-A4) and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus or chimpanzee CD3 and/or tumor antigen (e.g., MAGE-A4).
  • a human tumor antigen e.g., MAGE-A4
  • multispecific antigen-binding molecules comprising a first antigen-binding domain that binds human CD3 and cynomolgus CD3, and a second antigen-binding domain that specifically binds a human tumor antigen (e.g., MAGE-A4).
  • a human tumor antigen e.g., MAGE-A4
  • the present invention encompasses antigen-binding molecules conjugated to a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
  • a therapeutic moiety such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
  • Cytotoxic agents include any agent that is detrimental to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immunoconjugates are known in the art, (see for example, WO 05/103081).
  • the present invention provides an isolated nucleic molecule encoding the multispecific antigen-binding molecules disclosed herein, comprising a nucleic acid molecule comprising any of various different coding sequences disclosed herein, for example, the coding sequences of (5’ to 3’) a first polypeptide comprising, e.g., a first polypeptide comprising, from N-terminus to C-terminus (i) a first antigen-binding domain that specifically binds a T cell antigen, (ii) a first multimerizing domain, and (iii) a second antigen-binding domain that specifically binds a T cell antigen; and (b) a second polypeptide comprising, from N-terminus to C-terminus (i) a third antigen-binding domain that specifically binds a target antigen, and (ii) a second multimerizing domain, wherein the first and the second multimerizing domains associate with one another to form the molecule.
  • a first polypeptide comprising
  • the nucleic acid molecules disclosed herein may comprise a nucleotide sequence encoding an antigen binding portion(s) disclosed herein.
  • the nucleic acid molecules may comprise a nucleotide sequence encoding an antigen binding domain that may specifically target CD3.
  • the nucleic acid molecules may comprise a nucleotide sequence encoding an antigen binding domain that may specifically target MAGE-A4 polypeptide.
  • the polynucleotide encoding multispecific molecules herein is inserted into a vector.
  • the vector is a vehicle into which a polynucleotide encoding a protein may be covalently inserted so as to bring about the expression of that protein and/or the cloning of the polynucleotide.
  • Such vectors may also be referred to as "expression vectors".
  • the isolated polynucleotide may be inserted into a vector using any suitable methods known in the art, for example, without limitation, the vector may be digested using appropriate restriction enzymes and then may be ligated with the isolated polynucleotide having matching restriction ends.
  • Expression vectors can have the ability to incorporate and express heterologous or modified nucleic acid sequences coding for at least part of a gene product capable of being transcribed in a cell.
  • Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism.
  • control sequences which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism.
  • vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are discussed infra.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector may have the necessary 5' upstream and 3' downstream regulatory elements such as promoter sequences such as CMV, PGK and EFlalpha, promoters, ribosome recognition and binding TATA box, and 3' UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell.
  • promoter sequences such as CMV, PGK and EFlalpha
  • promoters ribosome recognition and binding TATA box
  • 3' UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell.
  • Other suitable promoters include the constitutive promoter of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and rous sarcoma virus promoter.
  • Human gene promoters may also be used, including, but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • inducible promoters are also contemplated as part of the vectors expressing chimeric antigen receptor. This provides a molecular switch capable of turning on expression of the polynucleotide sequence of interest or turning off expression. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.
  • the expression vector may have additional sequence such as 6x-histidine (SEQ ID NO: 273), c-Myc, and FLAG tags which are incorporated into the expressed multispecific molecule.
  • the expression vector may be engineered to contain 5' and 3' untranslated regulatory sequences that sometimes can function as enhancer sequences, promoter regions and/or terminator sequences that can facilitate or enhance efficient transcription of the nucleic acid(s) of interest carried on the expression vector.
  • An expression vector may also be engineered for replication and/or expression functionality (e.g., transcription and translation) in a particular cell type, cell location, or tissue type. Expression vectors may include a selectable marker for maintenance of the vector in the host or recipient cell.
  • the vectors are plasmid, autonomously replicating sequences, and transposable elements.
  • Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAG), or P1-derived artificial chromosome (PAG), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno- associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
  • retrovirus including lentivirus
  • adenovirus e.g., adeno-associated virus
  • herpesvirus e.g., herpes simplex virus
  • poxvirus baculovirus
  • papillomavirus papillomavirus
  • papovavirus e.g., SV40
  • expression vectors are Lenti-XTM Bicistronic Expression System (Neo) vectors (Clontrch), pCIneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST.TM., pLenti6/V5-DEST.TM., and pLenti6.2N5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • the coding sequences of the multispecific molecule disclosed herein can be ligated into such expression vectors for the expression of the chimeric protein in mammalian cells.
  • the nucleic acids encoding the multispecific molecule of the present disclosure are provided in a viral vector.
  • a viral vector can be that derived from retrovirus, lentivirus, or foamy virus.
  • viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle.
  • the viral vector can contain the coding sequence for the various chimeric proteins described herein in place of nonessential viral genes.
  • the vector and/or particle can be utilized for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
  • the viral vector containing the coding sequence for a multispecific molecule described herein is a retroviral vector or a lentiviral vector.
  • retroviral vector refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus.
  • lentiviral vector refers to a vector containing structural and functional genetic elements outside the LTRs that are primarily derived from a lentivirus.
  • the retroviral vectors for use herein can be derived from any known retrovirus (e.g., type c retroviruses, such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)).
  • type c retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)).
  • Retroviruses also include human T cell leukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family of retroviruses, such as Human Immunodeficiency Viruses, HIV-1 , HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immnodeficiency virus (EIV), and other classes of retroviruses.
  • retroviruses such as Human Immunodeficiency Viruses, HIV-1 , HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immnodeficiency virus (EIV), and other classes of retroviruses.
  • a lentiviral vector for use herein refers to a vector derived from a lentivirus, a group (or genus) of retroviruses that give rise to slowly developing disease.
  • Viruses included within this group include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi; a caprine arthritis-encephalitis virus; equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (Bl V); and simian immunodeficiency virus (SIV).
  • HIV human immunodeficiency virus
  • FMV feline immunodeficiency virus
  • Bl V bovine immune deficiency virus
  • SIV simian immunodeficiency virus
  • Preparation of the recombinant lentivirus can be achieved using the methods according to Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998
  • Retroviral vectors for use in the present disclosure can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.
  • Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in forming the vectors include, for example, genomic RNA and cDNAs available from commercially available sources, including the Type Culture Collection (ATCC), Rockville, Md. The sequences also can be synthesized chemically.
  • the vector may be introduced into a host cell to allow expression of the polypeptide within the host cell.
  • the expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art, as described herein.
  • the promoter sequences may be selected to promote the transcription of the polynucleotide in the vector. Suitable promoter sequences include, without limitation, T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter.
  • Enhancer sequences may be selected to enhance the transcription of the polynucleotide.
  • Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance.
  • Signal sequences may be selected to allow the expressed polypeptide to be transported outside of the host cell.
  • the vector may be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein.
  • the cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.
  • the origin of replication may be selected to promote autonomous replication of the vector in the host cell.
  • the present disclosure provides isolated host cells containing the vectors provided herein.
  • the host cells containing the vector may be useful in expression or cloning of the polynucleotide contained in the vector.
  • Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.
  • Suitable prokaryotic cells for this purpose include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E.
  • the multispecific of the present disclosure are introduced into a host cell using transfection and/or transduction techniques known in the art.
  • transfection and/or transduction
  • the terms, "transfection,” and, “transduction,” refer to the processes by which an exogenous nucleic acid sequence is introduced into a host cell.
  • the nucleic acid may be integrated into the host cell DNA or may be maintained extrachromosomally.
  • the nucleic acid may be maintained transiently or may be a stable introduction.
  • Transfection may be accomplished by a variety of means known in the art including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by means of viral infection rather than by transfection.
  • retroviral vectors are transduced by packaging the vectors into virions prior to contact with a cell.
  • a nucleic acid encoding a multispecific molecule by a retroviral vector can be transduced into a cell through infection and pro virus integration.
  • the term “genetically engineered” or “genetically modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell.
  • the terms, “genetically modified cells,” “modified cells,” and, “redirected cells,” are used interchangeably.
  • the present invention provides pharmaceutical compositions comprising the multispecific antigen-binding molecules of the present invention.
  • the pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • a multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • vesicles such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • the dose of antigen-binding molecule administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like.
  • the preferred dose is typically calculated according to body weight or body surface area.
  • the frequency and the duration of the treatment can be adjusted.
  • Effective dosages and schedules for administering a multispecific antigen-binding molecule may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991 , Pharmaceut. Res. 8:1351).
  • Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • a pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • reusable pens and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention.
  • Examples include, but are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, IN), NOVOPENTM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTM (sanofi-aventis, Frankfurt, Germany), to name only a few.
  • Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTARTM pen (sanofi-aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly), the SURECLICKTM Autoinjector (Amgen, Thousand Oaks, CA), the PENLETTM (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRATM Pen (Abbott Labs, Abbott Park IL), to name only a few.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida.
  • a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antigen-binding molecule or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • the amount of the aforesaid antigen-binding molecule contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antigen-binding molecule is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.
  • the present invention includes methods comprising administering to a subject in need thereof a therapeutic composition comprising a multispecific antigen-binding molecule that specifically binds a T-cell antigen (e.g., CD3) and a target antigen (e.g., MAGE-A4).
  • the therapeutic composition can comprise any of the multispecific antigen-binding molecules as disclosed herein and a pharmaceutically acceptable carrier or diluent.
  • a subject in need thereof means a human or non-human animal that exhibits one or more symptoms or indicia of cancer, or who otherwise would benefit from an inhibition or reduction in target antigen activity or a depletion of target-antigen positive cells (e.g., tumor cells).
  • the multispecific antigen-binding molecules of the invention are useful, inter alia, for treating any disease or disorder in which stimulation, activation and/or targeting of an immune response would be beneficial.
  • the multispecific antigen-binding molecules of the present invention may be used for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by target antigen expression or activity or the proliferation of target-antigen positive cells.
  • the mechanism of action by which the therapeutic methods of the invention are achieved includes killing of the cells expressing the target antigen in the presence of T cells.
  • the multispecific antigen-binding molecules of the present invention may be used to treat a disease or disorder associated with target antigen expression including, e.g., a cancer.
  • Analytic/diagnostic methods known in the art may be used to ascertain whether a patient harbors a tumor cell that is positive for the target antigen.
  • the cancer is selected from a solid tumor, cervical cancer, head and neck squamous cell carcinoma, melanoma, prostate cancer, acute myeloid leukemia, pancreatic cancer, colon cancer, acute lymphocytic leukemia, a non-Hodgkin’s lymphoma, gastric cancer, post-transplant lymphoproliferative disorder, ovarian cancer, lung cancer, squamous cell carcinoma, non-small cell lung cancer esophageal cancer, bladder cancer (e.g., urinary bladder squamous cell carcinoma), nasopharyngeal cancer, uterine cancer, liver cancer, testicular cancer, breast cancer, or synovial sarcoma.
  • the multispecific antigen-binding molecules of the invention may be useful for treating melanoma, lung cancer, bladder cancer, and/or synovial sarcoma.
  • the multispecific antigen-binding molecules of the invention may be useful for treating lung cancer, esophageal cancer, synovial sarcoma, ovarian cancer, melanoma, and/or gastric cancer.
  • the multispecific antigen-binding molecules of the present invention may be used to treat an individual diagnosed with or suspected of having, or at risk of developing a malignancy characterized at least in part by the expression of MAGE-A4 by cancer cells (e.g., MAGE-A4 expressing solid tumor cells), comprising administering to the individual a therapeutically effective amount of one or more of the multispecific antigen-binding molecules described elsewhere herein.
  • the present invention also includes methods for treating residual cancer in a subject.
  • residual cancer means the existence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy.
  • the present invention provides methods for treating a disease or disorder associated with target antigen expression (e.g., a cancer) comprising administering one or more of the multispecific antigen-binding molecules described elsewhere herein to a subject after the subject has been determined to have a target antigen positive cancer.
  • a disease or disorder associated with target antigen expression e.g., a cancer
  • the present invention includes methods for treating a cancer comprising administering a multispecific antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received other immunotherapy or chemotherapy.
  • the present invention provides methods which comprise administering a pharmaceutical composition comprising any of the exemplary multispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents.
  • additional therapeutic agents that may be combined with or administered in combination with an antigenbinding molecule of the present invention include, e.g., an anti-tumor agent (e.g. chemotherapeutic agents).
  • the second therapeutic agent may be a monoclonal antibody, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, a checkpoint inhibitor, or combinations thereof.
  • cytokine inhibitors including small-molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-17, IL-18, or to their respective receptors.
  • compositions of the present invention may also be administered as part of a therapeutic regimen comprising one or more therapeutic combinations selected from a monoclonal antibody that may interact with a different antigen on the cell surface, a bispecific antibody that has one arm that binds to an antigen on the tumor cell surface and the other arm binds to an antigen on a T cell, an antibody drug conjugate, a bispecific antibody conjugated with an anti-tumor agent, a checkpoint inhibitor, for example, one that targets, PD-1 or CTLA-4, or combinations thereof.
  • a therapeutic regimen comprising one or more therapeutic combinations selected from a monoclonal antibody that may interact with a different antigen on the cell surface, a bispecific antibody that has one arm that binds to an antigen on the tumor cell surface and the other arm binds to an antigen on a T cell, an antibody drug conjugate, a bispecific antibody conjugated with an anti-tumor agent, a checkpoint inhibitor, for example, one that targets, PD-1 or CTLA-4, or combinations thereof
  • the checkpoint inhibitors may be selected from PD-1 inhibitors, such as pembrolizumab (Keytruda), nivolumab (Opdivo), or cemiplimab (REGN2810).
  • the checkpoint inhibitors may be selected from PD-L1 inhibitors, such as atezolizumab (Tecentriq), avelumab (Bavencio), or Durvalumab (Imfinzi)).
  • the checkpoint inhibitors may be selected from CTLA-4 inhibitors, such as ipilimumab (Yervoy). Other combinations that may be used in conjunction with an antibody of the invention are described above.
  • the present invention also includes therapeutic combinations comprising any of the antigen-binding molecules mentioned herein and an inhibitor of one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvlll, cMet, IGF1 R, IL-10, B-raf, PDGFR-a, PDGFR- , FOLH1 (PSMA), PRLR, STEAP1 , STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, an siRNA, a peptibody, a nanobody, an antibody, a bispecific antibody or an antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules, such as diabodies, triabodies, tri
  • the antigen-binding molecules of the invention may also be administered and/or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs.
  • the antigen-binding molecules of the invention may also be administered as part of a treatment regimen that also includes radiation treatment and/or conventional chemotherapy.
  • the additional therapeutically active component(s) may be administered just prior to, concurrent with, or shortly after the administration of an antigen-binding molecule of the present invention; (for purposes of the present disclosure, such administration regimens are considered the administration of an antigen-binding molecule "in combination with" an additional therapeutically active component).
  • the present invention includes pharmaceutical compositions in which an antigen-binding molecule of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.
  • multiple doses of a multispecific antigen-binding molecule may be administered to a subject over a defined time course.
  • the methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an antigen-binding molecule of the invention.
  • sequentially administering means that each dose of an antigen-binding molecule is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • the present invention includes methods which comprise sequentially administering to the patient a single initial dose of an antigen-binding molecule, followed by one or more secondary doses of the antigen-binding molecule, and optionally followed by one or more tertiary doses of the antigen-binding molecule.
  • initial dose refers to the temporal sequence of administration of the antigen-binding molecule of the invention.
  • the "initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of the antigen-binding molecule, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of an antigen-binding molecule contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment.
  • two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").
  • each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1 , 1 %, 2, 2 1 / 2 , 3, 3 1 / 2 , 4, 4 1 / 2 , 5, 5%, 6, 6 1 / 2 , 7, 7 1 / 2 , 8, 8 1 / 2 , 9, 9 1 / 2 , 10, 10 1 / 2 , 11 , 11%, 12, 12%, 13, 13%, 14, 14 1 / 2 , 15, 15 1 / 2 , 16, 16 1 / 2 , 17, 17 1 / 2 , 18, 18 1 / 2 , 19, 19 1 / 2 , 20, 20%, 21 , 2134, 22, 22%, 23, 23%, 24, 24%, 25, 25%, 26, 26%, or more) weeks after the immediately preceding dose.
  • 1 to 26 e.g., 1 , 1 %, 2, 2 1 / 2 , 3, 3 1 / 2 , 4, 4 1 / 2 , 5, 5%, 6, 6 1 / 2 , 7, 7
  • the immediately preceding dose means, in a sequence of multiple administrations, the dose of antigen-binding molecule which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
  • the methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an antigen-binding molecule.
  • a single secondary dose is administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient.
  • only a single tertiary dose is administered to the patient.
  • two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
  • each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
  • Anti-MAGE-A4 antibodies were obtained by immunizing a genetically modified mouse (e.g., an engineered mouse comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions) with a human MAGE-A4 antigen and HLA-A2.
  • Anti-CD3 antibodies were obtained by immunizing a genetically modified mouse (e.g., an engineered mouse comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions) with a human CD3 antigen.
  • splenocytes were harvested from each mouse and either (1) fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for MAGE-A4 or CD3 specificity, or (2) B-cell sorted (as described in US Patent Pub. No. 2007/0280945A1) using a human MAGE-A4 fragment or a human CD3 fragment as the sorting reagent that binds and identifies reactive antibodies (antigen-positive B cells).
  • Chimeric antibodies to MAGE-A4 were initially isolated having a human variable region and a mouse constant region.
  • the antibodies were characterized and selected for desirable characteristics, including affinity, selectivity, etc.
  • mouse constant regions were replaced with a desired human constant region, for example wild-type or modified I gG 1 or lgG4 constant region, to generate a fully human anti-MAGE-A4 or anti-CD3 antibody. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
  • bsAbs bispecific antibodies
  • antibodies were reformatted as single chain variable fragments (scFvs) and combined with the above-described antibodies as shown in FIG. 2C.
  • Example 2 bsAb9930 controls A375 melanoma and SCaBER urinary bladder tumor growth in vivo
  • An A375 melanoma tumor cell line was expanded in T225 flasks in DMEM high glucose media supplemented with penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum until confluent. Trypsin-EDTA (0.25%) was used to detach the A375 cells from each flask to collect the cells. A375 tumor cells were then washed twice and resuspended in Hank’s Balanced Salt Solution. A cell aliquot was stained with ViaStainTM, a solution containing acridine orange and propidium iodide (AOPI), to determine the viable cell density using a Nexcelom Cellaca MX cell counter. A375 cells were then resuspended at a density of 2.5x10 7 viable cells per mL in Hank’s Balanced Salt Solution and 5x10 6 A375 cells were implanted subcutaneously into immunodeficient NSGTM mice.
  • the SCaBER urinary bladder tumor cell line was expanded in T225 flasks in MEM Earl’s Salts media supplemented with penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum until confluent. Trypsin-EDTA (0.25%) was used to detach cells from each flask for collection. SCaBER cells were then washed twice and resuspended in Hank’s Balanced Salt Solution. A cell aliquot was stained with ViaStainTM, a solution containing acridine orange and propidium iodide (AOPI), to determine the viable cell density using a Nexcelom Cellaca MX cell counter. SCaBER cells were then resuspended at a density of 5x10 7 viable cells per mL in Hank’s Balanced Salt Solution and 1x10 7 SCaBER cells were implanted subcutaneously into immunodeficient NSGTM mice.
  • ViaStainTM a solution containing acridine orange
  • PBMCs were prepared for engraftment into each NSGTM mouse. To do so, PBMCs were thawed in a warm water bath before first being washed in warmed OpTmizer media supplemented with CTS additive and Benzonase® nuclease at 25 Units/mL. Cells were then washed a second time with the same media minus the Benzonase nuclease.
  • PBMCs were resuspended in warmed RPMI media containing 10% fetal bovine serum and counted using ViaStainTM and the Nexcelom Cell Counter before being resuspended in Hank’s Balanced Salt Solution. Five million PBMCs were engrafted into each NSGTM mouse by intraperitoneal injection.
  • Study 1 Treatment protocol [0255] On Day 10 following A375 melanoma tumor cell implantation, tumor bearing NSGTM were randomized into treatment groups having a mean tumor volume of 125 mm 3 before receiving the first dose of the indicated biologies as defined in Table 8. Mice in each treatment group were treated twice weekly by intraperitoneal injection for a total of six doses with the final dosage being administered on Day 25 post-tumor cell implantation.
  • tumor bearing NSGTM mice were randomized into treatment groups having a mean tumor volume of 93 mm 3 before receiving a single dose of indicated biologies as defined in Table 9. All biologies were delivered by intraperitoneal injection. At 48 hours following dosage, spleen and tumors were collected to prepare single cell suspensions for flow cytometric analysis.
  • tumors were harvested, weighed, and dissociated using the Miltenyi Biotec Mouse Tumor Dissociation Kit in combination with the gentleMACSTM Tissue Dissociator. The instrument’s “Soft/Medium” digestion protocol was utilized. Following dissociation, cell suspensions were filtered through a MACS® 70 pM Smartstrainer and washed and resuspended with warmed RPMI media supplemented with 10% fetal bovine serum. To prepare single cell suspensions from the spleen, spleens were homogenized using a plunger from 5 mL syringe and filtered through a MACS® 70 uM Smartstrainer.
  • Spleen cells were then washed once in warmed RPMI media containing 10% FBS before centrifuging the cells at 1500 rpm for 4 minutes. Spleen cells were then washed in D-PBS before lysing red blood cells with ACK lysis buffer for 8 minutes at room temperature. Following red blood cell lysis, spleen cells were washed twice and resuspended in warmed RPMI media containing 10% fetal bovine serum.
  • Table 10 Flow panel used to characterize tumor infiltrating cells.
  • tumor bearing NSGTM were randomized into treatment groups having a mean tumor volume of 137 mm 3 before receiving the first dose via intraperitoneal injection of the indicated biologies as defined in Table 11 . Mice in each treatment group were treated every three days by intraperitoneal injection with the final dosage being administered on Day 43 post-tumor cell implantation. Table 11. Defined treatment groups and dosing protocol for study 3.
  • bsAb9930 represents a bispecific antibody formatted to target HLA-A2 molecules presenting the MAGE-A4 tumor derived peptides (286-294) and (230-239). The effector arms bind to CD3. This format is able to induce T cell activation and potent T cell mediated cytolytic activity of tumor cell lines expressing endogenous levels of MAGE-A4 peptide (see International Patent Pub. No. WO/2021/016585).
  • bsAb9930 was found to be capable of delaying A375 melanoma tumor growth when dosed twice weekly at 0.1 mg/kg (Fig.
  • bsAb9930 when bsAb9930 was co-administered with an anti-EGFR x anti-CD28 bispecific antibody designed to selectively activate T cells infiltrating into EGFR+ tumors, protection was enhanced, with one of five treated mice showing complete tumor regression and all treated mice showing delayed tumor growth kinetics relative to the isotype treated control group (Fig. 3A, Table 8). Efficacy was further enhanced when bsAb9930 was co-administered with both the anti-PD-1 antibody and the anti-EGFR x anti-CD28 bispecific antibody, as complete tumor regression was observed in all five treated mice (Fig. 3A, Table 8).
  • mice were treated as shown in Table 9, and 48 hours after the first dose, tumor-infiltrating and spleen T cells were characterized by flow cytometry to investigate their activation status (Table 10).
  • mice were treated with a bispecific antibody that binds to HLA-A2 in a peptide- independent manner and to CD3 to induce T cell effector function.
  • both bsAb9930 and the positive control bispecific monotherapy resulted in the selective activation of intra-tumoral T cells relative to the spleen as assessed by CD25 expression (Fig. 3B).
  • the positive control bispecific antibody can target every HLA- A2 complex on the tumor’s cell surface and bsAb9930 only targets a reduced number of HLA-A2 complexes presenting defined MAGE-A4 peptides, the proportion of activated T cells expressing CD25 in the tumor was greatest following treatment with the positive control relative to bsAb9930 monotherapy (Fig. 3B).
  • bsAb9930 was able to delay SCaBER urinary bladder tumor growth when dosed every three days at 0.1 mg/kg (Fig. 3C) and co-administered with an anti-PD-1 antibody and an anti-EGFR x anti-CD28 bispecific antibody designed to selectively activate T cells infiltrating into EGFR+ tumors.
  • Controls groups included tumor bearing mice being treated with sterile saline (Table 10, Group 1), an isotype control antibody (Table 10, Group 2), or a structurally matched bsAb control targeting an irrelevant protein in lieu of MAGE-A4 peptides presented by HLA-A2 and coadministered with an anti-PD-1 antibody and an anti-EGFR x anti-CD28 bispecific antibody (Table 10, Group 3).
  • Treatment group 3 in study 3 demonstrates that PD-1 blockade in combination with tumor-localized CD28 costimulation was insufficient to control SCaBER tumor growth in the absence of bsAb9930.
  • Example 3 Conventional TAAxCD3 bsAbs can be inefficient at redirecting T cells to kill tumor cell lines expressing endogenous MHC-peptide tumor neoantigens
  • a selected HLA-A2: MAGE-A4 (286-294) antibody was paired to a CD3 arm to generate a bispecific antibody.
  • the bispecific antibody s binding to Jurkat and A375 cells overexpressing the peptide of interest was assessed by flow cytometry.
  • the binding curves shown in Fig. 4A validate bispecific antibody binding to both Jurkat and A373 cells overexpressing the peptide of interest.
  • the cytotoxic potency of the HLA-A2: MAGE-A4 (286- 294) x CD3 bispecific antibody against A375 cells expressing endogenous level of MAGE-A4 peptide, or A375 cells engineered to overexpress HLA-A2/MAGE-A4 (286-294) peptide was also assessed by flow cytometry.
  • Target cells were incubated with a titration of antibody and human PBMC in a 10:1 E:T ratio for 96 hours (Fig. 4B). Results show a decreasing percentage of live cells with increasing Log10 [Antibody] (M) values.
  • a conventional xCD3 bispecific antibody targeting an MHC-peptide tumor neoantigen redirected T cells to kill tumor cells only when the peptide antigen was over expressed on the surface of the cells.
  • Example 4 2+2 bsAbs are the most potent at redirecting T cells to kill tumor cells expressing endogenous levels of peptide, and the potency relies on both the geometry and valency of these molecules, and is driven by two CD3 arms
  • Example 5 2+2 bsAb format allows for targeting two different peptides, and its potency is enhanced when combined with a co-stimulatory bispecific and anti-PD1
  • cytotoxic potency of a 2+2 bsAb targeting a single MAGE-A4 peptide or bsAb9930 against A375 cells expressing endogenous levels of MAGE-A4 peptides was assessed in-vitro by flow cytometry, as shown in Fig. 6A.
  • the specific cytotoxic potency of bsAb9930 as single agent or in combination with a CD28-based bispecific antibody (EGFRxCD28) and anti-PD1 was assessed in a flow cytometry-based cytotoxicity assay targeting A375 cells (Fig. 6B).
  • bsAb format is capable of targeting two different peptides and the potency of such bsAb format is enhanced when combined with a co-stimulatory bispecific.
  • Fig. 2D shows a cartoon of an exemplary mechanism of action for bsAb9930 in combination, for example, with a CD28-based bispecific antibody (EGFRxCD28) and an anti-PD1 antibody, in particular, targeting A375 cells.
  • EGFRxCD28 CD28-based bispecific antibody
  • anti-PD1 antibody in particular, targeting A375 cells.
  • bsAb9930 The specific cytotoxic potency of bsAb9930 was demonstrated in a flow cytometrybased cytotoxicity assay targeting a cell line that expresses HLA-A2 but does not express MAGE-A4 (J82 cells) (Fig. 7A).
  • T cell activation measured by the upregulation of the CD25 marker was not observed in the absence of tumor cells expressing the peptide antigen

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Abstract

La présente invention concerne des molécules de liaison à un antigène multispécifiques qui se lient à la fois à un antigène de lymphocyte T (par exemple, CDS) et à un antigène cible (par exemple, MAGE-A4), et leurs utilisations.
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