WO2024102962A1 - Cytotoxic bispecific antibodies binding to dr5 and muc16 and uses thereof - Google Patents

Cytotoxic bispecific antibodies binding to dr5 and muc16 and uses thereof Download PDF

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WO2024102962A1
WO2024102962A1 PCT/US2023/079313 US2023079313W WO2024102962A1 WO 2024102962 A1 WO2024102962 A1 WO 2024102962A1 US 2023079313 W US2023079313 W US 2023079313W WO 2024102962 A1 WO2024102962 A1 WO 2024102962A1
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seq
acid sequence
amino acid
binding molecule
heavy chain
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PCT/US2023/079313
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French (fr)
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Viktor S. Goldmakher
Iosif M. GERSHTEYN
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Immuvia Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—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
    • C07K16/3076—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 against structure-related tumour-associated moieties
    • C07K16/3092—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 against structure-related tumour-associated moieties against tumour-associated mucins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the human MUC16 gene (mucin 16, NCBI Entrez Gene: 94025) encodes the type I transmembrane protein MUC16 or mucin 16, a high molecular weight, heavily glycosylated protein. It is not expressed in most normal tissues, except for the apical surface in the epithelium of the upper respiratory tract, ocular surface, mesothelium lining body cavities (pleural, peritoneal, and pelvic cavities), internal organs, and male and female reproductive organs (Lee et al., Pharmaceuticals, 2021, 14, 1053). MUC16 is highly expressed in many different types of cancer, including most ovarian and endometrial tumors (Kabawat et al. Am. J. Clin.
  • MUC16 is highly expressed in idiopathic pulmonary fibrosis (IPF) (Ballester et al. Int. J. Mol. Sci. 2021; 22:6502) and, likely, other systemic fibrosis diseases (Zhang et al. Prog. Mol. Biol. Transl. Sci. 2019; 162:241-252).
  • IPF idiopathic pulmonary fibrosis
  • MUC16 is expressed in pathologic hyperplastic alveolar type II cells and in lung fibroblasts from fibrotic foci, but not normal lung (Ballester et al. Int. J. Mol. Sci. 2021; 22:6502).
  • Antibody-drug conjugates that target MUC16 have been, and bispecific moi eties targeting MUC16 and either CD28 or CD3 are currently in early human clinical trials for treatment of various cancers (NCT01335958, NCT04590326, NCT03564340).
  • the MUC16 extracellular domain consists of an unstructured N-terminal domain and a tandem repeat (TR) region.
  • the TR region contains about 60 repeats of 156 amino acids.
  • the TR region is interspersed with approximately 16 homologous SEA (Sea urchin sperm protein, Enterokinase, Agrin) domains (White et al., Proteins, 2022;90:1210-1218).
  • SEA Stea urchin sperm protein, Enterokinase, Agrin domains
  • MUC16 protein is cleaved extracellularly by an unknown mechanism and subsequently shed into blood. This extracellular portion of MUC16 is a well- established serum biomarker for ovarian cancer (Bast et al., Int. J. Biol. Markers 1998, 13, 179-187).
  • Death receptor 5 is a cell membrane protein that, when bound to its ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), can induce cell death via apoptosis. Apoptosis can also be mediated by DR5 when bound to agonistic monoclonal antibodies (Dubuisson and Micheau, Antibodies 2017, 6, 16). In preclinical studies, stimulation of DR5 with either TRAIL or an anti-DR5 antibody was reported to induce cell death in a variety of tumor cells, but to date, in clinical trials neither TRAIL or its derivatives, nor anti-DR5 antibodies showed a significant therapeutic benefit (Lemke et al., Cell Death Differ. 2014, 21: 1350-1364; von Karstedt et al. Nat Rev Cancer. 2017, 17: 352- 366).
  • Bispecific molecules targeting DR5 and various second antigens are known in the art, and some are in early human clinical trials.
  • an anti-CDH17/anti-DR5 bispecific antibody (Garcia-Martinez et al., Mol. Cancer Then 2021, 20: 96-108; United States patent No. 10,858,438) is currently in clinical development.
  • Other second antigens targeted by such DR5 bispecifics include folate receptor alpha (US patent publication No. 20200283537), fibroblast-activation protein (FAP) (US patent No.
  • the present invention provides bispecific binding molecules that comprise a first antigen-binding domain that binds specifically to the extracellular domain of human MUC16 and a second antigen-binding domain that binds specifically to human DR5.
  • These bispecific molecules are efficient in killing cells that co-express MUC16 and DR5 and are more potent in killing such cells than the monospecific DR5 antibody from which the second antigen-binding domain is derived (e.g., an anti-DR5 antibody having the same set of three heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) and light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) as are present in the bispecific binding molecule).
  • the first antigen-binding domain (which binds the extracellular portion of human MUC16) binds to an epitope present in more than one tandem repeat/SEA segment within the extracellular domain of MUC16.
  • the first antigen-binding domain is competed for MUC16 binding by one or more of the following antibodies: OC125, H185 (Invitrogen Cat No. MA5-11579), Mi l (American Tissue Culture Collection Accession No. PTA-6206), OV197 (Fujirebio Diagnostic), 5E11 (Millipore Sigma Cat No. MABC1608-25UG), AR9.6, H1H8794, VK-8, B43.13 (also known as Oregovomab), or 3A5 (Sofituzumab).
  • the first antigen-binding domain binds to a polypeptide or peptide consisting essentially of the amino acid sequence of at least one of SEQ ID NOs:176-181, or an extracellular fragment of MUC16 isolated from the cell culture media of OVCAR-3 cells.
  • the first antigen-binding domain comprises six specific heavy and light-chain CDR sequences (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3).
  • CDR-H1 comprises the amino acid sequence of SEQ ID NO:1;
  • CRD-H2 comprises the amino acid sequence of any one of SEQ ID NOs.:2-5;
  • CDR2-H3 comprises the amino acid sequence of any one of SEQ ID NOs.:6-61 (CDR3-H3);
  • CDR-L1 comprises the amino acid sequence of SEQ ID NO:62;
  • CDR-L2 comprises the amino acid sequence of SEQ ID NO:63; and
  • CDR-L3 comprises the amino acid sequence of SEQ ID NO:65.
  • CDR-H1 comprises the amino acid sequence of SEQ ID NO:1;
  • CDR-H2 comprises the amino acid sequence of SEQ ID NO:2;
  • CDR-H3 comprises the amino acid sequence of SEQ ID NO:6;
  • CDR-L1 comprises the amino acid sequence of SEQ ID NO:62;
  • CDR-L2 comprises the amino acid sequence of SEQ ID NO:63; and
  • CDR-L3 comprises the amino acid sequence of SEQ ID NO:65.
  • Each of the above CDR sequences are according to the Kabat convention of determining CDRs.
  • the set of 6 CDR sequences that characterize the first binding moiety are selected from any of the following set forth in Table 1.
  • the first antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of either SEQ ID NOs.:104 or 105, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to either SEQ ID NOs.: 104 or 105.
  • the first antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of either SEQ ID NOs.: 104 or 105.
  • the first antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of any one of SEQ ID NOs: 106- 114, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to any one of SEQ ID NOs: 106-114.
  • the first antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO:97.
  • the first antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97.
  • the first antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of any one of SEQ ID NOs:98-103, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to any one of SEQ ID NOs:98-103.
  • the first antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of either SEQ ID NOs.: 104 or 105, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to either SEQ ID NOs.:104 or 105; and a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 97.
  • the first antigenbinding domain comprises a light chain variable region that comprises the amino acid sequence of either SEQ ID NOs.: 104 or 105; and aheavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97.
  • the first antigen-binding domain comprises a combination of a heavy chain variable region and a light chain variable region that are selected from any of the following combinations set forth in Table 2.
  • the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs.: 115-118, or a heavy chain comprising the same three heavy chain CDRs as, and an amino acid sequence having at least 90% sequence identity to, any one of SEQ ID NOs.:115-118.
  • the heavy chain comprises the amino acid sequence of any one of SEQ ID NOs.:115-118.
  • the first antigen-binding domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 120; or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to, SEQ ID NO: 119 or SEQ ID NO: 120.
  • the light chain comprises the amino acid sequence of any one of SEQ ID NOs.:119-120.
  • the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs.: 115-118, or a heavy chain comprising the same three heavy chain CDRs as, and an amino acid sequence having at least 90% sequence identity to, any one of SEQ ID NOs. : 115-118; and a light chain comprising the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 120; or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to, SEQ ID NO: 119 or SEQ ID NO: 120.
  • the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs.: 115-118; and a light chain comprising the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 120.
  • the first antigen-binding domain as described for any of the first through tenth aspects comprises two heavy chains and two light chains.
  • each of the two heavy chains has an identical amino acid sequence and each of the two light chains has an identical amino acids sequence.
  • the second antigen-binding domain which binds to human DR5 is competed for DR5 binding by: a) one or more of the following antibodies: Conatumumab, Drozitumab, Lexatumumab, LBY135, Tigatuzumab, and DS- 8273a; or b) TRAIL or a fragment of TRAIL that binds to DR5.
  • the second antigen-binding domain comprises six specific heavy and light-chain CDR sequences (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3).
  • CDR-H1 comprises the amino acid sequence of SEQ ID NO: 121;
  • CRD-H2 comprises the amino acid sequence of SEQ ID NO: 122;
  • CDR2-H3 comprises the amino acid sequence of SEQ ID NO: 123;
  • CDR-L1 comprises the amino acid sequence of SEQ ID NO: 124;
  • CDR-L2 comprises the amino acid sequence of SEQ ID NO: 125; and
  • CDR-L3 comprises the amino acid sequence of SEQ ID NO: 126.
  • the above CDR sequences are according to the Kabat convention of determining CDRs.
  • the set of 6 CDR sequences that characterize the first binding moiety are selected from any of the following set forth in Table 3.
  • the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 158.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 158.
  • the second antigenbinding domain comprises a heavy chain variable region that comprise the amino acid sequence of any one of SEQ ID NOs: 159-164, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NOs: 159-164.
  • the second antigen-binding domain comprises a heavy chain variable region that comprise the amino acid sequence of any one of SEQ ID NOs: 218-220, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NOs: 218-220.
  • the heavy chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 218.
  • the heavy chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 219.
  • the heavy chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 220.
  • the second antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 165.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 165.
  • the second antigen-binding domain comprises a light chain variable region that comprise the amino acid sequence of any one of SEQ ID NOs: 166-171, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to SEQ ID NOs: 166-171.
  • the second antigen-binding domain comprises a light chain variable region that comprise the amino acid sequence of any one of SEQ ID NOs: 215-217, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to SEQ ID NOs: 215-217.
  • the light chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 215.
  • the light chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 216.
  • the light chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 217.
  • the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of either SEQ ID NO: 158, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 158; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 165.
  • the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 158; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 165.
  • the second antigen-binding domain comprises a combination of a heavy chain variable region and a light chain variable region that are selected from any of the following combinations set forth in Table 4.
  • the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 218; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 215.
  • the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 219; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 215.
  • the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 219; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 216.
  • the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 220; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 217.
  • the second antigen-binding domain is a scFv fragment of an antibody characterized by any of the foregoing twelfth through sixteenth aspects.
  • An scFv fragment comprises a heavy chain variable region and a light chain variable region of bound to one another through a peptide linker.
  • the scFv fragment comprises, in N- to C-terminal order, a light chain variable region, a peptide linker, and a heavy chain variable region.
  • the scFv fragment comprises the amino acid sequence of any one of SEQ ID NO: 172, SEQ ID NOs: 210-214; or an amino acid sequence having the same three heavy chain CDRs and the same three light chain CDRs as, and at least 90% sequence identity to, any one of SEQ ID NO: 172 or SEQ ID NOs: 210- 214.
  • the scFv fragment comprises the amino acid sequence of SEQ ID NO: 172; or an amino acid sequence having the same three heavy chain CDRs and the same three light chain CDRs as, and at least 90% sequence identity to, SEQ ID NO: 172.
  • the scFv fragment comprises the amino acid sequence of SEQ ID NO: 172. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:210. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:211. In other specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:212. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:213. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:214.
  • bispecific binding molecules disclosed herein may comprise a first antigen-binding domain characterized by any of the foregoing first through eleventh aspects and a second antigen-binding domain characterized by any of the foregoing twelfth through seventeenth aspects. All possible combinations of those first antigen-binding domains and second antigen binding domains are within the scope of the present disclosure.
  • the N-terminus of the second antigenbinding domain is fused directly or through a peptide linker having a length of 4 to 20 amino acids to the C-terminus of one of the heavy chains of the first antigen-binding domain.
  • the N-terminus of the second antigen-binding domain is fused through a peptide linker having a length of 4 to 20 amino acids to the C-terminus of one of the heavy chains of the first antigen-binding domain.
  • the peptide linker has the amino acid sequence of SEQ ID NO: 173.
  • the second antigen binding domain is an scFv fragment of the seventeenth aspect.
  • the bispecific antigen-binding molecule comprises two scFv fragments of the seventeenth aspect that specifically bind human DR5.
  • the bispecific antigen-binding molecule comprises two scFv fragments of the seventeenth aspect that specifically bind human DR5, wherein each is bound to the C-terminus of a different heavy chain of the first antigen-binding domain.
  • each of the two scFv fragments have the identical amino acid sequence.
  • the bispecific binding molecule comprises: a) two antibody light chains, wherein each light chain has an amino acid sequence independently selected from SEQ ID NO: 119 and SEQ ID NO: 120; and b) two antibody heavy chain fusions, wherein each heavy chain fusion has an amino acid sequence independently selected from the formula: X-L-Y, wherein: X is the amino acid sequence of any one of SEQ ID NOs.: 115-118; L is the amino acid sequence of SEQ ID NO: 173; and Y is the amino acid sequence of any one of SEQ ID NO: 172 or SEQ ID NOs: 210-214. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 172.
  • Y is the amino acid sequence of SEQ ID NO: 210. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 211. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 212. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 213. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 214. In some embodiments of the nineteenth aspect, each light chain has the identical amino acid sequence, and each heavy chain fusion has the identical amino acid sequence.
  • each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 187.
  • each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:203. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:204. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:205. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:206.
  • each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:207. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 186.
  • each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:203. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:204. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:205.
  • each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:206. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:207.
  • a pharmaceutical composition comprising a bispecific binding molecule described herein and a pharmaceutically acceptable carrier.
  • Y is the amino acid sequence of SEQ ID NO: 172.
  • Y is the amino acid sequence of SEQ ID NO: 210.
  • Y is the amino acid sequence of SEQ ID NO: 211. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO: 212. In some embodiments of the twenty -first aspect, Y is the amino acid sequence of SEQ ID NO: 213. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO: 214. In some embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 174. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 175.
  • the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 186. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 187. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:203. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:204. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:205. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:206.
  • the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:207.
  • the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 182 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 182 and encoding the same amino acid sequence as SEQ ID NO: 182.
  • the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 183 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 183 and encoding the same amino acid sequence as SEQ ID NO: 183.
  • an expression vector comprising the nucleic acid sequence of the twenty-first aspect or any embodiments thereof.
  • the expression vector additionally comprises a first promoter that is operatively linked to and can drive expression of the nucleic acid sequence.
  • an expression vector of the twenty second aspect that additionally comprises a nucleic acid sequence encoding a light chain of the first antigen-binding domain of the bispecific binding molecule as described in any of the foregoing aspects of the invention; and a second promoter that is operatively linked to and can drive expression of the light-chain encoding nucleic acid sequence.
  • the nucleic acid sequence encoding a light chain of the first antigen-binding domain of the bispecific binding molecule comprises the nucleotide sequence of SEQ ID NO: 184 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 184 and encoding the same amino acid sequence as SEQ ID NO: 184.
  • the nucleic acid sequence encoding a light chain of the first antigen-binding domain of the bispecific binding molecule comprises the nucleotide sequence of SEQ ID NO: 185 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 185 and encoding the same amino acid sequence as SEQ ID NO: 185.
  • the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 182 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 182 and encoding the same amino acid sequence as SEQ ID NO: 182; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 184 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 184 and encoding the same amino acid sequence as SEQ ID NO: 184.
  • the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 183 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 183 and encoding the same amino acid sequence as SEQ ID NO: 183; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 185 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 185 and encoding the same amino acid sequence as SEQ ID NO: 185.
  • a host cell harboring: a) the expression vector of the twenty-second aspect or any embodiments thereof; and b) an expression vector comprising a nucleic acid sequence encoding the light chain of the first antigen-binding domain of the bispecific binding molecule as described in any of the foregoing aspects of the invention.
  • the expression vector comprising a nucleic acid sequence encoding the light chain additionally comprises a promoter that is operatively linked to and can drive expression of the lightchain encoding nucleic acid sequence.
  • the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 184 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 184 and encoding the same amino acid sequence as SEQ ID NO: 184.
  • the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 185 or a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 185 and encoding the same amino acid sequence as SEQ ID NO: 185.
  • the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 182 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 182 and encoding the same amino acid sequence as SEQ ID NO: 182; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 184 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 184 and encoding the same amino acid sequence as SEQ ID NO: 184.
  • the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 183 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 183 and encoding the same amino acid sequence as SEQ ID NO: 183; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 185 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 185 and encoding the same amino acid sequence as SEQ ID NO: 185.
  • a host cell harboring the expression vector of the twenty -third aspect or any embodiments thereof.
  • a method of manufacturing the bispecific binding molecule disclosed herein comprising the steps of a) culturing the host cell of the twenty-fourth or twenty -fifth aspects or any embodiments thereof under conditions allowing for expression of the nucleic acid molecule encoding the heavy chain fusion and expression of the nucleic acid molecule encoding the light chain, and association of the expressed heavy chain fusion and the expressed light chain into the bispecific binding molecule; and b) recovering the bispecific binding molecule from the culture.
  • the recovered bispecific binding molecule is further purified and/or modified and/or formulated.
  • the invention provides a method of treating a MUC16-related fibrotic disease disorder, inflammatory disorder, immune disorder, or autoimmune disorder, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the bispecific binding molecule of any of the foregoing aspects or embodiments thereof, or the pharmaceutical composition of the twentieth aspect.
  • the MUC16-mediated fibrotic disorder is pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, cirrhosis, kidney fibrosis, glial scar, myocardial fibrosis, arterial stiffness, arthrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, or adhesive capsulitis.
  • the invention provides a method of treating a cancer or malignancy characterized by over-expression of MUC16, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the bispecific binding molecule of any of the foregoing aspects or embodiments thereof, or the pharmaceutical composition of the twentieth aspect.
  • the cancer or malignancy is a gynecologic cancer (cancer of the female reproductive tract), including the cervix, endometrium, fallopian tubes, ovaries, uterus, and vagina; pancreatic cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, or lung cancer.
  • FIG. 1 is a schematic representation of exemplary bispecific binding molecules of the present invention.
  • a binding molecule comprising (i) an Ig molecule comprising two heavy chain fusions, each comprising a variable region (“VHMUCI6”) that specifically binds to the extracellular domain of MUC16, a constant region (“CHI”, “CH2” and “CH3”), a first peptide linker (“Linker l)”and a single-chain variable fragment (“scFv”) comprising, in N- to C-terminal order, a variable light chain region that specifically bind to DR5 (“VLDRS”), a second peptide linker (“Linker 2”) and a variable heavy chain region that specifically bind to DR5 (“VHDRS”); and (ii) two light chains, each comprising a variable region (“VLMUCI6”) specifically binding to the extracellular domain of MUC16 and a constant region (“CL”).
  • VHMUCI6 variable region that specifically binds to the extracellular domain of MUC16,
  • FIG. 2A panel A depicts an SDS-PAGE gel of antibody IMV-18 that was expressed in CHO cells, and then purified from culture supernatant by Protein A affinity chromatography, and then additionally purified by preparative size exclusion chromatography. Lane R - reducing gel, Lane N-R - non-reducing gel, Lane M - molecular weight markers.
  • Panel B depicts a SEC-HPLC chromatogram of the purified IMV-18 as measured at 214 nm and 280 nm.
  • FIG. 3A panel A is a series of plots comparing the cytotoxicity of IMV-18 (circles) and the anti-DR5 antibody Lexatumumab (triangles) using a CellTiter-Glo® assay on NCI-H292, CAOV-3 and OVCAR-3 cells, each of which expresses both MUC16 and DR5, after two days of exposure to either IMV-18 or Lexatumumab.
  • Panel B (FIG.
  • 3B is a series of plots comparing the cytotoxicity of a bispecific antibody targeting human CD38 and DR5 (“IMV-15”; circles), a bispecific antibody targeting human LIV-1 and DR5 (“IMV-20”; squares), or Lexatumumab (triangles) using a CellTiter-Glo® assay on MM.
  • Panel C (FIG.
  • 3C is a series of plots comparing the cytotoxicity of a bispecific antibody targeting human MUC16 and DR5 (“MCLX-SE”, squares), a bispecific antibody targeting fluorescein and DR5 (“FLX-SE”, triangles), and a monospecific antibody targeting human MUC16 (“MC- SE”, circles) on a variety of cell lines.
  • the cytotoxicity was assessed using a CellTiter- Glo® assay, and was performed on the cell lines HP AC, PK-59, NCI-H1975, OVCAR-3, NCC-StC-K140, and HDQ-P1 each of which expresses MUC16 and DR5 (Table 11).
  • HP AC cells express both MUC16 and DR5. Intravenous administration with the test agents was performed when the mean tumor volume reached approximately 145 mm 3 .
  • Panel B (FIG.
  • FIG. 4B shows the average body weight (X-axis; grams) after the indicated number of days (Y-axis) following administration of each agent administered in FIG. 4A.
  • Panel C shows tumor volume (Y-axis; mm 3 ) after the indicated number of days (X-axis) following administration of the agent in individual mice.
  • FIG 5, panel A is a series of plots comparing anti-proliferative effects of continuous exposure of different concentrations of either a bispecific antibody targeting human MUC16 and DR5 (“MCLX-SE”), a bispecific antibody targeting fluorescein and DR5 (“FLX-SE”), or a monospecific antibody targeting human MUC16 (“MC-SE”) on PK- 59 cells.
  • MCLX-SE bispecific antibody targeting human MUC16 and DR5
  • FLX-SE bispecific antibody targeting fluorescein and DR5
  • MC-SE monospecific antibody targeting human MUC16
  • Panel B is a time-course of apoptotic events in PK-59 cells exposed to a bispecific antibody targeting human MUC16 and DR5 (“MCLX-SE”), a bispecific antibody targeting fluorescein and DR5 (“FLX-SE”), or a monospecific antibody targeting human MUC16 (“MC-SE”); all at 41.2 pM, continuous exposure, or to medium only (control).
  • MCLX-SE bispecific antibody targeting human MUC16 and DR5
  • FLX-SE bispecific antibody targeting fluorescein and DR5
  • MC-SE monospecific antibody targeting human MUC16
  • Caspase-3/7 Green Dye for Apoptosis, Catalog No. 4440, Sartorius (green fluorescence).
  • Caspase-3/7 Green Dye is a fluorogenic substrate of caspase 3 and caspase 7, two caspases that become active during later stages of apoptosis.
  • Panel C (FIG. 5C) shows another set of data from the same experiment, plotting the number of apoptotic events per well in PK-59 cells exposed to various concentrations of these three antibodies for 8 hours.
  • PBS vehicle only
  • HCC827 cells express both MUC16 and DR5 on day 1.
  • PK-59 cells and NCI-H1975 cells express both MUC16 and DR5
  • SEM standard error of the mean
  • FIG. 7 is a plot of the effect of various anti-MUC16-anti-DR5 bispecific antibodies (IMV-18, MCLX-SE, IMV-AA, or MC-AA) alone or in combination with an excess of a non-targeting mouse IgGl antibody (muIgGl) over time on tumor growth in PK-59 pancreatic carcinoma xenograft mice.
  • FIG. 8 is a plot of the effect of varying amounts of the anti-MUC16-anti-DR5 bispecific antibody IMV-M over time on tumor growth in PK-59 pancreatic carcinoma xenograft mice. DETAILED DESCRIPTION OF THE INVENTION
  • consists essentially of (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this present disclosure, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and/or C-terminal ends of the recited sequence such that the function of polypeptide is not materially altered.
  • the total of ten or less additional amino acids can include the total number of additional amino acids on both ends added together.
  • human MUC16 when used herein includes any variants, isoforms, and species homologs of human MUC16 (mucin 16, NCBI Entrez Gene: 94025) which are expressed by cells on their surface.
  • recombinant human MUC16 when used herein includes human MUC16 (NCBI Entrez Gene: 94025) available on the world wide web at uniprot.org; UniProtKB - Q8WXI7), and the nucleic acid sequence encoding that protein.
  • MUC16 is also intended to include variants (e.g., allelic variants), and derivatives thereof.
  • Human MUC16 cDNA and human MUC16 protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI).
  • Human MUC16 variants include mucin-16 precursor (NM_001401501.2 and NP_001388430.1), and mucin-16 (NM_024690.2 and NP_078966.2).
  • Nucleic acid and polypeptide sequences of MUC16 orthologs in organisms other than humans are well-known.
  • Death Receptor 5 also known as DR5; tumor necrosis factor receptor superfamily member 10B, TNFRSF10B, TNF-related apoptosis-inducing ligand receptor 2, TRAIL receptor 2, TRAIL-R2 and CD262, TNF receptor superfamily member 10b, KILLER, KILLER/DR5, TRICK2, TRICK2A, TRICK2B, TRICKB, ZTNFR9.
  • This term includes any variants, isoforms and species homologs of human protein provided in UniProt 014763 available on the world wide web at uniprot. org/uniprot/014763, and the nucleic acid sequence encoding that protein.
  • analog when used herein includes any compounds discovered in a structure-activity relationship study (see for instance Schnecke V. and Bostrom J. Drug Discovery Today. 11 (1-2): 43-50 (2006), having a structure similar to that of another compound, but differing from it in respect to a certain component (see for instance Willett P. et al. J. Chem. Informat. Comp. Sci. 38: 983-996 (1998), Johnson A. M. and Maggiora G. M. Concepts and Applications of Molecular Similarity. New York: John Willey & Sons. ISBN 978-0-471-62175-1 (1990), NikolovaN. and Jaworska J. QSAR & Combinatorial Science.
  • variant when used herein in the context of a reference amino acid sequence (e.g., a specific SEQ ID NO), means an amino acid sequence that has one or more amino acid insertions, deletions or replacements compared to the reference sequence.
  • derivative when used herein in the context of a reference antibody, means an immunoglobulin that has been chemically or recombinantly modified to include a moiety having an activity that differs from that of the reference antibody without destroying the ability of the reference antibody to bind to the antigen to which it is directed.
  • the moiety may be a second antibody or antigen-binding portion thereof that recognizes a different antibody; a peptide or chemical “tag” that allows the antibody to be recognized (e.g., for purification), or identified (e.g., a fluorescent molecule); a small molecule that has activity against a cell targeted by the antibody (e.g., an anti-cancer agent); a radioisotope; etc.
  • derivative when used herein in the context of a compound or a small peptide, includes any compound that is derived from a similar compound by a chemical reaction and/or that at least theoretically can be formed from the precursor compound (see for instance Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. ISBN 0-19-850673-2).
  • immunoglobulin refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light chains and one pair of heavy chains, all four inter-connected by disulfide bonds.
  • the structure of immunoglobulins has been well characterized (see for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).
  • each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3.
  • Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region typically is comprised of one domain, CL.
  • the VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL is typically 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 (see also Chothia and Lesk J. Mol. Biol. 196, 901- 917 (1987)).
  • expression vector in the context of the present invention refers to a plasmid or virus designed for gene expression in cells.
  • Various types of expression vectors are well known in the art.
  • antibody in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of thereof, which can specifically bind to an antigen under typical physiological conditions. An antibody is the source of each of the two different binding moieties present in the bispecific binding molecules disclosed herein.
  • antibody and “immunoglobulin” are used interchangeably and include: 1) molecules that have all of the structural features of an immunoglobulin (e.g., a pair of light chains and a pair of heavy chains that are bound to one another through disulfide bonds) and specifically bind to an antigen or an epitope thereof, such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, recombinantly produced antibodies, veneered antibodies, humanized antibodies, and mouse antibodies; 2) molecules that only have part of the structure of an immunoglobulin, but retain the three heavy chain and three light chain CDRs and maintain the ability to specifically bind to an antigen or an epitope thereof, such as Fab, Fab’, F(ab’)2, F(ab)3, scFv, scFv-Fc, diabodies, triabodies, tetrabodies, and minibodies, (individually and collectively termed, “antigen-binding fragment(s)”);
  • an antibody derivative may be attached directly to the antibody or through a linker.
  • the term “antibody” includes monoclonal antibodies (including full length four-chain antibodies or full length heavy-chain only antibodies which have an immunoglobulin Fc region, antibody compositions with poly-epitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments (e.g., Fab, F(ab')2, and Fv)).
  • Antibodies contemplated herein include single-domain antibodies, such as heavy chain only antibodies.
  • the basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains ten antigen binding sites, while IgA antibodies comprise from 2 - 5 of the basic four-chain units which can polymerize to form polyvalent assemblages in combination with the J chain.
  • IgA antibodies comprise from 2 - 5 of the basic four-chain units which can polymerize to form polyvalent assemblages in combination with the J chain.
  • HCAb heavy chain-only antibody
  • dAb single-domain antibody
  • CDRs complementary determining regions
  • Camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e. g., Hamers-Casterman et al., Nature 363: 446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)).
  • a basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1, CDR2 and CDR3 refer to the complementarity determining regions 1, 2 and 3.
  • Antibodies present in the bispecific binding molecules of the invention that include heavy chain constant regions can be of any isotype.
  • isotype refers to the immunoglobulin class (for instance IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes.
  • chimeric antibody in the context of the present invention refers to an antibody made by fusing the antigen binding region (variable domains of the heavy and light chains, VH and VL) from one species, such as a mouse, with the constant domain (effector region) from a human antibody (see for instance Morrison S. L. et al. Proc. Natl. Acad. Sci. USA 81, 6851-6855 (1984)).
  • mouse antibody is intended to include antibodies having variable and constant regions derived from mouse germline immunoglobulin sequences.
  • humanized antibody as used herein, means a mouse antibody having the variable framework and constant regions replaced with the corresponding regions derived from human germline immunoglobulin sequences.
  • a “humanized” antibody refers to an antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., complementary determining regions (CDRs)) correspond to those of a non-human antibody, and all or substantially the entire framework regions (FRs) correspond to those of a human antibody.
  • HVRs complementary determining regions
  • FRs framework regions
  • substantially means that both the heavy chain and the light chain of the humanized antibody has at least 80% alignment identity with the top human germline V gene hit.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • the mouse, chimeric and humanized antibodies that may be present in the bispecific binding molecules of the invention may include amino acid residues not encoded by their respective germline immunoglobulin sequences due to e.g., mutations, substitutions, deletions, or insertions, introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. These antibodies are termed “variants” because of such amino acid alterations.
  • an “isolated antibody,” as used herein, is intended to refer to an antibody, which is substantially free of other antibodies having different antigenic specificities (for instance an isolated antibody that specifically binds to MUC16 is substantially free of antibodies that specifically bind antigens other than MUC16; an isolated bispecific antibody that specifically binds to MUC16 and DR5 is substantially free of antibodies that specifically bind antigens other than MUC16 and DR5).
  • An isolated antibody that specifically binds to an epitope, isoform or variant of human MUC16 may, however, have cross-reactivity to other related antigens, for instance from other species (such as MUC16 species homologs).
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • bispecific binding molecule is a molecule that is capable of specific binding to two different antigens (e.g., MUC16 and DR5), i.e., it is a molecule that has two different “antigen binding moieties”.
  • antigen-binding moiety refers to a part of an antibody or a bispecific binding molecule that comprises amino acids responsible for the specific binding between the antibody or a portion of the bispecific binding molecule and a specific antigen. In instances where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as “epitope” or “antigenic determinant.”
  • an antigen-binding moiety typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), it does not necessarily have to comprise both.
  • the second antigen binding moiety of the present bispecific binding molecules may consist only of a VH domain of an anti-DR5 antibody, but still to retain its ability to bind to and agonize DR5.
  • Each variable region or domain comprises three CDRs.
  • the generalized structure of antibodies or immunoglobulin molecules is well known to those of skill in the art.
  • bispecific antibody as used herein is a bispecific binding molecule, wherein each antigen binding moiety thereof is derived from an antibody.
  • the term "bispecific antibody” is intended to include any antibody which has two different binding specificities, including, among others, tetravalent antibody molecules comprising an IgGl or its fragment or a variant, with one antigen specificity, and an antigen-binding fragment of an antibody with another antigen specificity, such as VHH, Fab, Fab’, F(ab’)2, F(ab)3, scFv, scFv-Fc, diabodies, triabodies, tetrabodies, and minibodies.
  • bispecific antibodies also includes diabodies (see for instance Holliger, P. et al., PNAS USA 90, 6444-6448 (1993), Poljak, R. J. et al., Structure 2, 1121-1123 (1994)).
  • binding in the context of the binding of an antibody to a predetermined antigen typically is a binding with an affinity corresponding to a KD of about 10,000 nM or less, such as about 1,000 nM or less, such as about 100 nM or less, such as about 10 nM or less, such as about 1 nM or less, about 0.1 nM or less, or about 10 pM or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore instrument or an Octet instrument, using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to anon-specific antigen (e.g., bovine serum albumin, casein) other than the predetermined antigen,
  • anon-specific antigen
  • the amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for anon-specific antigen may be at least 10,000-fold.
  • ko refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koffvalue.
  • KA (M' 1 times sec' 1 ), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.
  • KD (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
  • Eso refers to the concentration of the antibody targeting an antigen, for example, MUC16 that binds to cells expressing this antigen, so that the cells reach 50% maximal fluorescence as determined by flow cytometry.
  • percent sequence identity refers to the percentage of amino acids or nucleotides in a candidate sequence that are identical with the amino acids in a reference amino acid sequence or nucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be determined using any mathematical algorithm to determine amino acid sequence homology known in the art. One such algorithm is the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • epitopes means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • the epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide).
  • the terms, used interchangeably, “inhibits growth” or “inhibits proliferation” is intended to include any measurable decrease in the cell growth (which is meant the same as cell proliferation), or decrease in cell number when contacted with an anti-MUC16 antibody, or with an antibody that binds to another antigen, or with a non-targeting antibody, or with a bispecific antibody, or with any other molecule, as compared to the growth of the same cells not in contact with an anti-MUC16 or an antibody that binds to another antigen, or with a nontargeting antibody, respectively, or with any other molecule, e.g., the inhibition of growth of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • Such a decrease in cell growth can occur by a variety of mechanisms, e.g., ADCC, ADCP, CDC, cell cycle arrest, and/or apopto
  • cytotoxicity or “cytotoxic effect” (e.g. referring to cells, such as tumor cells) is intended to include any measurable decrease in the cell growth, or decrease in cell number when contacted with an anti-MUC16 antibody or an antibody that binds another antigen, or with a non-targeting antibody, or with a bispecific binding molecule disclosed herein, or with any other molecule, as compared to the growth of the same cells in the absence of such an antibody or molecule e.g., the cytotoxic effect of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • cytotoxicity or cytotoxic effect refers to the decrease in cell growth or decrease in cell number of cells that are positive for MUC16 and for DR5 (“MUC16 + /DR5 + ”), or of cells that are positive for another antigen and for DR5.
  • Recombinant host cell (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. Such terms are intended to refer not only to the subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK293 cells, NS/0 cells, and lymphocytic cells.
  • solid tumors and “solid cancers”, used interchangeably, in the context of the present invention include but is not limited to the following malignancies: epithelial ovarian cancer or any other ovarian cancer, pancreatic ductal adenocarcinoma or any other pancreatic cancer, esophageal adenocarcinoma or any other esophageal cancer, gastric adenocarcinoma, or any other gastric cancer, colon cancer, colorectal cancer, invasive micropapillary carcinoma of the breast, or any other breast cancer, non-small cell lung cancer or any other lung cancer.
  • blood cancers in the context of the present invention include but is not limited to hematological malignancies: acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia lymphomas, Hodgkin's lymphomas (all four subtypes), non-Hodgkin's lymphomas (all subtypes), small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, MM, lymphoplasmacytic lymphoma, splenic margina zone lymphoma, plasma cell neoplasms, such as plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, M
  • Treatment refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.
  • An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of a bispecific binding molecule disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of that molecule to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • an “effective amount” is an amount that causes cytotoxicity in cells that are MUC16 + /DR5 + .
  • variable domain or variable region (Fv) of the antibody.
  • the variable domain comprises three so-called complementary-determining regions (CDR’s) spaced apart by framework regions (FR’s).
  • CDRs are the regions of an antibody that are responsible for the specificity (binding) of the antibody for (to) a particular antigen.
  • CDRs of the MUC16 binding domain and the DR5 binding domain is based on CDRs reported in the prior art disclosing various MUC 16 and DR5 antibodies. These CDRs are typically based upon the CDR definition of Kabat (E. A. Kabat, et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda (1983), IMGT (Lefranc M P, et al., Dev Comp Immunol. 2003 January; 27(l):55-77; Giudicelli V et al., Cold Spring Harb Protoc. 2011; 2011(6):695-715), Chothia (Chothia and Lesk, J. Mol. Biol.
  • variable domains or “variable region” or Fv as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen.
  • the variable domain of a light chain is abbreviated as “VL” and the variable domain of a heavy chain is abbreviated as “VH”.
  • the variable light and heavy chain domains have the same general structure, and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three HVRs (or CDRs).
  • the framework regions adopt a beta-sheet conformation and the CDRs may form loops connecting the beta-sheet structure.
  • the CDRs in each chain are held in their three- dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.
  • constant domains or “constant region” as used within the current application denotes the sum of the domains of an antibody other than the variable region.
  • constant domains and regions are well known in the state of the art and e.g., described by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91).
  • the “Fc part” or “Fc region” of an antibody is not involved directly in binding of an antibody to an antigen but exhibit various effector functions.
  • An “Fc part of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies.
  • antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM. According to the heavy chain constant regions the different classes of immunoglobulins are called a, 6, s, y, and p respectively.
  • IgGl immunoglobulin G
  • IgG2 antibody dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • CDC complement activation
  • binding sites are known in the state of the art and described e.g., by Boakle et al., Nature 282 (1975) 742- 743, Lukas et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse and Cebra, Mol. Immunol. 16 (1979) 907-917, Burton et al., Nature 288 (1980) 338-344, Tansen et al., Mol. Immunol. 37 (2000) 995-1004, Idusogie et al., J. Immunol. 164 (2000) 4178-4184, Hezareh et al., J.
  • Such binding sites are e.g., L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat, see below).
  • Most crucial among these residues in mediating Clq and Fcgamma receptor binding in IgGl are L234 and L235 (Hezareh et al., J. Virology 75 (2001) 12161-12168).
  • Antibodies of subclass IgGl and IgG3 usually show complement activation and Clq and C3 binding, whereas IgG2 and IgG4 do not activate the complement system and do not bind Clq and C3.
  • antibody molecule or “antibody” (used synonymously herein) do not only include antibodies as they may be found in nature, comprising e.g., two light chains and two heavy chains, or just two heavy chains as in camelid species, but furthermore encompasses all molecules comprising at least one paratope with binding specificity to an antigen and structural similarity to a variable domain of an immunoglobulin.
  • an antibody portion of the bispecific binding molecules provided herein may comprise a monoclonal antibody, a multi-specific antibody, a bispecific antibody, an antibody derivative, a human antibody, a recombinant antibody, a veneered antibody, a humanized antibody, a chimeric antibody, a fragment of an antibody, in particular a Fv, Fab, Fab', or F(ab')2 fragment, a single chain antibody, in particular a scFv, a Small Modular Immunopharmaceutical (SMIP), a domain antibody, a nanobody, a diabody.
  • SMIP Small Modular Immunopharmaceutical
  • the antibody may have an effector function, such as ADCC or CDC, that is usually mediated by the Fc part (antibody constant region) of the antibody, or it may have no effector function, e.g., by lacking a Fc part or having a blocked, masked Fc part, in essence a Fc part that is not or insufficiently recognized by immune cells or immune system components, like the complement system.
  • Monoclonal antibodies are monospecific antibodies that are identical in amino acid sequence. They may be produced by hybridoma technology from a hybrid cell line (called hybridoma) representing a clone of a fusion of a specific antibodyproducing B cell with a myeloma (B cell cancer) cell (Kohler G, Milstein C.
  • a “recombinant antibody” or “recombinant binding molecule” is an antibody or binding molecule which has been produced by a recombinantly engineered host cell. It is optionally isolated or purified.
  • a “chimeric antibody” is understood to be antibody comprising a sequence part (e.g., a variable domain) derived from one species (e.g., mouse) fused to a sequence part (e.g., the constant domains) derived from a different species (e.g., human).
  • a “humanized antibody” is an antibody comprising a variable domain originally derived from a non-human species, wherein certain amino acids have been mutated to make the overall sequence of that variable domain more closely resemble to a sequence of a human variable domain.
  • Antibody can also include fragments of immunoglobulins which retain antigen binding properties, like Fab, Fab', or F(ab')2 fragments. Such fragments may be obtained by fragmentation of immunoglobulins e.g., by proteolytic digestion, or by recombinant expression of such fragments. For example, immunoglobulin digestion can be accomplished by means of routine techniques, e.g., using papain or pepsin (WO 94/29348). Papain digestion of antibodies typically produces two identical antigen binding fragments, so- called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields an F(ab')2.
  • variable domains are each fused to an immunoglobulin constant domain, preferably of human origin.
  • the heavy chain variable domain may be fused to a CHI domain (a so-called Fd fragment), and the light chain variable domain may be fused to a CL domain.
  • Fab molecules may be produced by recombinant expression of respective nucleic acids in host cells, see below.
  • variable domains of immunoglobulins or molecules derived from such variable domains, in a different molecular context.
  • these antibody molecules are smaller in size compared to immunoglobulins and may comprise a single amino acid chain or several amino acid chains.
  • a single-chain variable fragment scFv
  • S serine
  • G glycine
  • VHH single domain antibodies or “nanobodies” harbor an antigen-binding site in a single Ig-like domain (WO 94/04678; WO 03/050531, Ward et al., Nature. 1989 Oct. 12; 341:544-546; Revets et al., Expert Opin Biol Then 5:111-24, 2005).
  • One or more single domain antibodies with binding specificity for the same or a different antigen may be linked together.
  • Diabodies are bivalent antibody molecules consisting of two amino acid chains comprising two variable domains (WO 94/13804, Holliger et al., Proc. Natl. Acad. Sci. USA. 1993; 90:6444-8).
  • SMIP Small Modular Immunopharmaceutical
  • the present disclosure provides bispecific binding molecules having at least one antigen binding domain (a first antigen binding domain) that binds specifically to the extracellular domain of MUC16 and at least one antigen binding domain (a second antigen binding domain) that binds specifically to DR5.
  • the bispecific binding molecules provided can cause apoptosis of MUC16-expressing cells that also express DR5.
  • the second antigen binding domain when bound to DR5, stimulates the apoptotic activity of DR5.
  • the first antigen binding domain creates specificity enhancing the stimulated apoptotic activity of DR5 to MUC16-expressing cells.
  • MUC16 is a highly glycosylated protein having 14,507 amino acids. It consists of a 14,451 amino acid extracellular domain, a 21 amino acid transmembrane domain and a 35 amino acid C-terminal cytoplasmic domain.
  • a bispecific antibody comprising an anti-MUC16 antibody targeting multiple MUC16 epitopes present in the extracellular domain of MUC16 within its TR domain, is effective in potentiating the agonistic effect of the second, anti-DR5 binding domain.
  • Applicant believes that this potentiating effect stems from clustering DR5 on the cell surface by multiple bispecific antibody molecules bound to the same MUC16 molecule. Accordingly, Applicant believes that a bispecific antibody comprising an anti- MUC16 antibody targeting a unique MUC16 epitope (i.e., an epitope only present once per MUC16 molecule) outside of the MUC16 TR/SEA domain, will be inefficient in potentiating the agonistic effect of the second, anti-DR5 binding domain. Furthermore, the ability of MUC16 to potentiate the agonistic effect caused by the binding of the second antigen binding domain to DR5 is not common to other cell surface proteins.
  • an antibody targeting CD44v6 (a splice variant of CD44 that is known to be a tumor associated antigen with a preferential expression pattern in tumor over normal tissues) did not potentiate the agonistic effect of the death receptor binding molecule when it is fused to an anti-DR5 antibody. See United States Patent No. 10,858,438.
  • the bispecific binding molecules are derived from antibodies.
  • Techniques for making bispecific binding molecules include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knobin-hole” engineering (see, e.g., US5731168).
  • Bispecific binding molecules of the invention may also be made by engineering electrostatic steering effects for making antibody Fc- heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US4676980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., Immunol., 148: 1547-1553 (1992)); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci.
  • the first antigen binding domain binds to the extracellular domain of MUC16.
  • the first antigen binding domain binds to an epitope present in one or more tandem repeat/SEA segments within the extracellular domain of MUC16.
  • the first antigen binding domain will be derived from an antibody known to bind the extracellular domain of MUC16.
  • the first antigen binding domain will have the same heavy and light chain CDRs as those present in an antibody that binds the extracellular domain of MUC16.
  • the first antigen binding domain will have the same heavy and/or light chain variable regions as those present in an antibody that binds the extracellular domain of MUC16.
  • the first antigen binding domain will have the same heavy and/or light chains as those present in an antibody that binds the extracellular domain of MUC16.
  • Antibodies that bind the extracellular domain of MUC16 are known in the art and include, but are not limited to, OC125, Hl 85 (Invitrogen Cat No. MA5-11579), Mi l (American Tissue Culture Collection Accession No. PTA-6206), OV197 (Fujirebio Diagnostic), 5E11 (Millipore Sigma Cat No. MABC1608-25UG), AR9.6, H1H8794, VK-8, B43.13 (also known as Oregovomab), or 3A5 (Sofituzumab).
  • an antibody against MUC16 can be generated using standard monoclonal antibody techniques. For these newly generated antibodies, determining if they bind the TR/SEA portion of the extracellular domain may be achieved by methods known in the art. One way of making such determination is if the new MUC16 antibody is competed for binding to MUC16 or an extracellular fragment thereof by one or more of the known antibodies set forth above that bind to the TR/SEA portion of the extracellular domain of MUC16. Another way of determining if an anti-MUC16 antibody binds the TR/SEA portion of the extracellular domain is by ELISA or another well-known antigenantibody binding assay using a peptide or polypeptide or its derivative from the TR/SEA portion of the MUC16 extracellular domain as the antigen. These antigens include peptides or polypeptides consisting essentially of the amino acid sequence of at least one of SEQ ID NOs: 176-181, or an extracellular fragment of MUC16 isolated from the cell culture media of OVCAR-3 cells.
  • the first antigen binding domain comprises a heavy chain and a light chain, wherein the amino acid sequences of the heavy and light chain are derived from an antibody that binds the extracellular domain of MUC16 and the specificity for MUC16 is determined by CDRs present in each of the heavy and light chains.
  • the amino acid sequence of the heavy and/or light chain differs from that of the antibody from which it derives to improve one or more properties for use in humans. Typically, these differences involve a conservative substitution of less than five amino acids. See Tables 5-8.
  • the second antigen binding domain binds to DR5.
  • the second antigen binding domain is an amino acid sequence that is derived from an antibody that binds to DR5.
  • the parent antibody also agonizes DR5.
  • the second antigen binding domain will have the same heavy and light chain CDRs as those present in an antibody that binds DR5.
  • the second antigen binding domain will have the same heavy and/or light chain variable regions as those present in an antibody that binds DR5.
  • Antibodies that bind DR5 are known in the art and include, but are not limited to,
  • the amino acid sequence of the second antigen binding domain is derived from an scFv that binds to and agonizes DR5 and the specificity for DR5 is determined by CDRs present in the scFv.
  • an antibody against DR5 can be generated using standard monoclonal antibody techniques. For these newly generated antibodies, determining if they bind DR5 may be achieved by methods known in the art. One way of making such determination is if the new DR5 antibody is competed for binding to DR5 by one or more of the known antibodies set forth above that bind to DR5. Another way of determining if an anti-DR5 antibody binds DR5 is if it is competed for DR5 binding by TRAIL or a fragment or a derivative of TRAIL that binds to DR5. Still another way of determining if an antibody binds to DR5 is by ELISA or another well-known antigen-antibody binding assay using DR5 or a peptide or polypeptide fragment of DR5 as the antigen.
  • the bispecific binding molecule comprises two polypeptide chains each of which comprises a variable heavy chain region and heavy chain CDRs, and two polypeptide chains each of which comprises a variable light chain region and light chain CDRs, wherein the combination of such polypeptide chains causes the bispecific molecule to bind specifically to the TR/SEA region of MUC16.
  • the two polypeptide chains each comprising a variable heavy chain region have identical amino acid sequences.
  • the two polypeptide chains each comprising a variable light chain region have identical amino acid sequences.
  • the combination of the two variable heavy chain regions (or the CDRs therein) and the two variable light chain regions (or the CDRs therein) define the first antigen binding domain.
  • the polypeptide chains comprising the variable heavy chain region are derived from the heavy chain of an antibody that binds to the TR/SEA region of MUC16. In some embodiments, the polypeptide chains comprising the variable light chain region are derived from the light chain of an antibody that binds to the TR/SEA region of MUC16. In some embodiments, both the polypeptide chains comprising the variable heavy chain region and the polypeptide chains comprising the light chain region are derived from the same MUC16 antibody.
  • polypeptide chains that comprise a MUC16-specific variable heavy chain region further comprise the second antigen binding domain. In some embodiments the polypeptide chains that comprise a MUC16-specific variable light chain region further comprise the second antigen binding domain.
  • the location of the second antigen binding domains with respect to the first antigen binding domain can vary and includes all of the following: 1) the N-terminus of the second antigen binding domain is bound to the C-terminus of the heavy chain of the first antigen binding domain directly or through a peptide linker; 2) the N-terminus of the second antigen binding domain bound to the C-terminus of the light chain of the first antigen binding domain directly or through a peptide linker; 3) the N-terminus of the heavy chain of the first antigen binding domain bound to the C-terminus of the second antigen binding domain directly or through a peptide linker; 4) the N-terminus of the light chain of the first binding antigen site bound to the C-terminus of the second antigen binding domain directly or through a peptide linker; 5) the second antigen binding domain bound to an amino acid other than the C- or N-terminal amino acid of the heavy chain of the first antigen binding domain directly or through a linker (e.g., by reaction with a side
  • variable light chain region differs from a specified portion of a bispecific binding molecule described herein by one or more suitable amino acid residue alterations, that is substitutions, deletions, insertions, and/or terminal sequence additions, for instance in the constant domain, and/or the variable regions (or any one or more CDRs thereof).
  • a variant of a VL, VH, HC, LC or scFv region of a bispecific binding molecule of the invention retains at least a substantial proportion (a KD that at least about 100-fold greater or 10-fold greater or less) of the affinity/avidity and/or the specificity/selectivity of the parent antibodies to either MUC16 or to DR5 (i.e., a KD that at no more than about 100-fold greater, no more than 50-fold greater, no more than 40-fold greater, no more than 30-fold greater, no more than 20-fold greater, no more than 10-fold greater or less than 10-fold greater).
  • such variant sequences may be associated with greater affinity, selectivity and/or specificity (i. e. , a lower KD) than the parent antibody to either of the antigens.
  • Such functional variants have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the referenced portion of a specific sequence at the amino acid level.
  • sequence of variants may differ from the sequence of the parent antibody sequences through mostly conservative substitutions; for instance, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more (e.g., about 65-99%) of the substitutions in the variant are conservative amino acid residue replacements.
  • conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following tables:
  • Residue Amino Acids that may be substituted for one another.
  • the amino acid sequence alterations should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to disrupt secondary structure that characterizes the function of the parent sequence), but may be associated with advantageous properties, such as changing the functional or pharmacokinetic properties of the antibodies, for example increasing the halflife, altering the immunogenicity, providing a site for covalent or non-covalent binding to another molecule, reducing susceptibility to proteolysis, reducing susceptibility to oxidation, reducing susceptibility to deamidation, or altering the glycosylation pattern.
  • the amino acid alterations will not be in the CDR regions, unless the change is such that the CDRs defined by at least one convention are maintained.
  • the CDRs of the bispecific binding molecules set forth herein are based on those defined in the prior art disclosing the anti-MUC16 antibody or the DR5 antibody from which this binding moiety was derived. Applicant has indicated which specific CDR convention was used to elucidate these CDRs (e.g., see Tables 1 and 3). Those of ordinary skill in the art will understand that given the amino acid sequence of a variable region of the bispecific binding molecules set forth herein can easily determine the CDR sequences therein by these or any other conventions used to define CDR, such as, for example, IMGT, Kabat, Chothia, or Contact using web-based tools are available for determining the CDRs in such variable regions based on any known convention.
  • Such tools include those found at www. abysis.org/ abysis/sequence_input/key_annotation/key_annotation. cgi and at www.novoprolabs.com/tools/cdr. Accordingly, the disclosure herein of a set of three CDRs in a heavy or light chain variable region based upon one CDR convention is considered the equivalent of that same set of CDRs as determined by any other convention.
  • the bispecific binding molecules disclosed herein comprise anti-MUC16 antibody constant regions or parts thereof.
  • a VL domain may have attached, at its C terminus, antibody light chain constant domains including human CK or CX chains.
  • a specific antigen-binding domain based on a VH domain may have attached all or part of an immunoglobulin heavy chain derived from any antibody isotope, e.g, IgG, IgA, IgE, and IgM and any of the isotope sub-classes, which include but are not limited to, IgGl and IgG4.
  • the amino acid sequences of the C-terminal fragments of constant regions are well known in the art, as are DNA sequences that encode for such amino acids.
  • the first antigen binding domain that binds specifically to MUC16 is an immunoglobulin (Ig) molecule (having the conventional Y shaped structure of a full-length antibody comprising two heavy and two light chains) and the second antigen binding domain that binds specifically to DR5 comprises at least one or more scFv binding elements.
  • Ig immunoglobulin
  • the one or more scFvs specifically binding to DR5 is fused to the Ig molecule (e.g., human IgGl) specifically binding to MUC16 by a peptide linker.
  • the peptide linker has a length of about 4 to 20 amino acids.
  • the N-terminus of the scFv is fused to the C- terminus of the heavy chain of the Ig molecule or the C-terminus of the light chain of the Ig molecule.
  • the Ig molecule is an IgG.
  • GS mini-linker a small linker sequence comprising glycine and serine
  • the number of amino acids in the linker can vaiy from 4 (GGGS, SEQ ID NO.:221), 6 (GGSGGS, SEQ ID NO.:222), 10 (GGGGSGGGGS, SEQ ID NO.:223), 12 (GGGS GGGS GGGS, SEQ ID NO.:173), 15 (GGGGSGGGGSGGGGS, SEQ ID NO.:224) or more.
  • the GS mini-linker between the scFv molecule and the C-terminus of the heavy chain of the IgG molecule is GGGSGGGSGGGS (SEQ ID NO: 173).
  • the present invention provides a bispecific binding molecule comprising (i) an Ig molecule that specifically binds to MUC16 comprising two heavy and two light chains, and (ii) two scFv molecules, each specifically binding to DR5.
  • each heavy chain of the Ig molecule has one scFv molecule fused to its C-terminus, thereby forming a bispecific tetravalent binding protein.
  • the present invention provides a bispecific binding molecule comprising:
  • a heavy chain variable domain specific for MUC16 e.g., murine, humanized, or human VH domain
  • constant domains of an IgG e.g., human IgGl
  • a peptide linker e.g., a GS mini linker
  • a light chain variable domain specific for MUC16 e.g., murine, humanized or human VL domain
  • a light chain constant domain e.g., a human kappa chain
  • the binding activity of the bispecific binding molecules disclosed herein can be measured using various methods.
  • One method is enzyme-linked immunosorbent assay (ELISA).
  • ELISA is a biochemistry assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. Antigens from the sample are attached to a surface. Then, a further specific antibody or bispecific binding molecule is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added.
  • Fluorescence-activated cell sorting also called flow cytometry, provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.
  • the ECso is the concentration of the antibody or bispecific binding molecule that induces a response halfway between the baseline and maximum after some specified exposure time on a defined concentration of antigen by ELISA (enzyme-linked immuno-sorbent assay) or cell expressing the antigen by FACS.
  • Surface plasmon resonance is a label-free method of determining binding activity wherein the binding of a molecule in the soluble phase (the “analyte”) is directly measured to a “ligand” molecule immobilized on a sensor surface.
  • the binding of the ligand is monitored by an optical phenomenon termed surface plasmon.
  • SPR signal expressed in resonance units, RU
  • association (‘on rate’, k a ) and dissociation rates (‘off rate’, ko) are obtained from the signal obtained during the association and dissociation and the equilibrium dissociation constant (‘binding constant’, KD) can be calculated therefrom.
  • the signal given in resonance units (RU) depends on the size of the ligand present in the analyte, however in case the experimental conditions are the same, i.e. the ligand is the same molecule at the same condition the obtained RU can indicate affinity, wherein the higher the obtained signal in RU the higher the binding. Affinity may be expressed for example in half-maximal effective concentration (ECso) or the equilibrium dissociation constant (KD).
  • “Half maximal effective concentration” also called “ECso” refers to the concentration of a drug, antibody or toxicant which induces a response, such as binding or a cytotoxicity effect halfway between the baseline and maximum after a specified exposure time. ECso and affinity are inversely related, the lower the ECso value the higher the affinity of the antibody.
  • the binding molecule of the present invention binds to the MUC16 or DR5 target antigens with a KD value ranging from 1 pM to 100 pM, preferably 1 pM to 1 pM, as determined e.g., by ELISA or by surface plasmon resonance analysis (Malmqvist M., Curr. Opin. Immunol.
  • Antibody or bispecific binding molecule affinity can also be measured using kinetic exclusion assay (KinExA) technology (Darling, R. J., and Brault P-A. Assay and Drug Development Technologies. 2004, 2: 647-657).
  • KinExA kinetic exclusion assay
  • the ability of the bispecific binding molecules disclosed herein to cause cell death or apoptosis in MUC16 + /DR5 + cells may be detected by LIVE/DEAD Cell Viability Assay (Thermo Fisher Scientific) or a similar assay, or by CellTiter-Glo assay (Promega) or a similar assay, or by AlamarBlue and/or inducing inhibition of proliferation of MUC16- expressing cells in the range of concentrations between 1 pM and 1 pM; typically, between 10 nM and 0.01 nM.
  • the bispecific molecules of the invention induce cell death, wherein at least 30% of the MUC16 + /DR5 + cells in a sample or a patient undergo cell death. In some aspects of these embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells undergo cell death.
  • the bispecific binding molecules of the present invention can induce DR5-mediated apoptosis in one or more cancer cell types that are MUC16 + /DR5 + , such as the cancer cell line OVCAR-3, with more than 50% inhibition of cell viability at a concentration of 3 nM or less.
  • the bispecific binding molecules described herein induce apoptosis in cancer cells and therefore can be used in the therapy of cancers which express both MUC16 and DR5.
  • Methods of identifying whether a particular tumor expresses MUC16 and DR5 are well known in the art.
  • immunohistochemistry can be used to determine whether tumor tissue expresses MUC16 and DR5 (e.g., using the bispecific binding molecules described herein or any known anti-MUC16 and/or anti-DR5 antibody) and hence would be suitable for treatment with the binding molecule of the invention.
  • the bispecific binding molecules disclosed herein may be produced through the use of nucleic acid sequences that encode the amino acid sequences thereof.
  • the nucleic acids sequences may comprise DNA or RNA and may be wholly or partially synthetic or recombinant.
  • Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence and encompasses an RNA molecule with the specified sequence in which U is substituted for T unless context requires otherwise.
  • the nucleotide sequence is an RNA sequence that contains one or more modified ribonucleic acids (e.g., N6-methyladenosine (m6A), pseudouridine ( ⁇
  • modified ribonucleic acids e.g., N6-methyladenosine (m6A), pseudouridine ( ⁇
  • the bispecific binding molecules disclosed herein are made up of four polypeptide chains.
  • the bispecific binding molecule disclosed herein are made up of two identical heavy chain fusions and two identical light chains, wherein each heavy chain fusion comprises the heavy chain of the first antigen binding domain (e.g., a MUC16 antibody that specifically binds to the extracellular domain of MUC16) linked by short amino acid linker to the scFv of the second antigen binding domain (a scFv that specifically binds DR5); and each light chain comprises the light chain of the first binding moiety (e.g., a MUC16 antibody that specifically binds to the extracellular domain of MUC16).
  • the first antigen binding domain e.g., a MUC16 antibody that specifically binds to the extracellular domain of MUC16
  • a scFv that specifically binds DR5
  • the bispecific binding molecule disclosed herein are made up of two identical heavy chains and two identical light chain fusions, wherein each light chain fusion comprises the light chain of the first antigen binding domain linked by short amino acid linker to the scFv of the second antigen binding domain; and each heavy chain comprises the heavy chain of the first antigen binding domain.
  • fusion refers to a fusion of either a light or heavy chain of the first binding moiety directly or through a small peptide linker to the scFv of the second antigen binding domain; and the term “chain” refers to either a heavy or light chain, respectively, of the first binding domain.
  • an isolated nucleic acid sequence that encodes a fusion of the heavy chain of the first antigen binding domain linked by short amino acid linker to the scFv of the second antigen binding domain is provided, wherein the heavy chain of the first antigen binding domain is the amino acid sequence of any one of SEQ ID NOs: 115-118, the linker is the amino acid sequence of SEQ ID NO: 173 and the scFv of the second antigen binding domain is the amino acid sequence of SEQ ID NO: 172.
  • this fusion has the amino acid sequence of SEQ ID NO: 174. In other embodiments, this fusion has the amino acid sequence of SEQ ID NO: 175.
  • this fusion has the amino acid sequence of SEQ ID NO: 186. In other embodiments, this fusion has the amino acid sequence of SEQ ID NO: 187.
  • the nucleic acid sequence that encodes the heavy chain fusion comprises the nucleic acid sequence of SEQ ID NO: 182, or a sequence having at least 70% identity thereto and encoding the same amino acid sequence encoded by SEQ ID NO: 182.
  • the nucleic acid sequence that encodes the fusion comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 182 and encoding the same amino acid sequence encoded by SEQ ID NO: 182.
  • the nucleic acid sequence that encodes the fusion comprises the nucleic acid sequence of SEQ ID NO: 183, or a sequence having at least 70% identity thereto and encoding the same amino acid sequence encoded by SEQ ID NO: 183.
  • the nucleic acid sequence that encodes the fusion comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 183 and encoding the same amino acid sequence encoded by SEQ ID NO: 183.
  • a nucleic acid sequence encoding a chain and/or a fusion polypeptide of the bispecific binding molecule is constructed by chemical synthesis using an oligonucleotide synthesizer.
  • oligonucleotides can be designed based on the amino acid sequence of the desired chain or fusion polypeptide and codon optimization based on the host cell preferences. Standard methods can routinely be applied to synthesize and isolate polynucleotide sequences encoding the chain or fusion polypeptide of the bispecific binding molecule.
  • the bispecific binding molecule comprises two identical copies of a chain and two identical copies of a fusion as described above (e.g., two identical copies of a heavy chain fusion and two identical copies of a light chain, or two identical copies of a light chain fusion and two identical copies of a heavy chain)
  • the chain and the fusion must be separately expressed in a host cell. This is achieved by inserting the nucleotide sequences encoding each of the chain and the fusion into one or more expression vectors such that the sequences are operatively linked to transcriptional and translational control sequences.
  • expression vectors containing an isolated nucleic acid sequence(s) disclosed herein are also part of the present invention.
  • the nucleotide sequences encoding the fusion and the chain are present in the same expression vector.
  • the fusion and chain-encoding nucleic acid sequences may be operatively linked to the same or different transcriptional and translational control sequences.
  • the nucleotide sequences encoding the fusion and the chain are present in different expression vectors.
  • the fusion and chain-encoding nucleic acid sequences may also be operatively linked to the same or different transcriptional and translational control sequences.
  • binding molecules or antibodies of the invention For manufacturing the binding molecules or antibodies of the invention, the skilled artisan may choose from a great variety of expression systems well known in the art, e.g. those reviewed by Kipriyanov and Le Gall, Curr. Opin. Drug Discov. Devel. 2004; 7:233- 242.
  • Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like.
  • the expression vector and expression control sequences are selected to be compatible with the host cell.
  • Convenient vectors are those that encode a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that a VH or VL-encoding nucleotide sequence disclosed herein can be easily inserted and expressed, as described above.
  • the constant chain is usually kappa or lambda for the antibody light chain, for the antibody heavy chain, it can be, without limitation, any IgG isotype (IgGl, IgG2, IgG3, IgG4) or other immunoglobulins, including allelic variants.
  • the recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain (e.g., the heavy and light chains of the binding molecules or antibodies described herein) from a host cell.
  • the isolated nucleic acid sequence encoding the heavy or light chain may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the mature chain.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous peptide from a non-immunoglobulin protein.
  • the nucleic acid sequence encoding the heavy or light chains of the binding molecules described herein may already contain a signal peptide sequence.
  • the recombinant expression vectors may carry regulatory sequences including promoters, enhancers, termination and polyadenylation signals and other expression control elements that control the expression of the antibody chains in a host cell.
  • the choice of expression control sequence and expression vector will depend upon the choice of host. Examples for promoter sequences (exemplified for expression in mammalian cells) are promoters and/or enhancers derived from (CMV) (such as the CMV Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.
  • adenovirus major late promoter AdMLP
  • polyoma bovine papilloma virus
  • cytomegalovirus adenovirus major late promoter
  • strong mammalian promoters such as native immunoglobulin and actin promoters.
  • polyadenylation signals are BGH poly A, SV40 late or early poly A; alternatively, 3'UTRs of immunoglobulin genes etc. can be used.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and fdamentous single-stranded DNA phages.
  • the recombinant expression vectors may also carry sequences that regulate replication of the vector in host cells (e. g. origins of replication) and selectable marker genes.
  • the recombinant expression vectors may also be viral vectors that are known in the art including but not limited to AAV, lentivirus, or other retroviral vectors.
  • Nucleic acid molecules encoding the heavy chain or an antigen-binding portion thereof and/or the light chain or an antigen-binding portion thereof of a binding molecule or antibody described herein, and vectors comprising these nucleic acid molecules can be introduced into host cells, e.g. bacterial cells or higher eukaryotic cells, e.g. mammalian cells, according to transfection methods well known in the art, including but not limited to liposome-mediated transfection, poly cation-mediated transfection, protoplast fusion, microinjections, calcium phosphate precipitation, electroporation or transfer by viral vectors.
  • one or both of the heavy and light chains is fused to the second antigen binding moiety directly or through an amino acid linker.
  • the nucleic acid molecules encoding the chain and the fusion polypeptides of the binding molecules described herein are present on two separate expression vectors that are co-transfected into the host cell, preferably a mammalian cell.
  • a host cell comprising an expression vector comprising a nucleic acid molecule encoding a chain and an expression vector comprising a nucleic acid molecule encoding a fusion polypeptide of the binding molecules described herein.
  • Mammalian cell lines available as hosts for expression are well known in the art and include, inter alia, Chinese hamster ovary (CHO, CHO-DG44) cells, NSO, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human carcinoma cells (e. g., Hep G2), A549 cells, 3T3 cells, HEK-293 and HEK-293T cells, the COS-7 lines of monkey kidney cells described by Gluzman (Cell 23:175 (1981)), or the derivatives/progenies of any such cell line.
  • mammalian cells including but not limited to human, mice, rat, monkey and rodent cells lines, or other eukaryotic cells, including but not limited to yeast, insect and plant cells, or prokaryotic cells such as bacteria may be used.
  • the binding molecules of the invention are produced by culturing the host cells for a period sufficient to allow for expression of the binding molecule in the host cells.
  • the invention relates to a recombinant eukaryotic or prokaryotic host cell, which harbors an expression vector set forth above.
  • the host cell is eukaryotic.
  • the host cell is a mammalian host cell.
  • the mammalian host cell is a NSO murine myeloma cell, a PER.C6® human cell or a Chinese hamster ovary (CHO) cell. In some aspects the host cell is a hybridoma.
  • the disclosure also provides a method for making an anti-MUC16/anti-DR5 bispecific binding molecule comprising culturing a host cell (e.g., a hybridoma or transformed mammalian host cell) capable of expressing both the chain and fusion polypeptide of the bispecific binding molecule under suitable conditions and optionally provides a method for isolating the resulting expression products secreted from the host cell. And the disclosure additionally provides the bispecific binding molecule isolated using the disclosed methods.
  • the bispecific binding molecules are preferably recovered from the culture medium as a secreted polypeptide or from host cell lysates if, for example, expressed without a secretory signal.
  • bispecific binding molecules described herein it is necessary to purify the bispecific binding molecules described herein using standard protein purification methods used for recombinant proteins and host cell proteins in a way that substantially homogenous preparations of the binding molecule or antibody as described herein are obtained.
  • state-of-the art purification methods useful for obtaining the binding molecules and antibodies of the invention include, as a first step, removal of cells and/or particulate cell debris from the culture medium or lysate.
  • the binding molecule or antibody is then purified from contaminant soluble proteins, polypeptides, and nucleic acids, for example, by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or on a cation exchange resin.
  • the purified binding molecule may be dried, e.g., lyophilized, as described below for therapeutic applications.
  • the disclosure also optionally provides the bispecific binding molecule antibody produced using this method and pharmaceutical compositions comprising the bispecific binding molecule antibody and a pharmaceutically acceptable carrier.
  • compositions comprising a bispecific binding molecule disclosed herein; and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a compound of the present invention.
  • aqueous and nonaqueous carriers examples include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers.
  • Other carriers are well known in the pharmaceutical arts.
  • compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alphatocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetra acetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxy anisole (BHA), butylated
  • compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol, or sodium chloride.
  • isotonicity agents such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol, or sodium chloride.
  • compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives, or buffers, which may enhance the shelflife or effectiveness of the pharmaceutical composition.
  • adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives, or buffers, which may enhance the shelflife or effectiveness of the pharmaceutical composition.
  • the compounds of the present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Methods for the preparation of such formulations are generally known to those skilled in the art. See e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • the bispecific binding molecules provided herein can be administered to a human or other subject in an amount sufficient to produce a therapeutic effect.
  • Such “therapeutically effective amount” is the minimum amount necessary to prevent, ameliorate, or treat clinical symptoms of any of the diseases or conditions set forth below, in particular the minimum amount which is effective to ameliorate or treat these disorders.
  • the actual dosage levels of the bispecific binding molecule in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the bispecific binding molecule effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the therapeutically effective amount of the bispecific binding molecules of the invention applicable per day is usually from 0.001 mg/kg to 100 mg/kg, preferably from 0.1 mg/kg to 20 mg/kg.
  • the antibody molecules of the invention will generally be administered in an amount between 0.005 and 20.0 mg per kilogram of body weight and dose, preferably between 0.05 and 10.0 mg/kg/dose, and more preferably between 0.5 and 10 mg/kg/dose, either continuously (e.g. by infusion) or more preferably as single doses.
  • the administration interval may be, for example, twice a week, weekly, or monthly doses, but can significantly vary, especially, depending on the before-mentioned parameters. Thus, in some cases it may be sufficient to use less than the minimum dose given above, whereas in other cases the upper limit may have to be exceeded.
  • Administration may e.g., be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target.
  • the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • an administration regimen could include long-term, weekly treatment.
  • long-term is meant at least two weeks and preferably months, or years of duration.
  • the actual pharmaceutically effective amount or therapeutic dosage will of course depend on factors known by those skilled in the art such as age and weight of the patient, route of administration and severity of disease.
  • the binding molecule of the invention will be administered at dosages and in a manner which allows a pharmaceutically effective amount to be delivered based upon patient's unique condition.
  • the pharmaceutical composition may be administered by any suitable route and mode. Suitable routes of administering a compound of the present invention in vivo and in vitro are well known in the art and may be selected by those of ordinary skill in the art.
  • the mode of application is parenteral, by infusion or injection (intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal), but other modes of application such as by inhalation, transdermal, intranasal, buccal, oral, may also be applicable.
  • a pharmaceutical composition of the present invention is administered parenterally.
  • a bispecific binding molecule of the invention is used in combination with a device useful for its administration, such as a syringe, injector pen, micropump, or another device.
  • a binding molecule of the invention is comprised in a kit of parts, for example also including a package insert with instructions for the use of the binding molecule.
  • the efficacy of the binding molecules of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease involved.
  • suitable assays and animal models will be clear to the skilled person, and for example include the assays and animal models used in the Examples below.
  • binding molecules of the invention may be used on their own or in combination with other pharmacologically active ingredients, such as state-of-the-art or standard-of-care compounds, such as e.g., cytostatic, or cytotoxic substances, cell proliferation inhibitors, anti-angiogenic substances, steroids, immune modulators/checkpoint inhibitors, and the like.
  • pharmacologically active ingredients such as state-of-the-art or standard-of-care compounds, such as e.g., cytostatic, or cytotoxic substances, cell proliferation inhibitors, anti-angiogenic substances, steroids, immune modulators/checkpoint inhibitors, and the like.
  • a further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a binding molecule of the invention, together with a pharmaceutically acceptable carrier and optionally one or more further active ingredients.
  • the invention provides a method of treatment of a disease or disorder involving cells co-expressing MUC16 and DR5 comprising administering to a subject in need thereof, a bispecific binding molecule or bispecific binding moleculecontaining pharmaceutical composition of the invention.
  • the disclosure provides methods for treating and/or ameliorating conditions associated with a MUC16-mediated activity in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bispecific binding molecule provided herein.
  • the bispecific binding molecule is administered alone.
  • the bispecific binding molecule is administered as a combination therapy.
  • methods of reducing MUC16 activity in a subject comprising administering an effective amount of a bispecific binding molecule to a subject in need thereof.
  • the above method is used to treat a MUC16-related fibrotic disease disorder, inflammatory disorder, immune disorder, or autoimmune disorder.
  • the MUC16-mediated fibrotic disorder is pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, cirrhosis, kidney fibrosis, glial scar, myocardial fibrosis, arterial stiffness, arthrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, or adhesive capsulitis.
  • the disorder to be treated is idiopathic pulmonary fibrosis.
  • the above method is used to treat a cancer or malignancy characterized by over-expression of MUC16.
  • cancer or malignancy is a gynecologic cancer (cancer of the female reproductive tract), including the cervix, endometrium, fallopian tubes, ovaries, uterus, and vagina; pancreatic cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, or lung cancer (such as non-small cell lung cancer).
  • the solid tumor to be treated is selected from ovarian and pancreatic tumors.
  • subject is intended to include human and non-human animals, which respond to the bispecific antigen binding molecule.
  • Subjects may for instance include human patients having disorders that may be corrected or ameliorated by modulating MUC16 function, such as enzymatic activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and/or induction of lysis and/or eliminating/reducing the number of MUC16 expressing cells.
  • the bispecific binding molecule may be used to elicit in vivo or in vitro one or more of the following biological activities: modulating MUC16 function (such as apoptosis, permeabilization of plasma membrane, reduction in cell number, enzymatic activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and/or induction of lysis), killing a cell expressing MUC16, mediating phagocytosis or ADCC of a cell expressing MUC16 in the presence of human effector cells, and by mediating CDC of a cell expressing MUC16 in the presence of complement or by killing MUC16 expressing cells by apoptosis.
  • modulating MUC16 function such as apoptosis, permeabilization of plasma membrane, reduction in cell number, enzymatic activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and/or induction of lysis
  • MUC16 function such as apoptosis, permeabilization of plasma membrane,
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • an “effective amount” for cancer therapy is an amount sufficient to stabilize the progression of disease for a period of time.
  • a therapeutically effective amount of an antibody of the invention may decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • an “effective amount” for idiopathic pulmonary fibrosis may result in an improvement in any one or more accepted tests, such as changes from baseline in St. George's respiratory questionnaire SGRQ, dyspnea score, cough score and 6-minute walk test (grade and distance), changes in lung function (forced vital capacity (FVC); diffusing capacity of the lungs for carbon monoxide (DLCO)) compared with baseline, changes in abnormal values of routine safety tests (blood routine, urine routine, blood biochemistry, electrocardiogram (ECG), etc.) compared with baseline, changes in chest high-resolution computed tomography (HRCT) scores from baseline, changes of lung tumor markers from baseline, frequency and severity of acute exacerbations of IPF.
  • routine safety tests blood routine, urine routine, blood biochemistry, electrocardiogram (ECG), etc.
  • HRCT chest high-resolution computed tomography
  • a bispecific binding molecule disclosed herein may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in patients wherein it is difficult to locate a tumor that is known to be present due to other biological factors.
  • a bispecific binding molecule disclosed herein may also be administered prophylactically in order to reduce the risk of developing a fibrotic disease or disorder.
  • the bispecific binding molecule disclosed herein may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Such administration may be simultaneous, separate, or sequential. For simultaneous administration, the agents may be administered as one compositions or as separate compositions, as appropriate.
  • Kits comprising anti-MUC16/anti-DR5 antibodies
  • kits that include a bispecific binding molecule disclosed herein (including variants and derivatives thereof) in suitable packaging, and written material and that can be used to perform the methods described herein.
  • the written material can include any of the following information: instructions for use, discussion of clinical studies, listing of side effects, scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like.
  • the written material can indicate or establish the activities and/or advantages of the composition, and/or describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information can be based on the results of various studies, for example, studies using experimental animals involving in vivo models and/or studies based on human clinical trials.
  • the kit can further contain another therapy (e.g., another agent) and/or written material such as that described above that serve to provide information regarding the other therapy (e.g., the other agent).
  • kits comprises at least one purified bispecific binding molecule disclosed herein in one or more containers.
  • the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and/or any necessary software for analysis and presentation of results.
  • GFTFSTF (SEQ ID NO: 79) [AR9.6 CDR H-2]
  • Example 1 Design of binding molecules recognizing MUC16 and DR5
  • the present inventors have developed binding molecules that bind both MUC16 and DR5 and that induce cell death in cancer cells expressing both MUC16 and DR5.
  • the molecular design used in these experiments has an IgG antibody that has specificity for MUC16, with a scFv that has specificity for DR5 coupled to the C terminus of the heavy chain of the IgG through a small peptide linker.
  • a schematic representation of the design is shown in FIG. 1.
  • bispecific antigen binding molecules based on the format depicted in FIG. 1 were prepared (IMV-18, MCLX-SE, IMV-AA, and MC- AA) using methods well known to the skilled in the art. Each of these bispecific antigen binding molecules had the same anti-MUC16 CDRs and anti-DR5 CDRs.
  • the anti-MUC 16 CDRs were: CDR-H1 - SEQ ID NO:1; CDR-H2 - SEQ ID NO:2; CDR-H3 - SEQ ID NO:6; CDR-L1 - SEQ ID NO:62; CDR-L2 - SEQ ID NO:63; and CDR-L3 - SEQ ID NO:65.
  • the anti-DR5 CDRs were: CDR-H1 - SEQ ID NO: 121; CDR2-H2 - SEQ ID NO: 122; CDR-H3 - SEQ ID NO: 123; CDR-L1 -SEQ ID NO: 124; CDR-L2 - SEQ ID NO:
  • bispecific molecules that targeted DR5 and a cell surface antigen different from MUC16, as well as monospecific antibodies to compare activities. See Table 10. All of these other bispecific molecules comprised the same anti-DR5 scFv.
  • Example 2 Expression and purification of bispecific tetravalent molecules recognizing human MUC16, or another antigen, and human DR5
  • antibodies or immunoglobulin chains can readily produce said antibodies or immunoglobulin chains, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, Calif) and following the manufacturer's instructions. Alternatively, antibodies, immunoglobulin chains and antibody -like binding proteins of the invention can be synthesized by recombinant DNA techniques as is well- known in the art.
  • these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.
  • the expression vectors were constructed, and transfection of cells and antibody production and purification were carried out by methods well known in the art. Genes for both the heavy chain-scFv fusion and the light chain of the bispecific binding compounds set forth above were designed and optimized for expression in CHO cells, and the sequences were introduced into a protein expression vector of BioIntron, Building 5, No.388 Galileo Road, Zhangjiang High-Tech Park, Shanghai, China. CHO cells were transiently transfected with the expression vector and cultured for 5-7 days. Cultures were then harvested, and a two-step purification was performed.
  • Recombinant binding molecules or antibodies were purified from culture supernatant by Protein A affinity chromatography, and then additionally purified by preparative size exclusion chromatography (SEC) by methods well known in the art. Upon purification, the antibodies were analyzed for monomer purity and for the presence of fragments or degradation, by methods well known in the art, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with protein staining, and analytical SEC-HPLC, and their concentration was determined by UV spectroscopy. The extinction coefficient was calculated as in Pace et al, Protein Sci. 1995;4:2411-23. All purified proteins contained no fragments or other impurities and were 97.5-100% monomeric. An example of this analysis is depicted in FIG. 2.
  • IMV-M and its variants (IMV-Mvl, IMV-Mv2, IMV-Mv3 and IMY-Mv4) in which the scFv portion of the heavy chain fusion was stabilized by a disulfide bond between two cysteines, one each introduced into specific positions in the VL and VH of the scFv (see Table 9), some were purified using only Protein A affinity chromatography without the need for the additional SEC step. The final yields of each of these bispecific antibodies as determined is set below.
  • Example 3 Cytotoxicity of the bispecific molecules recognizing human MUC16 and human DR5.
  • NCI-H292 lung carcinoma CRL-1848TM
  • CAOV-3 ovarian adenocarcinoma HBV-75
  • NIH:0VCAR-3 also known as OVCAR- 3 ovarian adenocarcinoma
  • HTB-161 an adherent cell line, MM.
  • NCI-H292, CAOV-3, OVCAR-3, PK-59, HCC827, NCI-H1975, NCC-StC-K140, HDQ-P1 and HP AC express MUC16 transcripts and MUC16 protein. All these cell lines for which data for MUC16 protein expression are available, express MUC16 protein.
  • Proteomics data reporting that MUC16 protein is expressed by OVCAR-3, NCI-H292, HCC827, NCI-H1975 and NCC-StC-K140 are shown in Table 11, and, in addition, flow cytometric or Western blot data demonstrating MUC16 protein expression by 0VCAR3, HP AC and CAOV-3 cells are reported in Chen et al., Cancer Res. 2007, 67, 4924-5998; Haridas et al, PLoS ONE 2011; 6: e26839; and Kline et al., Oncotarget 2017; 8, 52045-5260. MM.
  • CD38 transcripts Table 11
  • CD38 protein Moreno et al, Clin Cancer Res. 2019, 25, 3176-3187
  • MM.1S, SU- DHL-8, Ramos, RPMI-8226, and Molp-8 cell lines express LIV-1 transcripts (Table 11).
  • TR - transcripts TR - transcripts
  • PE - protein expression proteomics
  • NA - not available PE for LIV-1 and for DR5 were not available for these cell lines.
  • the TR data are presented as log2(transcripts per million +1); the PE data are presented as log2 (relative data), as described in Nusinow et al., 2020, Cell 180, 387-402.
  • a) Adherent cells Cells were plated in a 96-well tissue culture plate at 2.5 x 10 3 cells/well and were allowed to adhere. The next day, the test-reagents were added, and cells were incubated for two additional days with the test antibodies, and CellTiter-Glo® (Promega) assay was performed in accordance with the manufacturer’s protocol. Each condition was in triplicate. Untreated cells (negative control) were exposed to culture medium only. The values were normalized to untreated control.
  • the surviving fractions of NCI-H292, Caov-3 OVCAR3 cells were 0.48, 0.20 and 0.21, respectively.
  • the parental anti-DR5 antibody lexatumumab was markedly less potent in killing these three cell lines, and most cells survived exposure to the concentration of lexatumumab as high as 10 nM with the surviving fractions of 0.94, 0.95, and 0.81, respectively.
  • binding of the monospecific antibody to DR5 without additional targeting of MUC16 was not sufficient to induce a strong DR5- mediated cytotoxic effect, while the anti-MUC16/anti-DR5 bispecific antibody had high cytotoxic potency.
  • HP AC pancreatic adenocarcinoma (CRL-2119TM; ATCC), PK-59 pancreatic carcinoma (RCB1901; RIKEN); HCC827 lung adenocarcinoma (CRL-2868; ATCC), NCI- H1975 lung adenocarcinoma (CRL-5908; ATCC); NIH:OVCAR-3, also known as OVCAR-3 ovarian adenocarcinoma (HTB-161; ATCC); NCC-StC-K140 gastric carcinoma (RCB2224; RIKEN); and HDQ-P1 breast ductal carcinoma (ACC 494; DSMZ).
  • Cells are plated in a 96-well tissue culture plate at 2.5 x 10 3 cells/well and are allowed to adhere. The next day, the test-reagents (IMV-18 (Table 9), MCLX-SE (Table 9), 11D10X (Table 10) and 11D10-SE (Table 10), parental anti-MUC16 antibody 3A5 (SEQ ID NOs:115 and 119), parental anti-MUC16 antibody MC-SE (SEQ ID NOs:116 and 120), the non-targeting bispecific antibody FLX-SE (Table 10), or IMV-15) are added to different culture plates, and cells are incubated for two additional days. CellTiter-Glo® (Promega) assays are performed in accordance with the manufacturer’s protocol. Each condition is assayed in triplicate. Untreated cells (negative control) are exposed to culture medium only. The values are normalized to untreated control.
  • mice The anti-tumor activity of IMV-18, a bispecific antibody targeting MUC16 and DR5 was examined in a xenograft model of HP AC pancreatic adenocarcinoma cell line (CRL-2119TM, American Type Culture Collection, www.atcc.org) in immunodeficient mice.
  • HPAC cells express both MUC16 and DR5 (Table 11).
  • mice were treated with IMV-21, a bispecific antibody employing the same anti-DR5 scFv, but targeting a different antigen, CD74.
  • BALB/c nude female mice, 6 -8-week-old, 18 to 21 g were used in this study.
  • HPAC cells Prior to inoculation into mice, HPAC cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 in F12/D MEM medium supplemented with 5% fetal bovine serum, 0.5 mM sodium pyruvate, 0.002 mg/ml insulin, 5 microgram'ml transferrin, 40 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor.
  • the tumor cells were routinely sub-cultured before reaching confluence by trypsin-EDTA treatment, not to exceed 4-5 passages. For tumor inoculation, the cells growing in an exponential growth phase were harvested and counted.
  • mice were inoculated subcutaneously on the right flank with inoculated subcutaneously with 5 x 10 6 HPAC cells in the presence of Corning® Matrigel® Matrix in accordance with the manufacturer’s protocol.
  • Treatment of tumorbearing mice with the test agents were started when the mean tumor volume reached about 145 mmf Based on the tumor volume, mice were randomly assigned to respective groups, five mice per group, such that the average starting tumor size was the same for each treatment group. The average body weight of mice in each group wns no less than 18g.
  • Mice were injected intravenously once (day 1, FIG. 4) with either IMV-21 (5 mg/kg in phosphate buffered saline (PBS)), or IMV-18 (5 mg/kg in PBS), or vehicle only (PBS).
  • tumor volume a x b x b/2, where “a” and “b” are long and short diameters of a tumor, respectively.
  • FIG. 4A treatment with IMV-18 caused marked delay in average tumor growth in mice, compared with the vehicle control, while treatment with IMV-21 did not cause any delay in tumor growth.
  • the body weight of mice did not decrease indicating absence of systemic toxicity of these molecules for the mice (FIG. 4B). Plots of tumor growth in individual mice (FIG.
  • mice treated with vehicle only or with IMV-21 demonstrate that while in mice treated with vehicle only or with IMV-21, growth of HPAC xenografts was robust; in mice treated with IMV-21 there was no delay in tumor growth. Two out of five mice treated with IMV-18 experienced marked delay in tumor growth, and in the remaining three mice, tumors disappeared with no regrowth for the entire duration of the experiment.
  • IMV-21 a bispecific antibody, composed of an IgG antibody targeting a distinct antigen (CD74), and of the anti-DR5 scFv identical to that in IMV-18, was inactive, demonstrates that the anti-tumor activity of IMV-18 was MUC16-dependent.
  • Example 5 Anti-proliferative effects and apoptosis in PK-59 ceils induced by the bispecific molecules recognizing human MUC16 and human DR5.
  • Caspase-3/7 Green Dye is a fluorogenic substrate of caspase 3 and caspase 7, two caspases that become active during later stages of apoptosis [Elmore S., Toxicol Pathol, 2007; 35(4): 495-516], In the presence of this dye, healthy cells are not fluorescent, while cells that are undergoing apoptosis become green fluorescent. Exposure of cells to Nuclight Rapid Red Dye makes their nuclei red fluorescent. The green fluorescence (green) and red fluorescence (red) objects per well were counted even 7 two hours using IncuCyte S3 (Sartorius AG, sartorius.com). Each condition was in triplicate.
  • FIG. 5A The anti -proliferative effect of the bispecific anti-MUC16/anti-DR5 antibody MCLX-SE is shown in FIG. 5A. Average numbers of cells with red fluorescent nucleus per well (average of three wells) (Y-axis in LoglO scale) were plotted versus time (X-axis; hours). In the absence of any test-agents (Medium) cells proliferated exponentially (linear semi-exponential plot) without slowing down, with the doubling time of 35 hours. In the presence of MCLX-SE, at the lowest tested concentration of 41.2 pM, proliferation of cells was drastically slowed down within the first 24 hours of exposure to a near complete arrest.
  • FIG. 5B and FIG. 5C The induction of apoptosis in cells by the bispecific anti-MUC16/anti-DR5 antibody MCLX-SE is shown in FIG. 5B and FIG. 5C.
  • FIG. 5B upon exposure to 41.2 pM of bispecific anti-MLiC16/anti-DR5 antibody MCLX-SE, a majority’ of cells rapidly underwent apoptosis. By 18 hours of exposure about 1900 of cells contained activated caspase 3 and/or caspase 7. In contrast, only a small fraction of cells underwent apoptosis during their exposure to the comparator bispecific antibody FLX-SE, which targeted DR5 on the cell surface, but did not target MUC16.
  • MC-SE another comparator antibody, a mono-specific antibody targeting MUC16, did not induce any significant apoptosis of the cells.
  • the comparator antibody FLX-SE induced apoptosis of only a minor fraction of cells even at the highest tested concentration of 10 nM
  • the other comparator antibody, MC-SE did not cause any significant apoptosis at any concentration (FIG. 5C).
  • Example 6 Evaluation of the anti-tumor activity of MCLX-SE in MUC16- positive/DRS positive xenograft models
  • the anti -tumor activity of MCLX-SE was examined in immunodeficient mice in the following xenograft models using MUC 16-positive cell lines that express both MUC16 and DR5 above the low-expression threshold (Table 11).
  • the cell lines tested w'ere HP AC pancreatic adenocarcinoma cell line (CRL-2119TM), HCC827 (HCC827 lung adenocarcinoma (CRL-2868)), PK-59 cells (pancreatic adenocarcinoma), and NCI-H1975 cells (lung adenocarcinoma).
  • some mice were treated with either FLX-SE (Table 10), or with MC-SE.
  • BALB/c nude female mice were used in this study.
  • MUC16 protein in xenografts was examined by immunohistochemistry.
  • the following reagents were used: rabbit monoclonal anti-MUC16 antibody, Abeam # 110640MXB (primary antibody); Polyclonal Goat anti-Rabbit Immunoglobulins, Dako (Agilent Technologies), cat# 4003 (secondary antibody); Dako antibody dilute solution, Dako#S2022; Wash buffer, Dako#K8007; Citrate 6.0 (MXB Biotechnologies, Fuzhou, Fujian, China #MVS-0066. Tumor tissues embedded in paraffin were cut with a microtome to the thickness of 4 pm, and then processed by a protocol well known in the arts.
  • sections were immersed in 3% hydrogen peroxide solution for 5 min. To avoid nonspecific staining, the sections were then incubated in blocking serum for 30 min containing normal goat serum at room temperature, followed by an overnight incubation with a rabbit monoclonal anti- MUC16 antibody (Abeam #110640, 1:500 dilution). Then the sections were exposed to secondary antibody conjugated to HRP. The nuclei were stained with hematoxylin. Slides were then scanned using Aperio Scanner: Versa 8 (Leica), at 200X magnification. Images were then opened with HALO, using the pen tool to select an annotation layer. The necrotic areas were excluded in the annotation layer.
  • Human DR5 recombinant protein (TRAIL R2, catalog number TR2-H5229; amino acids 56-182) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86 400-682-2521, www.acrobiosystems.cn.
  • a high-binding ELISA plate is coated with human DR5, 1 pg/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour.
  • the blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed.
  • various agents IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10.
  • the wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc-gamma polyclonal antibody, washed, and the bound polyclonal antibody is detected with 3, 3', 5,5'- tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm.
  • TMB 3, 3', 5,5'- tetramethylbenzidine
  • Stop solution ThermoFisher Scientific
  • Normal polyclonal human IgG is used to detect non-specific binding of human IgG to the plate coated with recombinant human DR5.
  • Example 8 Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE to human recombinant MUC16.
  • Human recombinant MUC16 (catalog number CA5-H52H6, amino acids 13810- 14451) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86400-682-2521, www.acrobiosystems.cn.
  • a high-binding ELISA plate is coated with human MUC16, 1 microgram/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour.
  • the blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX- SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed.
  • various agents IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX- SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10.
  • the wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc- gamma polyclonal antibody, washed and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • Example 9 Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE to human recombinant CD74.
  • Human recombinant CD74 (catalog number CD4-H524c, amino acids 73-232) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86400-682-2521, www.acrobiosystems.cn.
  • a high- binding ELISA plate is coated with human CD74, 1 microgram/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour.
  • the blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti- MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed.
  • various agents IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti- MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10.
  • the wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc-gamma polyclonal antibody, washed and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • Example 10 Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE to human recombinant CD38.
  • Human CD38 (catalog number CD8-H5224, amino acids 43-300) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86400-682-2521, www.acrobiosystems.cn.
  • a high-binding ELISA plate is coated with human CD38, 1 microgram/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour.
  • the blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti- MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed.
  • various agents IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti- MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10.
  • the wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc-gamma polyclonal antibody, washed and detected with 3.3'.5.5'-tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm.
  • TMB horse radish peroxidase-conjugated anti-human IgG Fc-gamma polyclonal antibody
  • Example 11 Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE to human recombinant LIV-1/SLC39A6.
  • Human LIV-1 (catalog number LV1-H5223, amino acids 29-325) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86400-682-2521, www.acrobiosystems.cn.
  • a high-binding ELISA plate is coated with human LIV-1, 1 microgram/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour.
  • the blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti- MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed.
  • various agents IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti- MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10.
  • the wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc-gamma polyclonal antibody, washed and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • Example 12 The anti-tumor activity of the anti-MUC16-anti-DR5 bispecific antibodies does not depend on its interaction with Fc receptors gamma in a pancreatic tumor xenograft model.
  • the anti -tumor activities of four variants of the anti-MUC16-anti-DR5 bispecific antibody (IMV-18, MCLX-SE, IMV-AA and MC-AA) against a pancreatic carcinoma xenograft model were compared. These variants (described in Table 9) were nearly identical except for several point mutations in the Fc region that affected the affinities of these variants towards Fc receptors gamma on immune cells of nude mice, as described in [Shivange et al.
  • IMV-18 contains a wild-type human IgGl Fc; MCLX-SE has enhanced affinity towards Fc gamma receptor 2B, IMV-AA has impeded affinity towards all Fc receptors gamma, and MC-AA has impeded affinity towards Fc receptors gamma except restored affinity towards Fc gamma receptor 2B.
  • these antibodies were co-injected with an excess (30 mg/kg) of a non-targeting mouse IgGl antibody (muIgGl, Table 10), which contains a wild-type human IgGl Fc. This co-inj ection was done to allow muIgGl to compete with the anti-MUC16-anti-DR5 bispecific antibody for binding to Fc gamma receptors.
  • MUC16-positive/DR5 positive xenograft pancreatic carcinoma model PK-59 (RCB1901, Cell Bank, RIKEN BioResource Research Center (BRC) 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan) was used in this study.
  • BALB/c nude were injected subcutaneously with the PK-59 cells and the resulting tumor-bearing mice were treated with one of the anti-MUC16-anti-DR5 bispecific antibodies (or an appropriate control) once the mean tumor volume reached about 100-150 mm 3 . Based on the tumor volume, mice were randomly assigned to respective groups, such that the average starting tumor size was the same for each treatment group.
  • mice were injected intravenously once on day 1 with one of the anti-MUC16-anti-DR5 bispecific antibodies (5 mg/kg) or vehicle (PBS), or with an anti-MUC16-anti-DR5 bispecific antibody (5 mg/kg) and muIgGl (30 mg/kg), four mice per group.
  • tumor volume a x b x b/2, where “a” and “b” are long and short diameters of a tumor, respectively.
  • all anti-MUC16/anti/DR5 bispecific antibodies were equally active in their ability to delay the growth of PK-59 xenografts and irrespective of the presence or absence of an excess of mouse IgGl antibody.
  • IVM-M displays dose-dependent anti-tumor activity in a pancreatic tumor xenograft model.
  • mice were subcutaneously injected with PK- 59 pancreatic carcinoma cells.
  • IVM-M treatment began when the mean tumor volume reached about 100-150 mm 3 .
  • mice were randomly assigned to respective groups, such that the average starting tumor size was the same for each treatment group.
  • Mice were injected intravenously once on day 1 with IMV-M at either 1 mg/kg, 2.5 mg/kg or 5 mg/kg, or vehicle only (PBS), four mice per group.
  • any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web at tigr.org and/or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.
  • TIGR The Institute for Genomic Research
  • NCBI National Center for Biotechnology Information

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Abstract

Bispecific binding molecules are disclosed, that bind to human MUC16 and to human death receptor 5 and can kill cancer cells expressing MUC16 and death receptor 5. Also disclosed are pharmaceutical compositions comprising the antibodies, and therapeutic methods for using the antibodies.

Description

CYTOTOXIC BISPECIFIC ANTIBODIES BINDING
TO DR5 AND MUC16 AND USES THEREOF
RELATED APPLICATIONS
This patent application claims the benefit of U.S. Provisional Patent Applications having serial numbers 63/424,323, filed on November 10, 2022, and 63/443,606, filed on February 6, 2023, the contents of each of which are hereby incorporated herein by reference in their entirety.
BACKGROUND
The human MUC16 gene (mucin 16, NCBI Entrez Gene: 94025) encodes the type I transmembrane protein MUC16 or mucin 16, a high molecular weight, heavily glycosylated protein. It is not expressed in most normal tissues, except for the apical surface in the epithelium of the upper respiratory tract, ocular surface, mesothelium lining body cavities (pleural, peritoneal, and pelvic cavities), internal organs, and male and female reproductive organs (Lee et al., Pharmaceuticals, 2021, 14, 1053). MUC16 is highly expressed in many different types of cancer, including most ovarian and endometrial tumors (Kabawat et al. Am. J. Clin. Pathol., 79:98-104; Suh et al., Chemo Open Access 2017, 6:2), pancreatic adenocarcinomas (Haridas et al. PLoS One 2011, 6, e26839; Jiang et al. Appl. Immunohistochem. Mol. Morphol. 2017, 25:620-623; Streppel et al. 2012, Hum. Pathol. 43, 1755-63), a fraction of esophageal, gastric, and colorectal adenocarcinomas (Streppel et al. Hum. Pathol. 2012, 43(10): 1755-1763), breast carcinomas (Moritani et al. Hum. Pathol. 2008, 39, 666-671) and non-small cell lung cancer (Chen et al. BMC Cancer 2019, 19:171; Kanwal et al. Oncotarget, 2018, 9: 12226-12239; Lee et al. Pharmaceuticals 2021, 14, 1053; Liu et al. Dig Dis Sci. 2022 Jun;67:2195-2208). In addition, MUC16 is highly expressed in idiopathic pulmonary fibrosis (IPF) (Ballester et al. Int. J. Mol. Sci. 2021; 22:6502) and, likely, other systemic fibrosis diseases (Zhang et al. Prog. Mol. Biol. Transl. Sci. 2019; 162:241-252). In IPF, MUC16 is expressed in pathologic hyperplastic alveolar type II cells and in lung fibroblasts from fibrotic foci, but not normal lung (Ballester et al. Int. J. Mol. Sci. 2021; 22:6502). Antibody-drug conjugates that target MUC16 have been, and bispecific moi eties targeting MUC16 and either CD28 or CD3 are currently in early human clinical trials for treatment of various cancers (NCT01335958, NCT04590326, NCT03564340). The MUC16 extracellular domain consists of an unstructured N-terminal domain and a tandem repeat (TR) region. The TR region contains about 60 repeats of 156 amino acids. The TR region is interspersed with approximately 16 homologous SEA (Sea urchin sperm protein, Enterokinase, Agrin) domains (White et al., Proteins, 2022;90:1210-1218). Some amount of the MUC16 protein is cleaved extracellularly by an unknown mechanism and subsequently shed into blood. This extracellular portion of MUC16 is a well- established serum biomarker for ovarian cancer (Bast et al., Int. J. Biol. Markers 1998, 13, 179-187).
Death receptor 5 (DR5) is a cell membrane protein that, when bound to its ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), can induce cell death via apoptosis. Apoptosis can also be mediated by DR5 when bound to agonistic monoclonal antibodies (Dubuisson and Micheau, Antibodies 2017, 6, 16). In preclinical studies, stimulation of DR5 with either TRAIL or an anti-DR5 antibody was reported to induce cell death in a variety of tumor cells, but to date, in clinical trials neither TRAIL or its derivatives, nor anti-DR5 antibodies showed a significant therapeutic benefit (Lemke et al., Cell Death Differ. 2014, 21: 1350-1364; von Karstedt et al. Nat Rev Cancer. 2017, 17: 352- 366).
Bispecific molecules targeting DR5 and various second antigens are known in the art, and some are in early human clinical trials. For example, an anti-CDH17/anti-DR5 bispecific antibody (Garcia-Martinez et al., Mol. Cancer Then 2021, 20: 96-108; United States patent No. 10,858,438) is currently in clinical development. Other second antigens targeted by such DR5 bispecifics include folate receptor alpha (US patent publication No. 20200283537), fibroblast-activation protein (FAP) (US patent No. 9,926,379), melanoma- associated chondroitin sulfate proteoglycan and roundabout homolog 4 (PCT patent publication WO2011039126A1), and lymphotoxin-beta receptor (Michaelson et al., mAbs (2009), 1:128-141). The anti-FAP/anti-DR5 bispecific antibody RG7386 was examined in a Phase 1 clinical trial (NCT02558140), but the trial was discontinued, and the development of RG7386 was abandoned. An anti-CD44v6/anti-DR5 bispecific did not potentiate the agonistic effect of DR5 in CD44v6-expressing cells (US Patent No. 10,858,438 B2), demonstrating that the ability of such anti-DR5 bispecific antibody to kill antigenexpressing cells is not common to all cell surface antigens. The mixed results in the prior art indicate an ongoing need to develop additional anti-DR5 bispecific molecules that demonstrate the ability to kill diseased cells, such as cancer cells. Moreover, the prior art results demonstrate that one cannot reasonably predict whether a bispecific antibody targeting DR5 together with a tumor-associated antigen will be effective in triggering apoptosis in a cancer cell. Thus, there is a persistent unmet need for cancer therapeutics.
SUMMARY OF THE INVENTION
The present invention provides bispecific binding molecules that comprise a first antigen-binding domain that binds specifically to the extracellular domain of human MUC16 and a second antigen-binding domain that binds specifically to human DR5. These bispecific molecules are efficient in killing cells that co-express MUC16 and DR5 and are more potent in killing such cells than the monospecific DR5 antibody from which the second antigen-binding domain is derived (e.g., an anti-DR5 antibody having the same set of three heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) and light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) as are present in the bispecific binding molecule).
Hence, in a first aspect of the invention, the first antigen-binding domain (which binds the extracellular portion of human MUC16) binds to an epitope present in more than one tandem repeat/SEA segment within the extracellular domain of MUC16.
In a second aspect of the invention, the first antigen-binding domain is competed for MUC16 binding by one or more of the following antibodies: OC125, H185 (Invitrogen Cat No. MA5-11579), Mi l (American Tissue Culture Collection Accession No. PTA-6206), OV197 (Fujirebio Diagnostic), 5E11 (Millipore Sigma Cat No. MABC1608-25UG), AR9.6, H1H8794, VK-8, B43.13 (also known as Oregovomab), or 3A5 (Sofituzumab).
In a third aspect of the invention, the first antigen-binding domain binds to a polypeptide or peptide consisting essentially of the amino acid sequence of at least one of SEQ ID NOs:176-181, or an extracellular fragment of MUC16 isolated from the cell culture media of OVCAR-3 cells.
In a fourth aspect of the invention, the first antigen-binding domain comprises six specific heavy and light-chain CDR sequences (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In one embodiment of the fourth aspect, CDR-H1 comprises the amino acid sequence of SEQ ID NO:1; CRD-H2 comprises the amino acid sequence of any one of SEQ ID NOs.:2-5; CDR2-H3 comprises the amino acid sequence of any one of SEQ ID NOs.:6-61 (CDR3-H3); CDR-L1 comprises the amino acid sequence of SEQ ID NO:62; CDR-L2 comprises the amino acid sequence of SEQ ID NO:63; and CDR-L3 comprises the amino acid sequence of SEQ ID NO:65. In a more specific embodiment of the fourth aspect, CDR-H1 comprises the amino acid sequence of SEQ ID NO:1; CDR-H2 comprises the amino acid sequence of SEQ ID NO:2; CDR-H3 comprises the amino acid sequence of SEQ ID NO:6; CDR-L1 comprises the amino acid sequence of SEQ ID NO:62; CDR-L2 comprises the amino acid sequence of SEQ ID NO:63; and CDR-L3 comprises the amino acid sequence of SEQ ID NO:65. Each of the above CDR sequences are according to the Kabat convention of determining CDRs.
In an alternate embodiment of the fourth aspect, the set of 6 CDR sequences that characterize the first binding moiety (and the CDR convention they correspond to) are selected from any of the following set forth in Table 1.
Table 1. Alternate CDR sets that characterize the first antigen-binding domain
Figure imgf000005_0001
In a fifth aspect of the invention, the first antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of either SEQ ID NOs.:104 or 105, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to either SEQ ID NOs.: 104 or 105. In a more specific embodiment of the fifth aspect, the first antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of either SEQ ID NOs.: 104 or 105. In an alternate embodiment of the fifth aspect, the first antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of any one of SEQ ID NOs: 106- 114, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to any one of SEQ ID NOs: 106-114.
In a sixth aspect of the invention, the first antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO:97. In a more specific embodiment of the sixth aspect, the first antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97. In an alternate embodiment of the sixth aspect, the first antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of any one of SEQ ID NOs:98-103, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to any one of SEQ ID NOs:98-103.
In a seventh aspect of the invention the first antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of either SEQ ID NOs.: 104 or 105, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to either SEQ ID NOs.:104 or 105; and a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 97. In a more specific embodiment of the seventh aspect, the first antigenbinding domain comprises a light chain variable region that comprises the amino acid sequence of either SEQ ID NOs.: 104 or 105; and aheavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97.
In an alternate embodiment of the seventh aspect, the first antigen-binding domain comprises a combination of a heavy chain variable region and a light chain variable region that are selected from any of the following combinations set forth in Table 2.
Table 2. Alternate heavy and light chain variable region combinations
Figure imgf000006_0001
Figure imgf000007_0001
In an eighth aspect of the invention, the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs.: 115-118, or a heavy chain comprising the same three heavy chain CDRs as, and an amino acid sequence having at least 90% sequence identity to, any one of SEQ ID NOs.:115-118. In some embodiments of this aspect the heavy chain comprises the amino acid sequence of any one of SEQ ID NOs.:115-118.
In a ninth aspect of the invention, the first antigen-binding domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 120; or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to, SEQ ID NO: 119 or SEQ ID NO: 120. In some embodiments of this aspect the light chain comprises the amino acid sequence of any one of SEQ ID NOs.:119-120.
In a tenth aspect of the invention, the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs.: 115-118, or a heavy chain comprising the same three heavy chain CDRs as, and an amino acid sequence having at least 90% sequence identity to, any one of SEQ ID NOs. : 115-118; and a light chain comprising the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 120; or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to, SEQ ID NO: 119 or SEQ ID NO: 120. In a more specific embodiment, of the tenth aspect, the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs.: 115-118; and a light chain comprising the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 120.
In an eleventh aspect of the invention, the first antigen-binding domain as described for any of the first through tenth aspects, comprises two heavy chains and two light chains. In some embodiments of the eleventh aspect, each of the two heavy chains has an identical amino acid sequence and each of the two light chains has an identical amino acids sequence.
In a twelfth aspect of the invention, the second antigen-binding domain, which binds to human DR5, is competed for DR5 binding by: a) one or more of the following antibodies: Conatumumab, Drozitumab, Lexatumumab, LBY135, Tigatuzumab, and DS- 8273a; or b) TRAIL or a fragment of TRAIL that binds to DR5.
In a thirteenth aspect of the invention, the second antigen-binding domain comprises six specific heavy and light-chain CDR sequences (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In one embodiment of the thirteenth aspect, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 121; CRD-H2 comprises the amino acid sequence of SEQ ID NO: 122; CDR2-H3 comprises the amino acid sequence of SEQ ID NO: 123; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 124; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 125; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 126. The above CDR sequences are according to the Kabat convention of determining CDRs.
In an alternate embodiment of the thirteenth aspect, the set of 6 CDR sequences that characterize the first binding moiety (and the CDR convention they correspond to) are selected from any of the following set forth in Table 3.
Table 3. Alternate CDR sets that characterize the second antigen-binding domain
Figure imgf000008_0001
Figure imgf000009_0001
In a fourteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 158. In a more specific embodiment of the fourteenth aspect, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 158. In an alternate embodiment of the fourteenth aspect, the second antigenbinding domain comprises a heavy chain variable region that comprise the amino acid sequence of any one of SEQ ID NOs: 159-164, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NOs: 159-164. In another alternate embodiment of the fourteenth aspect, the second antigen-binding domain comprises a heavy chain variable region that comprise the amino acid sequence of any one of SEQ ID NOs: 218-220, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NOs: 218-220. In a more specific embodiment, the heavy chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 218. In another more specific embodiment, the heavy chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 219. In still another more specific embodiment, the heavy chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 220.
In a fifteenth aspect of the invention, the second antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 165. In a more specific embodiment of the fifteenth aspect, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 165. In an alternate embodiment of the fifteenth aspect, the second antigen-binding domain comprises a light chain variable region that comprise the amino acid sequence of any one of SEQ ID NOs: 166-171, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to SEQ ID NOs: 166-171. In another alternate embodiment of the fifteenth aspect, the second antigen-binding domain comprises a light chain variable region that comprise the amino acid sequence of any one of SEQ ID NOs: 215-217, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to SEQ ID NOs: 215-217. In a more specific embodiment, the light chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 215. In another more specific embodiment, the light chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 216. In still another more specific embodiment, the light chain variable region of the second antigen binding domain comprises the amino acid sequence of SEQ ID NO: 217.
In a sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of either SEQ ID NO: 158, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 158; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to SEQ ID NO: 165. In a more specific embodiment of the sixteenth aspect, the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 158; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 165.
In an alternate embodiment of the sixteenth aspect, the second antigen-binding domain comprises a combination of a heavy chain variable region and a light chain variable region that are selected from any of the following combinations set forth in Table 4.
Table 4. Alternate variable heavy and light chain combinations of the second antigenbinding domain
Figure imgf000010_0001
Figure imgf000011_0001
In still another alternate embodiment of the sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 218; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 215. In still another alternate embodiment of the sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 219; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 215. In still another alternate embodiment of the sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 219; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 216. In still another alternate embodiment of the sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 220; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 217.
In a seventeenth aspect of the invention, the second antigen-binding domain is a scFv fragment of an antibody characterized by any of the foregoing twelfth through sixteenth aspects. An scFv fragment comprises a heavy chain variable region and a light chain variable region of bound to one another through a peptide linker. In some embodiments of this aspect, the scFv fragment comprises, in N- to C-terminal order, a light chain variable region, a peptide linker, and a heavy chain variable region. In some embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of any one of SEQ ID NO: 172, SEQ ID NOs: 210-214; or an amino acid sequence having the same three heavy chain CDRs and the same three light chain CDRs as, and at least 90% sequence identity to, any one of SEQ ID NO: 172 or SEQ ID NOs: 210- 214. In more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 172; or an amino acid sequence having the same three heavy chain CDRs and the same three light chain CDRs as, and at least 90% sequence identity to, SEQ ID NO: 172. In even more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 172. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:210. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:211. In other specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:212. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:213. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO:214.
Given the above, it will be readily apparent to those of ordinary skill in the art that the bispecific binding molecules disclosed herein may comprise a first antigen-binding domain characterized by any of the foregoing first through eleventh aspects and a second antigen-binding domain characterized by any of the foregoing twelfth through seventeenth aspects. All possible combinations of those first antigen-binding domains and second antigen binding domains are within the scope of the present disclosure.
In an eighteenth aspect of the invention, the N-terminus of the second antigenbinding domain is fused directly or through a peptide linker having a length of 4 to 20 amino acids to the C-terminus of one of the heavy chains of the first antigen-binding domain. In some embodiments of the eighteenth aspect, the N-terminus of the second antigen-binding domain is fused through a peptide linker having a length of 4 to 20 amino acids to the C-terminus of one of the heavy chains of the first antigen-binding domain. In more specific embodiments of the eighteenth aspect, the peptide linker has the amino acid sequence of SEQ ID NO: 173. In other more specific embodiments of the eighteenth aspect, the second antigen binding domain is an scFv fragment of the seventeenth aspect. In yet other specific embodiments of the eighteenth aspect, the bispecific antigen-binding molecule comprises two scFv fragments of the seventeenth aspect that specifically bind human DR5. In some even more specific embodiments of the eighteenth aspect, the bispecific antigen-binding molecule comprises two scFv fragments of the seventeenth aspect that specifically bind human DR5, wherein each is bound to the C-terminus of a different heavy chain of the first antigen-binding domain. In some further embodiments, each of the two scFv fragments have the identical amino acid sequence. In a nineteenth aspect of the invention the bispecific binding molecule comprises: a) two antibody light chains, wherein each light chain has an amino acid sequence independently selected from SEQ ID NO: 119 and SEQ ID NO: 120; and b) two antibody heavy chain fusions, wherein each heavy chain fusion has an amino acid sequence independently selected from the formula: X-L-Y, wherein: X is the amino acid sequence of any one of SEQ ID NOs.: 115-118; L is the amino acid sequence of SEQ ID NO: 173; and Y is the amino acid sequence of any one of SEQ ID NO: 172 or SEQ ID NOs: 210-214. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 172. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 210. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 211. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 212. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 213. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 214. In some embodiments of the nineteenth aspect, each light chain has the identical amino acid sequence, and each heavy chain fusion has the identical amino acid sequence. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:203. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:204. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:205. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:206. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:207. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:203. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:204. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:205. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:206. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:207.
In a twentieth aspect of the invention is provided a pharmaceutical composition comprising a bispecific binding molecule described herein and a pharmaceutically acceptable carrier.
In twenty-first aspect of the invention is provided an isolated nucleic acid sequence encoding a heavy chain fusion having an amino acid sequence the formula: X-L-Y, wherein: X is the amino acid sequence of any one of SEQ ID NOs.: 115-118; L is the amino acid sequence of SEQ ID NO: 173; and Y is the amino acid sequence of any one of SEQ ID NO: 172 or SEQ ID NOs: 210-214. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO: 172. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO: 210. In some embodiments of the twenty -first aspect, Y is the amino acid sequence of SEQ ID NO: 211. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO: 212. In some embodiments of the twenty -first aspect, Y is the amino acid sequence of SEQ ID NO: 213. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO: 214. In some embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 174. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 175. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 186. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 187. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:203. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:204. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:205. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:206. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:207. In still other embodiments of this aspect, the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 182 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 182 and encoding the same amino acid sequence as SEQ ID NO: 182. In yet other embodiments of this aspect, the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 183 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 183 and encoding the same amino acid sequence as SEQ ID NO: 183.
In a twenty-second aspect of the invention is provided an expression vector comprising the nucleic acid sequence of the twenty-first aspect or any embodiments thereof. In some embodiments of the twenty-second aspect, the expression vector additionally comprises a first promoter that is operatively linked to and can drive expression of the nucleic acid sequence. In a twenty -third aspect of the invention is provided an expression vector of the twenty second aspect that additionally comprises a nucleic acid sequence encoding a light chain of the first antigen-binding domain of the bispecific binding molecule as described in any of the foregoing aspects of the invention; and a second promoter that is operatively linked to and can drive expression of the light-chain encoding nucleic acid sequence. In one more specific embodiment of this aspect, the nucleic acid sequence encoding a light chain of the first antigen-binding domain of the bispecific binding molecule comprises the nucleotide sequence of SEQ ID NO: 184 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 184 and encoding the same amino acid sequence as SEQ ID NO: 184. In another more specific embodiment of this aspect, the nucleic acid sequence encoding a light chain of the first antigen-binding domain of the bispecific binding molecule comprises the nucleotide sequence of SEQ ID NO: 185 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 185 and encoding the same amino acid sequence as SEQ ID NO: 185. In an even more specific embodiment of this aspect, the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 182 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 182 and encoding the same amino acid sequence as SEQ ID NO: 182; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 184 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 184 and encoding the same amino acid sequence as SEQ ID NO: 184. In another even more specific embodiment of this aspect, the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 183 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 183 and encoding the same amino acid sequence as SEQ ID NO: 183; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 185 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 185 and encoding the same amino acid sequence as SEQ ID NO: 185.
In a twenty -fourth aspect of the invention is provided a host cell harboring: a) the expression vector of the twenty-second aspect or any embodiments thereof; and b) an expression vector comprising a nucleic acid sequence encoding the light chain of the first antigen-binding domain of the bispecific binding molecule as described in any of the foregoing aspects of the invention. In some embodiments of the twenty-fourth aspect, the expression vector comprising a nucleic acid sequence encoding the light chain additionally comprises a promoter that is operatively linked to and can drive expression of the lightchain encoding nucleic acid sequence. In a more specific embodiment of this aspect, the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 184 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 184 and encoding the same amino acid sequence as SEQ ID NO: 184. In another more specific embodiment of this aspect, the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 185 or a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 185 and encoding the same amino acid sequence as SEQ ID NO: 185. In an even more specific embodiment of this aspect, the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 182 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 182 and encoding the same amino acid sequence as SEQ ID NO: 182; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 184 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 184 and encoding the same amino acid sequence as SEQ ID NO: 184. In another even more specific embodiment of this aspect, the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 183 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 183 and encoding the same amino acid sequence as SEQ ID NO: 183; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 185 or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 185 and encoding the same amino acid sequence as SEQ ID NO: 185.
In a twenty-fifth aspect of the invention is provided a host cell harboring the expression vector of the twenty -third aspect or any embodiments thereof.
In a twenty-sixth aspect of the invention is provided a method of manufacturing the bispecific binding molecule disclosed herein comprising the steps of a) culturing the host cell of the twenty-fourth or twenty -fifth aspects or any embodiments thereof under conditions allowing for expression of the nucleic acid molecule encoding the heavy chain fusion and expression of the nucleic acid molecule encoding the light chain, and association of the expressed heavy chain fusion and the expressed light chain into the bispecific binding molecule; and b) recovering the bispecific binding molecule from the culture. In some embodiments of the twenty-sixth aspect the recovered bispecific binding molecule is further purified and/or modified and/or formulated. In a twenty-seventh aspect, the invention provides a method of treating a MUC16-related fibrotic disease disorder, inflammatory disorder, immune disorder, or autoimmune disorder, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the bispecific binding molecule of any of the foregoing aspects or embodiments thereof, or the pharmaceutical composition of the twentieth aspect. In some embodiments of the twenty-seventh aspect, the MUC16-mediated fibrotic disorder is pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, cirrhosis, kidney fibrosis, glial scar, myocardial fibrosis, arterial stiffness, arthrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, or adhesive capsulitis.
In a twenty-eighth aspect, the invention provides a method of treating a cancer or malignancy characterized by over-expression of MUC16, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the bispecific binding molecule of any of the foregoing aspects or embodiments thereof, or the pharmaceutical composition of the twentieth aspect. In some embodiments of the twentyeighth aspect, the cancer or malignancy is a gynecologic cancer (cancer of the female reproductive tract), including the cervix, endometrium, fallopian tubes, ovaries, uterus, and vagina; pancreatic cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, or lung cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of exemplary bispecific binding molecules of the present invention. Depicted is a binding molecule comprising (i) an Ig molecule comprising two heavy chain fusions, each comprising a variable region (“VHMUCI6”) that specifically binds to the extracellular domain of MUC16, a constant region (“CHI”, “CH2” and “CH3”), a first peptide linker (“Linker l)”and a single-chain variable fragment (“scFv”) comprising, in N- to C-terminal order, a variable light chain region that specifically bind to DR5 (“VLDRS”), a second peptide linker (“Linker 2”) and a variable heavy chain region that specifically bind to DR5 (“VHDRS”); and (ii) two light chains, each comprising a variable region (“VLMUCI6”) specifically binding to the extracellular domain of MUC16 and a constant region (“CL”). The resulting molecule is a symmetric, bispecific and tetraval ent antibody-like molecule.
FIG. 2, panel A (FIG. 2A) depicts an SDS-PAGE gel of antibody IMV-18 that was expressed in CHO cells, and then purified from culture supernatant by Protein A affinity chromatography, and then additionally purified by preparative size exclusion chromatography. Lane R - reducing gel, Lane N-R - non-reducing gel, Lane M - molecular weight markers. Panel B (FIG. 2B) depicts a SEC-HPLC chromatogram of the purified IMV-18 as measured at 214 nm and 280 nm.
FIG. 3, panel A (FIG. 3A) is a series of plots comparing the cytotoxicity of IMV-18 (circles) and the anti-DR5 antibody Lexatumumab (triangles) using a CellTiter-Glo® assay on NCI-H292, CAOV-3 and OVCAR-3 cells, each of which expresses both MUC16 and DR5, after two days of exposure to either IMV-18 or Lexatumumab. Panel B (FIG. 3B) is a series of plots comparing the cytotoxicity of a bispecific antibody targeting human CD38 and DR5 (“IMV-15”; circles), a bispecific antibody targeting human LIV-1 and DR5 (“IMV-20”; squares), or Lexatumumab (triangles) using a CellTiter-Glo® assay on MM. IS, SU-DHL-8, Ramos, RPMI-8226 and MOLP-8 cell lines, each of which expresses CD38, LIV-1, and DR5 (Table 11), after two-day exposure to the various agents. Panel C (FIG. 3C) is a series of plots comparing the cytotoxicity of a bispecific antibody targeting human MUC16 and DR5 (“MCLX-SE”, squares), a bispecific antibody targeting fluorescein and DR5 (“FLX-SE”, triangles), and a monospecific antibody targeting human MUC16 (“MC- SE”, circles) on a variety of cell lines. The cytotoxicity was assessed using a CellTiter- Glo® assay, and was performed on the cell lines HP AC, PK-59, NCI-H1975, OVCAR-3, NCC-StC-K140, and HDQ-P1 each of which expresses MUC16 and DR5 (Table 11).
FIG. 4, panel A (FIG. 4A) is a plot depicting average tumor volume (Y-axis; mm3) after the indicated number of days (X-axis) in a HP AC xenograft model in BALB/c nude mice (n=5 for each group) intravenously administered a single dose of vehicle only (PBS) (group 1 (Gl)), a bispecific antibody targeting CD74 and DR5 (“IMV-21”; 5 mg/kg in PBS; group 2 (G2)), or IMV-18 (5 mg/kg in PBS; group 3 (G3)). HP AC cells express both MUC16 and DR5. Intravenous administration with the test agents was performed when the mean tumor volume reached approximately 145 mm3. Panel B (FIG. 4B) shows the average body weight (X-axis; grams) after the indicated number of days (Y-axis) following administration of each agent administered in FIG. 4A. Panel C (FIG. 4C) shows tumor volume (Y-axis; mm3) after the indicated number of days (X-axis) following administration of the agent in individual mice.
FIG 5, panel A (FIG. 5 A) is a series of plots comparing anti-proliferative effects of continuous exposure of different concentrations of either a bispecific antibody targeting human MUC16 and DR5 (“MCLX-SE”), a bispecific antibody targeting fluorescein and DR5 (“FLX-SE”), or a monospecific antibody targeting human MUC16 (“MC-SE”) on PK- 59 cells. Cells were stained with Incucyte® Nuclight Rapid Red Dye for nuclear labeling, and counted every two hours as red fluorescent objects, using IncuCyte S3 (Sartorius AG, sartorius.com). Y-axis is plotted using logarithmic (base 10) scale. For each condition, average values from three wells were plotted. Each plot (except the control plot “Medium”) compares the time-course of proliferation of cells continuously exposed to either MC-SE (circles), FLX-SE (squares), or MCLX-SE (triangles) at the same concentration. Cells exposed to medium only (plot “Medium”) continued to proliferate with the same rate without slowing down (same doubling time) for the duration of the experiment, forming a straight line in this semi-logarithmic plot. Cells exposed to the parental monospecific anti- MUC16 antibody MC-SE at any concentration in this concentration range of 41.2 pM - 10 nM continued to proliferate without slowing down with the rate similar to that of the cells exposed to medium only, demonstrating that the cells survived. Panel B (FIG. 5B) is a time-course of apoptotic events in PK-59 cells exposed to a bispecific antibody targeting human MUC16 and DR5 (“MCLX-SE”), a bispecific antibody targeting fluorescein and DR5 (“FLX-SE”), or a monospecific antibody targeting human MUC16 (“MC-SE”); all at 41.2 pM, continuous exposure, or to medium only (control). Cells were stained with Incucyte® Caspase-3/7 Green Dye for Apoptosis, Catalog No. 4440, Sartorius (green fluorescence). Caspase-3/7 Green Dye is a fluorogenic substrate of caspase 3 and caspase 7, two caspases that become active during later stages of apoptosis. Panel C (FIG. 5C) shows another set of data from the same experiment, plotting the number of apoptotic events per well in PK-59 cells exposed to various concentrations of these three antibodies for 8 hours.
FIG. 6, panel A (FIG. 6A) is a plot of tumor volume over time in a HP AC xenograft model in BALB/c nude mice (n=5 for each group) intravenously administered a dose of vehicle only (PBS) (circles) on day 1; a dose of a bispecific antibody targeting MUC16 and DR5 “MCLX-SE”; 5 mg/kg on day 1, and then a dose of 5 mg/kg on day 13 (up-facing triangles); a dose of 5 mg/kg of a bispecific antibody targeting fluorescein and DR5 “FLX- SE”; 5 mg/kg (down-facing triangles) on day 1; or a dose of 5 mg/kg of a monospecific antibody targeting MUC16 “MC-SE” (squares) on day 1. The mean and standard error of the mean (SEM) values are shown (n = 5). HP AC cells express both MUC16 and DR5 (Table 11). Panel B (FIG. 6B) is a plot of tumor volume over time in a HCC827 xenograft model in BALB/c nude mice (n=5 for each group) intravenously administered a dose of vehicle only (PBS) (circles) on day 1; a dose of a bispecific antibody targeting MUC16 and DR5 “MCLX-SE”; 5 mg/kg on day 1, and then a dose of 5 mg/kg on day 15 (up-facing triangles); a dose of 5 mg/kg of a bispecific antibody targeting fluorescein and DR5 “FLX- SE”; 5 mg/kg (down-facing triangles) on day 1; or a dose of 5 mg/kg of a monospecific antibody targeting MUC16 “MC-SE” (squares). HCC827 cells express both MUC16 and DR5 on day 1. The mean and standard error of the mean (SEM) values are shown (n = 5). Note that in FIG. 6B some error bars overlap and obscure others at the same time point (e.g., See several time points for Vehicle, FLX-SE and MC-SE). Panels C (FIG. 6C) and D (FIG. 6D) are plots of tumor volumes over time in a PK-59 xenograft model, and an NCI-H1975 xenograft model, respectively; both in BALB/c nude mice (n=4 for each group) intravenously administered a dose of vehicle only (PBS) (circles) on day 1, or a dose of a bispecific antibody targeting MUC16 and DR5 “MCLX-SE”; 5 mg/kg on day 1 (squares). PK-59 cells and NCI-H1975 cells express both MUC16 and DR5 The mean and standard error of the mean (SEM) values are shown (n = 4). Note that at some data points in each of FIGS. 6A-6D the error bars are obscured by the data point itself.
FIG. 7 is a plot of the effect of various anti-MUC16-anti-DR5 bispecific antibodies (IMV-18, MCLX-SE, IMV-AA, or MC-AA) alone or in combination with an excess of a non-targeting mouse IgGl antibody (muIgGl) over time on tumor growth in PK-59 pancreatic carcinoma xenograft mice.
FIG. 8 is a plot of the effect of varying amounts of the anti-MUC16-anti-DR5 bispecific antibody IMV-M over time on tumor growth in PK-59 pancreatic carcinoma xenograft mice. DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
Terms not specifically defined herein should be given the meanings that would be given to them by one of skill in the art considering the disclosure and the context. As used in the specification, however, unless specified to the contrary, the following terms have the meaning indicated and the following conventions are adhered to.
As used in the description of the present disclosure and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").
The terms “about” and “approximately” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, + 0.5%, or even ± 0.1% of the specified amount.
The transitional phrase “consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03.
The term “consists essentially of’ (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this present disclosure, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and/or C-terminal ends of the recited sequence such that the function of polypeptide is not materially altered. The total of ten or less additional amino acids can include the total number of additional amino acids on both ends added together.
The term “human MUC16” when used herein includes any variants, isoforms, and species homologs of human MUC16 (mucin 16, NCBI Entrez Gene: 94025) which are expressed by cells on their surface. The term “recombinant human MUC16” when used herein includes human MUC16 (NCBI Entrez Gene: 94025) available on the world wide web at uniprot.org; UniProtKB - Q8WXI7), and the nucleic acid sequence encoding that protein. The term “MUC16” is also intended to include variants (e.g., allelic variants), and derivatives thereof. Representative human MUC16 cDNA and human MUC16 protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Human MUC16 variants include mucin-16 precursor (NM_001401501.2 and NP_001388430.1), and mucin-16 (NM_024690.2 and NP_078966.2). Nucleic acid and polypeptide sequences of MUC16 orthologs in organisms other than humans are well-known.
The term “Death Receptor 5” also known as DR5; tumor necrosis factor receptor superfamily member 10B, TNFRSF10B, TNF-related apoptosis-inducing ligand receptor 2, TRAIL receptor 2, TRAIL-R2 and CD262, TNF receptor superfamily member 10b, KILLER, KILLER/DR5, TRICK2, TRICK2A, TRICK2B, TRICKB, ZTNFR9. This term includes any variants, isoforms and species homologs of human protein provided in UniProt 014763 available on the world wide web at uniprot. org/uniprot/014763, and the nucleic acid sequence encoding that protein.
The term “analog” when used herein includes any compounds discovered in a structure-activity relationship study (see for instance Schnecke V. and Bostrom J. Drug Discovery Today. 11 (1-2): 43-50 (2006), having a structure similar to that of another compound, but differing from it in respect to a certain component (see for instance Willett P. et al. J. Chem. Informat. Comp. Sci. 38: 983-996 (1998), Johnson A. M. and Maggiora G. M. Concepts and Applications of Molecular Similarity. New York: John Willey & Sons. ISBN 978-0-471-62175-1 (1990), NikolovaN. and Jaworska J. QSAR & Combinatorial Science. 22: 1006-1026 (2003); specifically, with the aim to enable it to be chemically linked to an anti-MUC16 antibody, or to an anti-CD74 antibody, for instance, like depicted in Scheme 1 and described in (Sijbrandi N. J. et al. Cancer Res; 77; 257-67 (2017)), or like depicted in Scheme 2 and described in (Drake P.M. et al. Bioconjug. Chem. 25: 1331-1341 (2014)).
The term “variant” when used herein in the context of a reference amino acid sequence (e.g., a specific SEQ ID NO), means an amino acid sequence that has one or more amino acid insertions, deletions or replacements compared to the reference sequence.
The term “derivative” when used herein in the context of a reference antibody, means an immunoglobulin that has been chemically or recombinantly modified to include a moiety having an activity that differs from that of the reference antibody without destroying the ability of the reference antibody to bind to the antigen to which it is directed. For example, the moiety may be a second antibody or antigen-binding portion thereof that recognizes a different antibody; a peptide or chemical “tag” that allows the antibody to be recognized (e.g., for purification), or identified (e.g., a fluorescent molecule); a small molecule that has activity against a cell targeted by the antibody (e.g., an anti-cancer agent); a radioisotope; etc. The term “derivative” when used herein in the context of a compound or a small peptide, includes any compound that is derived from a similar compound by a chemical reaction and/or that at least theoretically can be formed from the precursor compound (see for instance Oxford Dictionary of Biochemistry and Molecular Biology. Oxford University Press. ISBN 0-19-850673-2).
The term “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light chains and one pair of heavy chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized (see for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically 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 (see also Chothia and Lesk J. Mol. Biol. 196, 901- 917 (1987)).
The term “expression vector” in the context of the present invention refers to a plasmid or virus designed for gene expression in cells. Various types of expression vectors are well known in the art. The term “antibody” in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of thereof, which can specifically bind to an antigen under typical physiological conditions. An antibody is the source of each of the two different binding moieties present in the bispecific binding molecules disclosed herein. Thus, the term “antibody” and “immunoglobulin” are used interchangeably and include: 1) molecules that have all of the structural features of an immunoglobulin (e.g., a pair of light chains and a pair of heavy chains that are bound to one another through disulfide bonds) and specifically bind to an antigen or an epitope thereof, such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, recombinantly produced antibodies, veneered antibodies, humanized antibodies, and mouse antibodies; 2) molecules that only have part of the structure of an immunoglobulin, but retain the three heavy chain and three light chain CDRs and maintain the ability to specifically bind to an antigen or an epitope thereof, such as Fab, Fab’, F(ab’)2, F(ab)3, scFv, scFv-Fc, diabodies, triabodies, tetrabodies, and minibodies, (individually and collectively termed, “antigen-binding fragment(s)”); 3) molecules that are heavy chain-only antibodies, which comprises heavy chains, but lacks the light chains, and single-domain antibodies; and 4) molecules of 1) to 3) that retain the ability to specifically bind to an antigen or an epitope thereof, and are modified (chemically or recombinantly) to further comprise a moiety having a different activity than the reference antibody or antigenbinding fragment, such as bispecific antibodies; multi-specific antibodies; and antibodies conjugated to a toxin, radioisotope, therapeutic agent, fluorescent tag, or peptide tag (individually and collectively termed, “antibody derivatives”). Depending upon the nature of the moiety and its desired function, the moiety in an antibody derivative may be attached directly to the antibody or through a linker. Each of the above antigen-binding fragments is well-known in the art. The term “antibody” includes monoclonal antibodies (including full length four-chain antibodies or full length heavy-chain only antibodies which have an immunoglobulin Fc region, antibody compositions with poly-epitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments (e.g., Fab, F(ab')2, and Fv)). Antibodies contemplated herein include single-domain antibodies, such as heavy chain only antibodies.
The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains ten antigen binding sites, while IgA antibodies comprise from 2 - 5 of the basic four-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The term “heavy chain-only antibody” (HCAb) refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in four-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) and sharks are known to produce HCAbs. The term “single-domain antibody” or “dAb” refers to a single antigenbinding polypeptide having three complementary determining regions (CDRs). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR- containing polypeptide. In some cases, single-domain antibodies are engineered from camelid or shark, and their heavy chain variable domains are referred herein as “VHHs”. Some VHHs may also be known as nanobodies. Camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e. g., Hamers-Casterman et al., Nature 363: 446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1, CDR2 and CDR3 refer to the complementarity determining regions 1, 2 and 3.
Antibodies present in the bispecific binding molecules of the invention that include heavy chain constant regions can be of any isotype. As used herein, "isotype" refers to the immunoglobulin class (for instance IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes.
The term “chimeric antibody” in the context of the present invention refers to an antibody made by fusing the antigen binding region (variable domains of the heavy and light chains, VH and VL) from one species, such as a mouse, with the constant domain (effector region) from a human antibody (see for instance Morrison S. L. et al. Proc. Natl. Acad. Sci. USA 81, 6851-6855 (1984)).
The term “mouse antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from mouse germline immunoglobulin sequences. The term “humanized antibody”, as used herein, means a mouse antibody having the variable framework and constant regions replaced with the corresponding regions derived from human germline immunoglobulin sequences.
A “humanized” antibody refers to an antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., complementary determining regions (CDRs)) correspond to those of a non-human antibody, and all or substantially the entire framework regions (FRs) correspond to those of a human antibody. In this context, “substantially” means that both the heavy chain and the light chain of the humanized antibody has at least 80% alignment identity with the top human germline V gene hit. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.
The mouse, chimeric and humanized antibodies that may be present in the bispecific binding molecules of the invention may include amino acid residues not encoded by their respective germline immunoglobulin sequences due to e.g., mutations, substitutions, deletions, or insertions, introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo. These antibodies are termed “variants” because of such amino acid alterations.
An “isolated antibody,” as used herein, is intended to refer to an antibody, which is substantially free of other antibodies having different antigenic specificities (for instance an isolated antibody that specifically binds to MUC16 is substantially free of antibodies that specifically bind antigens other than MUC16; an isolated bispecific antibody that specifically binds to MUC16 and DR5 is substantially free of antibodies that specifically bind antigens other than MUC16 and DR5). An isolated antibody that specifically binds to an epitope, isoform or variant of human MUC16 may, however, have cross-reactivity to other related antigens, for instance from other species (such as MUC16 species homologs). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. The term “bispecific binding molecule” as used herein is a molecule that is capable of specific binding to two different antigens (e.g., MUC16 and DR5), i.e., it is a molecule that has two different “antigen binding moieties”.
The term “antigen-binding moiety” refers to a part of an antibody or a bispecific binding molecule that comprises amino acids responsible for the specific binding between the antibody or a portion of the bispecific binding molecule and a specific antigen. In instances where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as “epitope” or “antigenic determinant.”
Though an antigen-binding moiety typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), it does not necessarily have to comprise both. For example, the second antigen binding moiety of the present bispecific binding molecules may consist only of a VH domain of an anti-DR5 antibody, but still to retain its ability to bind to and agonize DR5. Each variable region or domain comprises three CDRs. The generalized structure of antibodies or immunoglobulin molecules is well known to those of skill in the art.
The term “bispecific antibody” as used herein is a bispecific binding molecule, wherein each antigen binding moiety thereof is derived from an antibody. The term "bispecific antibody" is intended to include any antibody which has two different binding specificities, including, among others, tetravalent antibody molecules comprising an IgGl or its fragment or a variant, with one antigen specificity, and an antigen-binding fragment of an antibody with another antigen specificity, such as VHH, Fab, Fab’, F(ab’)2, F(ab)3, scFv, scFv-Fc, diabodies, triabodies, tetrabodies, and minibodies. The term "bispecific antibodies" also includes diabodies (see for instance Holliger, P. et al., PNAS USA 90, 6444-6448 (1993), Poljak, R. J. et al., Structure 2, 1121-1123 (1994)).
As used herein, the term “binding” in the context of the binding of an antibody to a predetermined antigen typically is a binding with an affinity corresponding to a KD of about 10,000 nM or less, such as about 1,000 nM or less, such as about 100 nM or less, such as about 10 nM or less, such as about 1 nM or less, about 0.1 nM or less, or about 10 pM or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore instrument or an Octet instrument, using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to anon-specific antigen (e.g., bovine serum albumin, casein) other than the predetermined antigen, a closely-related antigen, or in the case of bispecific or multi-specific antibodies, an antigen other than the antigen targeted by such other specificities. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for anon-specific antigen may be at least 10,000-fold.
The term “ko”, as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koffvalue.
The term “kA” (M'1 times sec'1), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.
The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
The term “ECso” (M) as used herein, refers to the concentration of the antibody targeting an antigen, for example, MUC16 that binds to cells expressing this antigen, so that the cells reach 50% maximal fluorescence as determined by flow cytometry.
The term “percent sequence identity” as used herein refers to the percentage of amino acids or nucleotides in a candidate sequence that are identical with the amino acids in a reference amino acid sequence or nucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be determined using any mathematical algorithm to determine amino acid sequence homology known in the art. One such algorithm is the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Another is the GAP program in the GCG software package (available on the world wide web at the GCG company website), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Still other algorithms that can be used to determine sequence identity include ClustalW, Clustal Omega, BLAST, BLAST-2, ALIGN-2 or Megalign (DNASTAR). The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide).
As used herein, the terms, used interchangeably, “inhibits growth” or “inhibits proliferation” (e.g. referring to cells, such as tumor cells) is intended to include any measurable decrease in the cell growth (which is meant the same as cell proliferation), or decrease in cell number when contacted with an anti-MUC16 antibody, or with an antibody that binds to another antigen, or with a non-targeting antibody, or with a bispecific antibody, or with any other molecule, as compared to the growth of the same cells not in contact with an anti-MUC16 or an antibody that binds to another antigen, or with a nontargeting antibody, respectively, or with any other molecule, e.g., the inhibition of growth of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Such a decrease in cell growth can occur by a variety of mechanisms, e.g., ADCC, ADCP, CDC, cell cycle arrest, and/or apoptosis and/or decrease in cell number.
As used herein, the term “cytotoxicity” or “cytotoxic effect” (e.g. referring to cells, such as tumor cells) is intended to include any measurable decrease in the cell growth, or decrease in cell number when contacted with an anti-MUC16 antibody or an antibody that binds another antigen, or with a non-targeting antibody, or with a bispecific binding molecule disclosed herein, or with any other molecule, as compared to the growth of the same cells in the absence of such an antibody or molecule e.g., the cytotoxic effect of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Such a cytotoxic effect can occur by a variety of mechanisms, e.g., ADCC, CDC, ADCP, cell cycle arrest, and/or apoptosis and/or decrease in the cell number. In some embodiments, cytotoxicity or cytotoxic effect refers to the decrease in cell growth or decrease in cell number of cells that are positive for MUC16 and for DR5 (“MUC16+/DR5+”), or of cells that are positive for another antigen and for DR5.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. Such terms are intended to refer not only to the subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK293 cells, NS/0 cells, and lymphocytic cells.
The terms “solid tumors” and “solid cancers”, used interchangeably, in the context of the present invention include but is not limited to the following malignancies: epithelial ovarian cancer or any other ovarian cancer, pancreatic ductal adenocarcinoma or any other pancreatic cancer, esophageal adenocarcinoma or any other esophageal cancer, gastric adenocarcinoma, or any other gastric cancer, colon cancer, colorectal cancer, invasive micropapillary carcinoma of the breast, or any other breast cancer, non-small cell lung cancer or any other lung cancer.
The terms “blood cancers" in the context of the present invention include but is not limited to hematological malignancies: acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia lymphomas, Hodgkin's lymphomas (all four subtypes), non-Hodgkin's lymphomas (all subtypes), small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, MM, lymphoplasmacytic lymphoma, splenic margina zone lymphoma, plasma cell neoplasms, such as plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodal marginal B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, hairy cell leukemia, primary effusion lymphoma.
“Treatment” refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states. An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a bispecific binding molecule disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of that molecule to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. In some embodiments, an “effective amount” is an amount that causes cytotoxicity in cells that are MUC16+/DR5+.
The antigen binding domain of an antibody resides in the so-called variable domain, or variable region (Fv) of the antibody. The variable domain comprises three so-called complementary-determining regions (CDR’s) spaced apart by framework regions (FR’s). The CDRs are the regions of an antibody that are responsible for the specificity (binding) of the antibody for (to) a particular antigen.
Within the context of this invention, reference to CDRs of the MUC16 binding domain and the DR5 binding domain is based on CDRs reported in the prior art disclosing various MUC 16 and DR5 antibodies. These CDRs are typically based upon the CDR definition of Kabat (E. A. Kabat, et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda (1983), IMGT (Lefranc M P, et al., Dev Comp Immunol. 2003 January; 27(l):55-77; Giudicelli V et al., Cold Spring Harb Protoc. 2011; 2011(6):695-715), Chothia (Chothia and Lesk, J. Mol. Biol. 1987, 196: 901-917), AbM (Martin and Allen, 2007, Bioinformatics tools for antibody engineering. In: Diibel S, editor. Handbook of Therapeutic Antibodies . Weinheim: Wiley -VCH Verlag GmbH (2008). p. 95- 117; Abhinandan and Martin, 2008, Protein Eng Des Sei. (2010) 23:689-97), or Contact (Padlan EA, et al., FASEBJOffPublFedAm Soc Exp Biol. (1995) 9:133-9).
Expressions “variable domains” or “variable region” or Fv as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen. The variable domain of a light chain is abbreviated as “VL” and the variable domain of a heavy chain is abbreviated as “VH”. The variable light and heavy chain domains have the same general structure, and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three HVRs (or CDRs). The framework regions adopt a beta-sheet conformation and the CDRs may form loops connecting the beta-sheet structure. The CDRs in each chain are held in their three- dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.
The term “constant domains” or “constant region” as used within the current application denotes the sum of the domains of an antibody other than the variable region. Such constant domains and regions are well known in the state of the art and e.g., described by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91).
The “Fc part” or “Fc region” of an antibody is not involved directly in binding of an antibody to an antigen but exhibit various effector functions. An “Fc part of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM. According to the heavy chain constant regions the different classes of immunoglobulins are called a, 6, s, y, and p respectively. Several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, and IgG4, IgAl, and IgA2. The Fc part of an antibody is directly involved in ADCC (antibody dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, Clq binding and Fc receptor binding. Complement activation (CDC) is initiated by binding of complement factor Clq to the Fc part of most IgG antibody subclasses. While the influence of an antibody on the complement system is dependent on certain conditions, binding to Clq is caused by defined binding sites in the Fc part. Such binding sites are known in the state of the art and described e.g., by Boakle et al., Nature 282 (1975) 742- 743, Lukas et al., J. Immunol. 127 (1981) 2555-2560, Brunhouse and Cebra, Mol. Immunol. 16 (1979) 907-917, Burton et al., Nature 288 (1980) 338-344, Thommesen et al., Mol. Immunol. 37 (2000) 995-1004, Idusogie et al., J. Immunol. 164 (2000) 4178-4184, Hezareh et al., J. Virology 75 (2001) 12161-12168, Morgan et al., Immunology 86 (1995) 319-324, EP 0307434. Such binding sites are e.g., L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat, see below). Most crucial among these residues in mediating Clq and Fcgamma receptor binding in IgGl are L234 and L235 (Hezareh et al., J. Virology 75 (2001) 12161-12168). Antibodies of subclass IgGl and IgG3 usually show complement activation and Clq and C3 binding, whereas IgG2 and IgG4 do not activate the complement system and do not bind Clq and C3.
The art has further developed antibodies and made them versatile tools in medicine and technology. Thus, in the context of the present invention the terms “antibody molecule” or “antibody” (used synonymously herein) do not only include antibodies as they may be found in nature, comprising e.g., two light chains and two heavy chains, or just two heavy chains as in camelid species, but furthermore encompasses all molecules comprising at least one paratope with binding specificity to an antigen and structural similarity to a variable domain of an immunoglobulin.
Thus, an antibody portion of the bispecific binding molecules provided herein may comprise a monoclonal antibody, a multi-specific antibody, a bispecific antibody, an antibody derivative, a human antibody, a recombinant antibody, a veneered antibody, a humanized antibody, a chimeric antibody, a fragment of an antibody, in particular a Fv, Fab, Fab', or F(ab')2 fragment, a single chain antibody, in particular a scFv, a Small Modular Immunopharmaceutical (SMIP), a domain antibody, a nanobody, a diabody. The antibody may have an effector function, such as ADCC or CDC, that is usually mediated by the Fc part (antibody constant region) of the antibody, or it may have no effector function, e.g., by lacking a Fc part or having a blocked, masked Fc part, in essence a Fc part that is not or insufficiently recognized by immune cells or immune system components, like the complement system. Monoclonal antibodies (mAb) are monospecific antibodies that are identical in amino acid sequence. They may be produced by hybridoma technology from a hybrid cell line (called hybridoma) representing a clone of a fusion of a specific antibodyproducing B cell with a myeloma (B cell cancer) cell (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256:495- 7.). Alternatively, monoclonal antibodies may be produced by recombinant expression in host cells (Norderhaug L, Olafsen T, Michaelsen T E, Sandlie I. (1997) J Immunol Methods 204 (1): 77-87; see also below). A “recombinant antibody” or “recombinant binding molecule” is an antibody or binding molecule which has been produced by a recombinantly engineered host cell. It is optionally isolated or purified.
For application in humans, it is often desirable to reduce immunogenicity of antibodies originally derived from other species, like mouse. This can be done by construction of chimeric antibodies, or by a process called “humanization”. In this context, a “chimeric antibody” is understood to be antibody comprising a sequence part (e.g., a variable domain) derived from one species (e.g., mouse) fused to a sequence part (e.g., the constant domains) derived from a different species (e.g., human). A “humanized antibody” is an antibody comprising a variable domain originally derived from a non-human species, wherein certain amino acids have been mutated to make the overall sequence of that variable domain more closely resemble to a sequence of a human variable domain. Methods of chimerization and humanization of antibodies are well-known in the art (Billetta R, Lobuglio AF. Int. Rev. Immunol. 1993; 10:165-76; Riechmann L, Clark M, Waldmann H, Winter G (1988). Nature: 332:323).
Furthermore, technologies have been developed for creating antibodies based on sequences derived from the human genome, for example by phage display or use of transgenic animals (WO 90/05144; D. Marks, H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D. Griffiths and G. Winter (1991) J. Mol. Biol., 222, 581-597; Knappik et al., J. Mol. Biol. 296: 57-86, 2000; S. Carmen and L. Jermutus, Briefings in Functional Genomics and Proteomics 2002 1(2): 189-203; Lonberg N, Huszar D. Int Rev Immunol. 1995; 13:65-93; Bruggemann M, Taussig M J. Curr Opin Biotechnol. 1997 August; 8:455- 458). Such antibodies are “human antibodies” in the context of the present invention.
Antibody can also include fragments of immunoglobulins which retain antigen binding properties, like Fab, Fab', or F(ab')2 fragments. Such fragments may be obtained by fragmentation of immunoglobulins e.g., by proteolytic digestion, or by recombinant expression of such fragments. For example, immunoglobulin digestion can be accomplished by means of routine techniques, e.g., using papain or pepsin (WO 94/29348). Papain digestion of antibodies typically produces two identical antigen binding fragments, so- called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields an F(ab')2. In Fab molecules, the variable domains are each fused to an immunoglobulin constant domain, preferably of human origin. Thus, the heavy chain variable domain may be fused to a CHI domain (a so-called Fd fragment), and the light chain variable domain may be fused to a CL domain. Fab molecules may be produced by recombinant expression of respective nucleic acids in host cells, see below.
Several technologies have been developed for placing variable domains of immunoglobulins, or molecules derived from such variable domains, in a different molecular context. Those should be also considered as “antibodies” in accordance with the present invention. In general, these antibody molecules are smaller in size compared to immunoglobulins and may comprise a single amino acid chain or several amino acid chains. For example, a single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine (S) or glycine (G) (WO 88/01649; WO 91/17271; Huston et al; Intemat. Rev. Immunol. 10, 1993, 195-217). “VHH”, or “Single domain antibodies” or “nanobodies” harbor an antigen-binding site in a single Ig-like domain (WO 94/04678; WO 03/050531, Ward et al., Nature. 1989 Oct. 12; 341:544-546; Revets et al., Expert Opin Biol Then 5:111-24, 2005). One or more single domain antibodies with binding specificity for the same or a different antigen may be linked together. Diabodies are bivalent antibody molecules consisting of two amino acid chains comprising two variable domains (WO 94/13804, Holliger et al., Proc. Natl. Acad. Sci. USA. 1993; 90:6444-8). Other examples of antibody-like molecules are immunoglobulin super family antibodies (IgSF; Srinivasan and Roeske, Current Protein Pept. Sci. 2005, 6: 185-96). A different concept leads to the so- called Small Modular Immunopharmaceutical (SMIP) which comprises a Fv domain linked to single-chain hinge and effector domains devoid of the constant domain CHI (WO 02/056910).
Bispecific Binding Molecules
The present disclosure provides bispecific binding molecules having at least one antigen binding domain (a first antigen binding domain) that binds specifically to the extracellular domain of MUC16 and at least one antigen binding domain (a second antigen binding domain) that binds specifically to DR5. The bispecific binding molecules provided can cause apoptosis of MUC16-expressing cells that also express DR5. The second antigen binding domain, when bound to DR5, stimulates the apoptotic activity of DR5. The first antigen binding domain creates specificity enhancing the stimulated apoptotic activity of DR5 to MUC16-expressing cells. Without being bound by theory, applicant believes that the ability of the first antigen binding domain to bind the extracellular domain of MUC16 and, therefore, to potentiate the agonistic effect caused by the second, anti-DR5, antigen binding domain, is critical. MUC16 is a highly glycosylated protein having 14,507 amino acids. It consists of a 14,451 amino acid extracellular domain, a 21 amino acid transmembrane domain and a 35 amino acid C-terminal cytoplasmic domain. As disclosed herein, applicant has shown that a bispecific antibody comprising an anti-MUC16 antibody targeting multiple MUC16 epitopes present in the extracellular domain of MUC16 within its TR domain, is effective in potentiating the agonistic effect of the second, anti-DR5 binding domain. Applicant believes that this potentiating effect stems from clustering DR5 on the cell surface by multiple bispecific antibody molecules bound to the same MUC16 molecule. Accordingly, Applicant believes that a bispecific antibody comprising an anti- MUC16 antibody targeting a unique MUC16 epitope (i.e., an epitope only present once per MUC16 molecule) outside of the MUC16 TR/SEA domain, will be inefficient in potentiating the agonistic effect of the second, anti-DR5 binding domain. Furthermore, the ability of MUC16 to potentiate the agonistic effect caused by the binding of the second antigen binding domain to DR5 is not common to other cell surface proteins. For example, an antibody targeting CD44v6 (a splice variant of CD44 that is known to be a tumor associated antigen with a preferential expression pattern in tumor over normal tissues) did not potentiate the agonistic effect of the death receptor binding molecule when it is fused to an anti-DR5 antibody. See United States Patent No. 10,858,438.
Also, applicant’s own experiments demonstrate that: a) a bispecific binding molecule that bound both CD38 and DR5 was both unable to kill cells that express both antigens; and b) a bispecific binding molecule that binds the cell surface antigen LIV-1 and DR5 binding site was ineffective in killing cells that express both LIV-1 and DR5. See Example 3. Based upon these results, the utility of MUC16 as an antigen able to potentiate DR5-mediated apoptosis by anti-MUC16/anti-DR5 bispecific binding molecules disclosed herein, could not have been reasonably expected. As stated above, until the present invention, the applicants were unaware of any binding molecules that could specifically bind to both DR5 and MUC16, much less even being suggested. Nonetheless, individually each protein and their associated genes, as well as antibodies specific for each, are known in the art and are well represented in biological databases.
In relation to the present invention, in some embodiments the bispecific binding molecules are derived from antibodies. Techniques for making bispecific binding molecules include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knobin-hole” engineering (see, e.g., US5731168). Bispecific binding molecules of the invention may also be made by engineering electrostatic steering effects for making antibody Fc- heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., US4676980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., Immunol., 148: 1547-1553 (1992)); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g. Gruber et al., I. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. I. Immunol. 147: 60 (1991).
As set forth above, the first antigen binding domain binds to the extracellular domain of MUC16. In some embodiments, the first antigen binding domain binds to an epitope present in one or more tandem repeat/SEA segments within the extracellular domain of MUC16. In some embodiments, the first antigen binding domain will be derived from an antibody known to bind the extracellular domain of MUC16. In some embodiments, the first antigen binding domain will have the same heavy and light chain CDRs as those present in an antibody that binds the extracellular domain of MUC16. In some embodiments, the first antigen binding domain will have the same heavy and/or light chain variable regions as those present in an antibody that binds the extracellular domain of MUC16. In some embodiments, the first antigen binding domain will have the same heavy and/or light chains as those present in an antibody that binds the extracellular domain of MUC16. Antibodies that bind the extracellular domain of MUC16 are known in the art and include, but are not limited to, OC125, Hl 85 (Invitrogen Cat No. MA5-11579), Mi l (American Tissue Culture Collection Accession No. PTA-6206), OV197 (Fujirebio Diagnostic), 5E11 (Millipore Sigma Cat No. MABC1608-25UG), AR9.6, H1H8794, VK-8, B43.13 (also known as Oregovomab), or 3A5 (Sofituzumab). These antibodies are described in one or more the following references: Aithal et al. Exp. Opin. Therapeutic Targets 2018: 22, 675-686; Bressan et al., Disease Markers 34 (2013) 257-267; White et al., Proteins, 2022;90:1210-1218; Argueso et al. Investigat. Ophthalmol. Visual Sci. 2003; 44, 2487-2495; Eric Nunez Aguilar “HARNESSING ANTIBODIES FOR TREATING PANCREATIC CANCER: AR9.6 - A MUC16 SPECIFIC MONOCLONAL ANTIBODY” A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology in the College of Science and Mathematics California State University, Fresno May 2021; Marcos-Silva et al. Glycobiology, 2015, vol. 25, no. 11, 1172-1182; United States Patent No. 7,989,595; and Chen et al. Cancer Res. 2007:67, 4924-4932.
In other embodiments, an antibody against MUC16 can be generated using standard monoclonal antibody techniques. For these newly generated antibodies, determining if they bind the TR/SEA portion of the extracellular domain may be achieved by methods known in the art. One way of making such determination is if the new MUC16 antibody is competed for binding to MUC16 or an extracellular fragment thereof by one or more of the known antibodies set forth above that bind to the TR/SEA portion of the extracellular domain of MUC16. Another way of determining if an anti-MUC16 antibody binds the TR/SEA portion of the extracellular domain is by ELISA or another well-known antigenantibody binding assay using a peptide or polypeptide or its derivative from the TR/SEA portion of the MUC16 extracellular domain as the antigen. These antigens include peptides or polypeptides consisting essentially of the amino acid sequence of at least one of SEQ ID NOs: 176-181, or an extracellular fragment of MUC16 isolated from the cell culture media of OVCAR-3 cells.
In certain embodiments, the first antigen binding domain comprises a heavy chain and a light chain, wherein the amino acid sequences of the heavy and light chain are derived from an antibody that binds the extracellular domain of MUC16 and the specificity for MUC16 is determined by CDRs present in each of the heavy and light chains. In certain embodiments, the amino acid sequence of the heavy and/or light chain differs from that of the antibody from which it derives to improve one or more properties for use in humans. Typically, these differences involve a conservative substitution of less than five amino acids. See Tables 5-8.
As set forth above, the second antigen binding domain binds to DR5. In certain embodiments, the second antigen binding domain is an amino acid sequence that is derived from an antibody that binds to DR5. In some embodiments the parent antibody also agonizes DR5. In some embodiments, the second antigen binding domain will have the same heavy and light chain CDRs as those present in an antibody that binds DR5. In some embodiments, the second antigen binding domain will have the same heavy and/or light chain variable regions as those present in an antibody that binds DR5.
Antibodies that bind DR5 are known in the art and include, but are not limited to,
Conatumumab (AMG655), Drozitumab (Apomab or PRO955780), Lexatumumab (HGS- TR2), LBY135, Tigatuzumab (CS-1008 or TRA-8), and DS-8273a. In certain embodiments, the amino acid sequence of the second antigen binding domain is derived from an scFv that binds to and agonizes DR5 and the specificity for DR5 is determined by CDRs present in the scFv.
In other embodiments, an antibody against DR5 can be generated using standard monoclonal antibody techniques. For these newly generated antibodies, determining if they bind DR5 may be achieved by methods known in the art. One way of making such determination is if the new DR5 antibody is competed for binding to DR5 by one or more of the known antibodies set forth above that bind to DR5. Another way of determining if an anti-DR5 antibody binds DR5 is if it is competed for DR5 binding by TRAIL or a fragment or a derivative of TRAIL that binds to DR5. Still another way of determining if an antibody binds to DR5 is by ELISA or another well-known antigen-antibody binding assay using DR5 or a peptide or polypeptide fragment of DR5 as the antigen.
In some embodiments, the bispecific binding molecule comprises two polypeptide chains each of which comprises a variable heavy chain region and heavy chain CDRs, and two polypeptide chains each of which comprises a variable light chain region and light chain CDRs, wherein the combination of such polypeptide chains causes the bispecific molecule to bind specifically to the TR/SEA region of MUC16. In some embodiments, the two polypeptide chains each comprising a variable heavy chain region have identical amino acid sequences. In some embodiments, the two polypeptide chains each comprising a variable light chain region have identical amino acid sequences. In some embodiments, the combination of the two variable heavy chain regions (or the CDRs therein) and the two variable light chain regions (or the CDRs therein) define the first antigen binding domain. In some embodiments, the polypeptide chains comprising the variable heavy chain region are derived from the heavy chain of an antibody that binds to the TR/SEA region of MUC16. In some embodiments, the polypeptide chains comprising the variable light chain region are derived from the light chain of an antibody that binds to the TR/SEA region of MUC16. In some embodiments, both the polypeptide chains comprising the variable heavy chain region and the polypeptide chains comprising the light chain region are derived from the same MUC16 antibody.
In some embodiments the polypeptide chains that comprise a MUC16-specific variable heavy chain region further comprise the second antigen binding domain. In some embodiments the polypeptide chains that comprise a MUC16-specific variable light chain region further comprise the second antigen binding domain.
The location of the second antigen binding domains with respect to the first antigen binding domain can vary and includes all of the following: 1) the N-terminus of the second antigen binding domain is bound to the C-terminus of the heavy chain of the first antigen binding domain directly or through a peptide linker; 2) the N-terminus of the second antigen binding domain bound to the C-terminus of the light chain of the first antigen binding domain directly or through a peptide linker; 3) the N-terminus of the heavy chain of the first antigen binding domain bound to the C-terminus of the second antigen binding domain directly or through a peptide linker; 4) the N-terminus of the light chain of the first binding antigen site bound to the C-terminus of the second antigen binding domain directly or through a peptide linker; 5) the second antigen binding domain bound to an amino acid other than the C- or N-terminal amino acid of the heavy chain of the first antigen binding domain directly or through a linker (e.g., by reaction with a side group of such heavy chain amino acid); 6) the second antigen binding domain bound to an amino acid other than the C- or N-terminal amino acid of the light chain of the first antigen binding domain directly or through a linker (e.g., by reaction with a side group of such light chain amino acid). In some specific embodiments, the N-terminus of the second antigen binding domain is bound to the C-terminus of the heavy chain of the first antigen binding domain directly or through a peptide linker of between 4 and 20 amino acids.
Although specific amino acid sequences are set forth herein for the variable light chain region, the variable heavy chain region, the light chain, and the heavy chain of the first antigen binding moiety, as well as the scFv of the second antigen binding moiety, the disclosure further includes variants thereof. Such a variant differs from a specified portion of a bispecific binding molecule described herein by one or more suitable amino acid residue alterations, that is substitutions, deletions, insertions, and/or terminal sequence additions, for instance in the constant domain, and/or the variable regions (or any one or more CDRs thereof). A variant of a VL, VH, HC, LC or scFv region of a bispecific binding molecule of the invention retains at least a substantial proportion (a KD that at least about 100-fold greater or 10-fold greater or less) of the affinity/avidity and/or the specificity/selectivity of the parent antibodies to either MUC16 or to DR5 (i.e., a KD that at no more than about 100-fold greater, no more than 50-fold greater, no more than 40-fold greater, no more than 30-fold greater, no more than 20-fold greater, no more than 10-fold greater or less than 10-fold greater). In some cases such variant sequences may be associated with greater affinity, selectivity and/or specificity (i. e. , a lower KD) than the parent antibody to either of the antigens.
Such functional variants have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the referenced portion of a specific sequence at the amino acid level.
The sequence of variants may differ from the sequence of the parent antibody sequences through mostly conservative substitutions; for instance, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more (e.g., about 65-99%) of the substitutions in the variant are conservative amino acid residue replacements. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following tables:
Table 5. Amino acid residue classes for conservative substitutions: type of Amino Acid
Residue Amino Acids that may be substituted for one another.
Figure imgf000042_0001
Table 6. Alternative conservative amino acid residue substitution classes: groupings of Amino Acid Residues Amino Acids that may be substituted for one another.
Figure imgf000042_0002
Table 7. Alternative Physical and Functional Classifications of Amino Acid Residues: type of Amino Acid Residue Amino Acids that may be substituted for one another.
Figure imgf000043_0001
Table 8. Most Conservative Amino Acid Substitutions: groupings of Amino Acid Residues Amino Acids that may be substituted for one another.
Figure imgf000043_0002
As explained above, the amino acid sequence alterations should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to disrupt secondary structure that characterizes the function of the parent sequence), but may be associated with advantageous properties, such as changing the functional or pharmacokinetic properties of the antibodies, for example increasing the halflife, altering the immunogenicity, providing a site for covalent or non-covalent binding to another molecule, reducing susceptibility to proteolysis, reducing susceptibility to oxidation, reducing susceptibility to deamidation, or altering the glycosylation pattern. In most embodiments, the amino acid alterations will not be in the CDR regions, unless the change is such that the CDRs defined by at least one convention are maintained.
In some embodiments, the CDRs of the bispecific binding molecules set forth herein are based on those defined in the prior art disclosing the anti-MUC16 antibody or the DR5 antibody from which this binding moiety was derived. Applicant has indicated which specific CDR convention was used to elucidate these CDRs (e.g., see Tables 1 and 3). Those of ordinary skill in the art will understand that given the amino acid sequence of a variable region of the bispecific binding molecules set forth herein can easily determine the CDR sequences therein by these or any other conventions used to define CDR, such as, for example, IMGT, Kabat, Chothia, or Contact using web-based tools are available for determining the CDRs in such variable regions based on any known convention. Such tools include those found at www. abysis.org/ abysis/sequence_input/key_annotation/key_annotation. cgi and at www.novoprolabs.com/tools/cdr. Accordingly, the disclosure herein of a set of three CDRs in a heavy or light chain variable region based upon one CDR convention is considered the equivalent of that same set of CDRs as determined by any other convention.
In certain embodiments, the bispecific binding molecules disclosed herein comprise anti-MUC16 antibody constant regions or parts thereof. For example, a VL domain may have attached, at its C terminus, antibody light chain constant domains including human CK or CX chains. Similarly, a specific antigen-binding domain based on a VH domain may have attached all or part of an immunoglobulin heavy chain derived from any antibody isotope, e.g, IgG, IgA, IgE, and IgM and any of the isotope sub-classes, which include but are not limited to, IgGl and IgG4. The amino acid sequences of the C-terminal fragments of constant regions are well known in the art, as are DNA sequences that encode for such amino acids.
In more specific embodiments, the first antigen binding domain that binds specifically to MUC16 is an immunoglobulin (Ig) molecule (having the conventional Y shaped structure of a full-length antibody comprising two heavy and two light chains) and the second antigen binding domain that binds specifically to DR5 comprises at least one or more scFv binding elements.
In other more specific embodiments, the one or more scFvs specifically binding to DR5 is fused to the Ig molecule (e.g., human IgGl) specifically binding to MUC16 by a peptide linker. In some embodiments the peptide linker has a length of about 4 to 20 amino acids. In some more specific embodiments, the N-terminus of the scFv is fused to the C- terminus of the heavy chain of the Ig molecule or the C-terminus of the light chain of the Ig molecule. In some embodiments, the Ig molecule is an IgG.
Methods of linking scFv molecules to the C-terminus of the heavy or the light chain of the IgG molecule is well known in the art. Typically a small linker sequence comprising glycine and serine (termed a GS mini-linker) is used. The number of amino acids in the linker can vaiy from 4 (GGGS, SEQ ID NO.:221), 6 (GGSGGS, SEQ ID NO.:222), 10 (GGGGSGGGGS, SEQ ID NO.:223), 12 (GGGS GGGS GGGS, SEQ ID NO.:173), 15 (GGGGSGGGGSGGGGS, SEQ ID NO.:224) or more. In a more specific embodiment, the GS mini-linker between the scFv molecule and the C-terminus of the heavy chain of the IgG molecule is GGGSGGGSGGGS (SEQ ID NO: 173).
In some embodiments, the present invention provides a bispecific binding molecule comprising (i) an Ig molecule that specifically binds to MUC16 comprising two heavy and two light chains, and (ii) two scFv molecules, each specifically binding to DR5. In a more specific embodiment, each heavy chain of the Ig molecule has one scFv molecule fused to its C-terminus, thereby forming a bispecific tetravalent binding protein.
In some embodiments, the present invention provides a bispecific binding molecule comprising:
(i) two heavy chains, each comprising in N- to C-terminus order: a heavy chain variable domain specific for MUC16 (e.g., murine, humanized, or human VH domain); constant domains of an IgG (e.g., human IgGl); a peptide linker (e.g., a GS mini linker); and a scFv specific for DR5; and
(ii) two light chains, each comprising in N to C-terminus order: a light chain variable domain specific for MUC16 (e.g., murine, humanized or human VL domain); and a light chain constant domain (e.g., a human kappa chain).
Methods for generating bispecific binding molecules
Using methods known in the art and described herein it would be routine for the person ordinarily skilled in the art to prepare the bispecific binding molecules described herein. Isolation of the binding domains from antibodies (e.g., from an anti-MUC16 antibody or an anti-DR5 antibody) is a routine practice and indeed further information on methods that can be used to generate antibodies and binding molecules as described herein are provided in the accompanying examples. Activity of Bispecific Molecules and Assays Therefor
The binding activity of the bispecific binding molecules disclosed herein (as well as for the parental MUC16 and DR5 antibodies from which these bispecific molecules may be derived), can be measured using various methods. One method is enzyme-linked immunosorbent assay (ELISA). ELISA is a biochemistry assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. Antigens from the sample are attached to a surface. Then, a further specific antibody or bispecific binding molecule is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. Fluorescence-activated cell sorting (FACS), also called flow cytometry, provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. In these assays, the ECso is the concentration of the antibody or bispecific binding molecule that induces a response halfway between the baseline and maximum after some specified exposure time on a defined concentration of antigen by ELISA (enzyme-linked immuno-sorbent assay) or cell expressing the antigen by FACS. Surface plasmon resonance is a label-free method of determining binding activity wherein the binding of a molecule in the soluble phase (the “analyte”) is directly measured to a “ligand” molecule immobilized on a sensor surface. In the sensor device the binding of the ligand is monitored by an optical phenomenon termed surface plasmon. In particular, when the “analyte” molecule dissociates from the “ligand” molecule, a decrease in SPR signal (expressed in resonance units, RU) is observed. Association (‘on rate’, ka) and dissociation rates (‘off rate’, ko) are obtained from the signal obtained during the association and dissociation and the equilibrium dissociation constant (‘binding constant’, KD) can be calculated therefrom. The signal given in resonance units (RU) depends on the size of the ligand present in the analyte, however in case the experimental conditions are the same, i.e. the ligand is the same molecule at the same condition the obtained RU can indicate affinity, wherein the higher the obtained signal in RU the higher the binding. Affinity may be expressed for example in half-maximal effective concentration (ECso) or the equilibrium dissociation constant (KD). “Half maximal effective concentration” also called “ECso” refers to the concentration of a drug, antibody or toxicant which induces a response, such as binding or a cytotoxicity effect halfway between the baseline and maximum after a specified exposure time. ECso and affinity are inversely related, the lower the ECso value the higher the affinity of the antibody. In one aspect, the binding molecule of the present invention binds to the MUC16 or DR5 target antigens with a KD value ranging from 1 pM to 100 pM, preferably 1 pM to 1 pM, as determined e.g., by ELISA or by surface plasmon resonance analysis (Malmqvist M., Curr. Opin. Immunol. 1993 5:282-6.). Antibody or bispecific binding molecule affinity can also be measured using kinetic exclusion assay (KinExA) technology (Darling, R. J., and Brault P-A. Assay and Drug Development Technologies. 2004, 2: 647-657).
The ability of the bispecific binding molecules disclosed herein to cause cell death or apoptosis in MUC16+/DR5+ cells, may be detected by LIVE/DEAD Cell Viability Assay (Thermo Fisher Scientific) or a similar assay, or by CellTiter-Glo assay (Promega) or a similar assay, or by AlamarBlue and/or inducing inhibition of proliferation of MUC16- expressing cells in the range of concentrations between 1 pM and 1 pM; typically, between 10 nM and 0.01 nM.
In some embodiments, the bispecific molecules of the invention induce cell death, wherein at least 30% of the MUC16+/DR5+ cells in a sample or a patient undergo cell death. In some aspects of these embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells undergo cell death.
In one embodiment, the bispecific binding molecules of the present invention can induce DR5-mediated apoptosis in one or more cancer cell types that are MUC16+/DR5+, such as the cancer cell line OVCAR-3, with more than 50% inhibition of cell viability at a concentration of 3 nM or less.
The bispecific binding molecules described herein induce apoptosis in cancer cells and therefore can be used in the therapy of cancers which express both MUC16 and DR5. Methods of identifying whether a particular tumor expresses MUC16 and DR5 are well known in the art. For example, immunohistochemistry can be used to determine whether tumor tissue expresses MUC16 and DR5 (e.g., using the bispecific binding molecules described herein or any known anti-MUC16 and/or anti-DR5 antibody) and hence would be suitable for treatment with the binding molecule of the invention.
Nucleic Acids. Cloning, and Expression Systems
The bispecific binding molecules disclosed herein may be produced through the use of nucleic acid sequences that encode the amino acid sequences thereof. The nucleic acids sequences may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence and encompasses an RNA molecule with the specified sequence in which U is substituted for T unless context requires otherwise. In some aspects, the nucleotide sequence is an RNA sequence that contains one or more modified ribonucleic acids (e.g., N6-methyladenosine (m6A), pseudouridine (\|/), Nl- methylpseudouridine (ml\|/), or 5-methoxyuridine (5moU)). In some embodiments, the bispecific binding molecules disclosed herein are made up of four polypeptide chains. In some embodiments, the bispecific binding molecule disclosed herein are made up of two identical heavy chain fusions and two identical light chains, wherein each heavy chain fusion comprises the heavy chain of the first antigen binding domain (e.g., a MUC16 antibody that specifically binds to the extracellular domain of MUC16) linked by short amino acid linker to the scFv of the second antigen binding domain (a scFv that specifically binds DR5); and each light chain comprises the light chain of the first binding moiety (e.g., a MUC16 antibody that specifically binds to the extracellular domain of MUC16). In some embodiments, the bispecific binding molecule disclosed herein are made up of two identical heavy chains and two identical light chain fusions, wherein each light chain fusion comprises the light chain of the first antigen binding domain linked by short amino acid linker to the scFv of the second antigen binding domain; and each heavy chain comprises the heavy chain of the first antigen binding domain. For this section, the term “fusion” refers to a fusion of either a light or heavy chain of the first binding moiety directly or through a small peptide linker to the scFv of the second antigen binding domain; and the term “chain” refers to either a heavy or light chain, respectively, of the first binding domain.
In some embodiments of the present invention, an isolated nucleic acid sequence that encodes a fusion of the heavy chain of the first antigen binding domain linked by short amino acid linker to the scFv of the second antigen binding domain is provided, wherein the heavy chain of the first antigen binding domain is the amino acid sequence of any one of SEQ ID NOs: 115-118, the linker is the amino acid sequence of SEQ ID NO: 173 and the scFv of the second antigen binding domain is the amino acid sequence of SEQ ID NO: 172. In certain embodiments, this fusion has the amino acid sequence of SEQ ID NO: 174. In other embodiments, this fusion has the amino acid sequence of SEQ ID NO: 175. In other embodiments, this fusion has the amino acid sequence of SEQ ID NO: 186. In other embodiments, this fusion has the amino acid sequence of SEQ ID NO: 187. In some embodiments, the nucleic acid sequence that encodes the heavy chain fusion comprises the nucleic acid sequence of SEQ ID NO: 182, or a sequence having at least 70% identity thereto and encoding the same amino acid sequence encoded by SEQ ID NO: 182. In some more specific embodiments, the nucleic acid sequence that encodes the fusion comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 182 and encoding the same amino acid sequence encoded by SEQ ID NO: 182. In some embodiments, the nucleic acid sequence that encodes the fusion comprises the nucleic acid sequence of SEQ ID NO: 183, or a sequence having at least 70% identity thereto and encoding the same amino acid sequence encoded by SEQ ID NO: 183. In some more specific embodiments, the nucleic acid sequence that encodes the fusion comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 183 and encoding the same amino acid sequence encoded by SEQ ID NO: 183.
In some embodiments, a nucleic acid sequence encoding a chain and/or a fusion polypeptide of the bispecific binding molecule is constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired chain or fusion polypeptide and codon optimization based on the host cell preferences. Standard methods can routinely be applied to synthesize and isolate polynucleotide sequences encoding the chain or fusion polypeptide of the bispecific binding molecule.
In embodiments wherein the bispecific binding molecule comprises two identical copies of a chain and two identical copies of a fusion as described above (e.g., two identical copies of a heavy chain fusion and two identical copies of a light chain, or two identical copies of a light chain fusion and two identical copies of a heavy chain), the chain and the fusion must be separately expressed in a host cell. This is achieved by inserting the nucleotide sequences encoding each of the chain and the fusion into one or more expression vectors such that the sequences are operatively linked to transcriptional and translational control sequences. Such expression vectors containing an isolated nucleic acid sequence(s) disclosed herein are also part of the present invention. In some embodiments, the nucleotide sequences encoding the fusion and the chain are present in the same expression vector. In these embodiments, the fusion and chain-encoding nucleic acid sequences may be operatively linked to the same or different transcriptional and translational control sequences. In some embodiments, the nucleotide sequences encoding the fusion and the chain are present in different expression vectors. In these embodiments, the fusion and chain-encoding nucleic acid sequences may also be operatively linked to the same or different transcriptional and translational control sequences.
For manufacturing the binding molecules or antibodies of the invention, the skilled artisan may choose from a great variety of expression systems well known in the art, e.g. those reviewed by Kipriyanov and Le Gall, Curr. Opin. Drug Discov. Devel. 2004; 7:233- 242.
Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like. The expression vector and expression control sequences are selected to be compatible with the host cell. Convenient vectors are those that encode a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that a VH or VL-encoding nucleotide sequence disclosed herein can be easily inserted and expressed, as described above. The constant chain is usually kappa or lambda for the antibody light chain, for the antibody heavy chain, it can be, without limitation, any IgG isotype (IgGl, IgG2, IgG3, IgG4) or other immunoglobulins, including allelic variants.
The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain (e.g., the heavy and light chains of the binding molecules or antibodies described herein) from a host cell. The isolated nucleic acid sequence encoding the heavy or light chain may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the mature chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous peptide from a non-immunoglobulin protein. Alternatively, the nucleic acid sequence encoding the heavy or light chains of the binding molecules described herein may already contain a signal peptide sequence.
In addition to the nucleic acid sequences encoding the antibody chains (e.g., the heavy and light chains of the binding molecules or bispecific antibodies described herein), the recombinant expression vectors may carry regulatory sequences including promoters, enhancers, termination and polyadenylation signals and other expression control elements that control the expression of the antibody chains in a host cell. The choice of expression control sequence and expression vector will depend upon the choice of host. Examples for promoter sequences (exemplified for expression in mammalian cells) are promoters and/or enhancers derived from (CMV) (such as the CMV Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e. g., the adenovirus major late promoter (AdMLP)), polyoma, bovine papilloma virus, cytomegalovirus and strong mammalian promoters such as native immunoglobulin and actin promoters. Examples for polyadenylation signals are BGH poly A, SV40 late or early poly A; alternatively, 3'UTRs of immunoglobulin genes etc. can be used. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and fdamentous single-stranded DNA phages. The recombinant expression vectors may also carry sequences that regulate replication of the vector in host cells (e. g. origins of replication) and selectable marker genes. The recombinant expression vectors may also be viral vectors that are known in the art including but not limited to AAV, lentivirus, or other retroviral vectors.
Nucleic acid molecules encoding the heavy chain or an antigen-binding portion thereof and/or the light chain or an antigen-binding portion thereof of a binding molecule or antibody described herein, and vectors comprising these nucleic acid molecules can be introduced into host cells, e.g. bacterial cells or higher eukaryotic cells, e.g. mammalian cells, according to transfection methods well known in the art, including but not limited to liposome-mediated transfection, poly cation-mediated transfection, protoplast fusion, microinjections, calcium phosphate precipitation, electroporation or transfer by viral vectors. In some embodiments, one or both of the heavy and light chains is fused to the second antigen binding moiety directly or through an amino acid linker. In some embodiments, the nucleic acid molecules encoding the chain and the fusion polypeptides of the binding molecules described herein are present on two separate expression vectors that are co-transfected into the host cell, preferably a mammalian cell.
Hence, further embodiments provide a host cell comprising an expression vector comprising a nucleic acid molecule encoding a chain and an expression vector comprising a nucleic acid molecule encoding a fusion polypeptide of the binding molecules described herein.
Mammalian cell lines available as hosts for expression are well known in the art and include, inter alia, Chinese hamster ovary (CHO, CHO-DG44) cells, NSO, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human carcinoma cells (e. g., Hep G2), A549 cells, 3T3 cells, HEK-293 and HEK-293T cells, the COS-7 lines of monkey kidney cells described by Gluzman (Cell 23:175 (1981)), or the derivatives/progenies of any such cell line. Other mammalian cells, including but not limited to human, mice, rat, monkey and rodent cells lines, or other eukaryotic cells, including but not limited to yeast, insect and plant cells, or prokaryotic cells such as bacteria may be used. The binding molecules of the invention are produced by culturing the host cells for a period sufficient to allow for expression of the binding molecule in the host cells. In some embodiments, the invention relates to a recombinant eukaryotic or prokaryotic host cell, which harbors an expression vector set forth above. In some aspects of these embodiments, the host cell is eukaryotic. In some aspects the host cell is a mammalian host cell. In some aspects the mammalian host cell is a NSO murine myeloma cell, a PER.C6® human cell or a Chinese hamster ovary (CHO) cell. In some aspects the host cell is a hybridoma.
The disclosure also provides a method for making an anti-MUC16/anti-DR5 bispecific binding molecule comprising culturing a host cell (e.g., a hybridoma or transformed mammalian host cell) capable of expressing both the chain and fusion polypeptide of the bispecific binding molecule under suitable conditions and optionally provides a method for isolating the resulting expression products secreted from the host cell. And the disclosure additionally provides the bispecific binding molecule isolated using the disclosed methods. The bispecific binding molecules are preferably recovered from the culture medium as a secreted polypeptide or from host cell lysates if, for example, expressed without a secretory signal. It is necessary to purify the bispecific binding molecules described herein using standard protein purification methods used for recombinant proteins and host cell proteins in a way that substantially homogenous preparations of the binding molecule or antibody as described herein are obtained. By way of example, state-of-the art purification methods useful for obtaining the binding molecules and antibodies of the invention include, as a first step, removal of cells and/or particulate cell debris from the culture medium or lysate. The binding molecule or antibody is then purified from contaminant soluble proteins, polypeptides, and nucleic acids, for example, by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or on a cation exchange resin. As a final step in the process for obtaining a bispecific binding molecule as described herein, the purified binding molecule may be dried, e.g., lyophilized, as described below for therapeutic applications.
The disclosure also optionally provides the bispecific binding molecule antibody produced using this method and pharmaceutical compositions comprising the bispecific binding molecule antibody and a pharmaceutically acceptable carrier.
Pharmaceutical Compositions
The disclosure further provides a pharmaceutical composition comprising a bispecific binding molecule disclosed herein; and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a compound of the present invention.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical arts.
Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alphatocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetra acetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol, or sodium chloride.
The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives, or buffers, which may enhance the shelflife or effectiveness of the pharmaceutical composition. The compounds of the present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Methods for the preparation of such formulations are generally known to those skilled in the art. See e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
The bispecific binding molecules provided herein can be administered to a human or other subject in an amount sufficient to produce a therapeutic effect. Such “therapeutically effective amount” is the minimum amount necessary to prevent, ameliorate, or treat clinical symptoms of any of the diseases or conditions set forth below, in particular the minimum amount which is effective to ameliorate or treat these disorders. The actual dosage levels of the bispecific binding molecule in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the bispecific binding molecule effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The therapeutically effective amount of the bispecific binding molecules of the invention applicable per day is usually from 0.001 mg/kg to 100 mg/kg, preferably from 0.1 mg/kg to 20 mg/kg.
Generally, for the treatment and/or alleviation of the diseases, disorders and conditions mentioned herein and depending on the specific disease, disorder or condition to be treated, the potency of the bispecific binding molecule of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the antibody molecules of the invention will generally be administered in an amount between 0.005 and 20.0 mg per kilogram of body weight and dose, preferably between 0.05 and 10.0 mg/kg/dose, and more preferably between 0.5 and 10 mg/kg/dose, either continuously (e.g. by infusion) or more preferably as single doses. The administration interval may be, for example, twice a week, weekly, or monthly doses, but can significantly vary, especially, depending on the before-mentioned parameters. Thus, in some cases it may be sufficient to use less than the minimum dose given above, whereas in other cases the upper limit may have to be exceeded.
When administering large amounts, it may be advisable to divide them up into a number of smaller doses spread over the day. Administration may e.g., be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. If desired, the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
Depending on the specific binding molecule of the invention and its specific pharmacokinetic and other properties, it may be administered daily, every second, third, fourth, fifth or sixth day, weekly, monthly, and the like. An administration regimen could include long-term, weekly treatment. By “long-term” is meant at least two weeks and preferably months, or years of duration.
The actual pharmaceutically effective amount or therapeutic dosage will of course depend on factors known by those skilled in the art such as age and weight of the patient, route of administration and severity of disease. In any case the binding molecule of the invention will be administered at dosages and in a manner which allows a pharmaceutically effective amount to be delivered based upon patient's unique condition.
The pharmaceutical composition may be administered by any suitable route and mode. Suitable routes of administering a compound of the present invention in vivo and in vitro are well known in the art and may be selected by those of ordinary skill in the art. In some embodiments, the mode of application is parenteral, by infusion or injection (intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal), but other modes of application such as by inhalation, transdermal, intranasal, buccal, oral, may also be applicable. In some embodiments, a pharmaceutical composition of the present invention is administered parenterally.
In a further aspect, a bispecific binding molecule of the invention is used in combination with a device useful for its administration, such as a syringe, injector pen, micropump, or another device. In a further aspect, a binding molecule of the invention is comprised in a kit of parts, for example also including a package insert with instructions for the use of the binding molecule.
The efficacy of the binding molecules of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease involved. Suitable assays and animal models will be clear to the skilled person, and for example include the assays and animal models used in the Examples below.
The binding molecules of the invention may be used on their own or in combination with other pharmacologically active ingredients, such as state-of-the-art or standard-of-care compounds, such as e.g., cytostatic, or cytotoxic substances, cell proliferation inhibitors, anti-angiogenic substances, steroids, immune modulators/checkpoint inhibitors, and the like.
Hence a further aspect of the invention provides a pharmaceutical composition comprising a binding molecule of the invention, together with a pharmaceutically acceptable carrier and optionally one or more further active ingredients.
Therapeutic Uses
In some embodiments, the invention provides a method of treatment of a disease or disorder involving cells co-expressing MUC16 and DR5 comprising administering to a subject in need thereof, a bispecific binding molecule or bispecific binding moleculecontaining pharmaceutical composition of the invention. The disclosure provides methods for treating and/or ameliorating conditions associated with a MUC16-mediated activity in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bispecific binding molecule provided herein. In some embodiments, the bispecific binding molecule is administered alone. In other embodiments, the bispecific binding molecule is administered as a combination therapy. Also provided are methods of reducing MUC16 activity in a subject comprising administering an effective amount of a bispecific binding molecule to a subject in need thereof.
In some embodiments, the above method is used to treat a MUC16-related fibrotic disease disorder, inflammatory disorder, immune disorder, or autoimmune disorder. In some embodiments, the MUC16-mediated fibrotic disorder is pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, cirrhosis, kidney fibrosis, glial scar, myocardial fibrosis, arterial stiffness, arthrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, or adhesive capsulitis. In some aspects of these embodiments, the disorder to be treated is idiopathic pulmonary fibrosis.
In some embodiments, the above method is used to treat a cancer or malignancy characterized by over-expression of MUC16. In some embodiments, such cancer or malignancy is a gynecologic cancer (cancer of the female reproductive tract), including the cervix, endometrium, fallopian tubes, ovaries, uterus, and vagina; pancreatic cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, or lung cancer (such as non-small cell lung cancer). In some embodiments, the solid tumor to be treated is selected from ovarian and pancreatic tumors.
As used herein, the term "subject" is intended to include human and non-human animals, which respond to the bispecific antigen binding molecule. Subjects may for instance include human patients having disorders that may be corrected or ameliorated by modulating MUC16 function, such as enzymatic activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and/or induction of lysis and/or eliminating/reducing the number of MUC16 expressing cells.
For example, the bispecific binding molecule may be used to elicit in vivo or in vitro one or more of the following biological activities: modulating MUC16 function (such as apoptosis, permeabilization of plasma membrane, reduction in cell number, enzymatic activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and/or induction of lysis), killing a cell expressing MUC16, mediating phagocytosis or ADCC of a cell expressing MUC16 in the presence of human effector cells, and by mediating CDC of a cell expressing MUC16 in the presence of complement or by killing MUC16 expressing cells by apoptosis.
Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
An “effective amount” for cancer therapy is an amount sufficient to stabilize the progression of disease for a period of time. A therapeutically effective amount of an antibody of the invention may decrease tumor size, or otherwise ameliorate symptoms in a subject.
An “effective amount” for idiopathic pulmonary fibrosis may result in an improvement in any one or more accepted tests, such as changes from baseline in St. George's respiratory questionnaire SGRQ, dyspnea score, cough score and 6-minute walk test (grade and distance), changes in lung function (forced vital capacity (FVC); diffusing capacity of the lungs for carbon monoxide (DLCO)) compared with baseline, changes in abnormal values of routine safety tests (blood routine, urine routine, blood biochemistry, electrocardiogram (ECG), etc.) compared with baseline, changes in chest high-resolution computed tomography (HRCT) scores from baseline, changes of lung tumor markers from baseline, frequency and severity of acute exacerbations of IPF.
A bispecific binding molecule disclosed herein may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in patients wherein it is difficult to locate a tumor that is known to be present due to other biological factors.
A bispecific binding molecule disclosed herein may also be administered prophylactically in order to reduce the risk of developing a fibrotic disease or disorder.
The bispecific binding molecule disclosed herein may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Such administration may be simultaneous, separate, or sequential. For simultaneous administration, the agents may be administered as one compositions or as separate compositions, as appropriate.
Kits comprising anti-MUC16/anti-DR5 antibodies
This disclosure further provides kits that include a bispecific binding molecule disclosed herein (including variants and derivatives thereof) in suitable packaging, and written material and that can be used to perform the methods described herein. The written material can include any of the following information: instructions for use, discussion of clinical studies, listing of side effects, scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like. The written material can indicate or establish the activities and/or advantages of the composition, and/or describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information can be based on the results of various studies, for example, studies using experimental animals involving in vivo models and/or studies based on human clinical trials. The kit can further contain another therapy (e.g., another agent) and/or written material such as that described above that serve to provide information regarding the other therapy (e.g., the other agent).
In certain embodiments, a kit comprises at least one purified bispecific binding molecule disclosed herein in one or more containers. In some embodiments, the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and/or any necessary software for analysis and presentation of results.
Unless otherwise indicated, the practice of the disclosure employs conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Sequences:
(SEQ ID NO: 1) NDYAWN
(SEQ ID NO: 2) YISYSGYTTYNPSLKS
(SEQ ID NO: 3) YINYSGYTTYNPSLKS
(SEQ ID NO: 4) YINYAGYTTYNPSLKS
(SEQ ID NO: 5) YISYAGYTTYNPSLKS
(SEQ ID NO: 6) WTSGLDY
(SEQ ID NO: 7) WDGGLTY
(SEQ ID NO: 8) WAAGLTN
(SEQ ID NO: 9) WDAGLSY
(SEQ ID NO: 10) WDAGLTY
(SEQ ID NO: 11) WEAGLNH
(SEQ ID NO: 12) WEAGLNY
(SEQ ID NO: 13) WMAGLSD
(SEQ ID NO: 14) WSAGLDH
(SEQ ID NO: 15) WTAGLDY
(SEQ ID NO: 16) WTAGLTH
(SEQ ID NO: 17) WVAGLTN
(SEQ ID NO: 18) WAGGLEN
(SEQ ID NO: 19) WDGGLSY
(SEQ ID NO: 20) WDRGLTY
(SEQ ID NO: 21) WASGLSH
(SEQ ID NO: 22) WASGLSN
(SEQ ID NO: 23) WASGLSY
(SEQ ID NO: 24) WASGLTH
(SEQ ID NO: 25) WASGLTN
(SEQ ID NO: 26) WDSGLKY
(SEQ ID NO: 27) WDSGLNY
(SEQ ID NO: 28) WDSGLSS
(SEQ ID NO: 29) WDSGLSV
(SEQ ID NO: 30) WDSGLSY
(SEQ ID NO: 31) WDSGLTY
Figure imgf000061_0001
(SEQ ID NO: 64) GATSLET
(SEQ ID NO: 65) QQYWTTPFT
(SEQ ID NO: 66) [VK-8 H-CDR1 (from WO 2003076465)]
DYNMH
(SEQ ID NO: 67) [VK-8 H-CDR2]
YIYPYNGDTGYNQKFRN
(SEQ ID NO: 68) [VK-8 H-CDR3]
SGGFWYFDV
(SEQ ID NO: 69) [VK-8 L-CDR1]
RATPSVSYMH
(SEQ ID NO: 70) [VK-8 L-CDR2]
TTSNLAS
(SEQ ID NO: 71) [VK-8 L-CDR3]
QQWSRSPPT
(SEQ ID NO: 72) [OC125 CDR H-1 (from WO 2003076465)]
SYWMH
(SEQ ID NO: 73) [OC125 CDR H-2]
AIYPGNSDTSYNQKFKG
(SEQ ID NO: 74) [OC125 CDR H-3]
SYDWYFDV
(SEQ ID NO: 75) [OC125 CDR L-l]
RASQSIGTDMH
(SEQ ID NO: 76) [OC125 CDR L-2]
YASESIS
(SEQ ID NO: 77) [OC125 CDR L-3]
QQSYSWPLT
(SEQ ID NO: 78) [AR9.6 CDR H-l (from Eric Nunez Aguilar “HARNESSING ANTIBODIES FOR TREATING PANCREATIC CANCER: AR9.6 - A MUC16 SPECIFIC MONOCLONAL ANTIBODY” A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology in the College of Science and Mathematics California State University, Fresno May 2021)]
GFTFSTF (SEQ ID NO: 79) [AR9.6 CDR H-2]
SSGSST
(SEQ ID NO: 80) [AR9.6 CDR H-3]
SGYDYDPIYYALDY
(SEQ ID NO: 81) [AR9.6 CDR L-l]
RASESVDNYGISFMN
(SEQ ID NO: 82) [AR9.6 CDR L-2]
GASNQGS
(SEQ ID NO: 83) [AR9.6 CDR L-3]
QQTKEVPWT
(SEQ ID NO: 84) [H1H8794 CDR H-1 (From US 10738130)]
GFTFRDYS
(SEQ ID NO: 85) [H1H8794 CDR H-2]
VTFFNSAI
(SEQ ID NO: 86) [H1H8794 CDR H-3]
AREREPIVGGFDY
(SEQ ID NO: 87) [H1H8794 CDR L-l]
QSINSY
[H1H8794 CDR L-2]
AAS
(SEQ ID NO: 89) [H1H8794 CDR L-3]
QQSYSSPPIT
(SEQ ID NO: 90) [B43.13 CDR-H1 (derived from Sharm et al. 2014 Protein Expression and Purification 102:27-37]
NYWMN
(SEQ ID NO: 91) [B43.13 CDR-H2]
QIVPGGGDPNYNGKFKG
(SEQ ID NO: 92) [B43.13 CDR-H3]
WAHSYAMDY
(SEQ ID NO: 93) [B43.13 CDR-L1 vl]
KSSQSLLNSSTQKNYLA
(SEQ ID NO: 94) [B43.13 CDR-L1 v2]
Figure imgf000064_0001
QVQLQQSGAELVRPGSSVKISCKASDYAFSNYWMNWVKQRPGKGLEWIG QIVPGGGDPNYNGKFKGKATLTADKSSSTAYMQLSRLTSEDSAVYFCARWAHSYA MDYWGQGTSVTVSS
(SEQ ID NO: 104)
DIQMTQSPSSLSASVGDRVTITCKASDLIHNWLAWYQQKPGKAFKLLIYGATSLET
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWTTPFTFGQGTKVEIK
(SEQ ID NO: 105)
DIQMTQSPSSLSASVGDRVTITCKASDLIHNWLAWYQQKPGKAPKLLIYGATSLET
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWTTPFTFGQGTKVEIK
(SEQ ID NO: 106) [VK-8 VL vl]
DIQMTQSPAILSASPGEKVTMTCRATPSVSYMHWYQQKPGSSPKPWIYTTS
NLASGVPARFSGGGSGTSYSLTVSRVEAEDAATYYCQQWSRSPPTFGAGTKLEIK
(SEQ ID NO: 107) [VK-8 VL v2]
DIQMTQSPAILSASPGEKVTMTCRATPSVSYMHWYQQKPGSSPKPWIYTTS
NLASGVPARFSGGGSGTSYSLTVSRVEVEDAATYYCQQWSRSPPTFGAGTKLEIK
(SEQ ID NO: 108) [VK-8 VL v3]
DIQMTQSPAILSASPGEKVTMTCRATPSVSYMHWYQQKPGSSPKPLIYTTSN
LASGVPARFSGGGSGTSYSLTVSRVEAEDAATYYCQQWSRSPPTFGAGTKLEIK
(SEQ ID NO: 109) [VK-8 VL v4]
DIQMTQSPAILSASPGEKVTMTCRATPSVSYMHWYQQKPGSSPKPLIYTTSN
LASGVPARFSGGGSGTSYSLTVSRVEVEDAATYYCQQWSRSPPTFGAGTKLEIK
(SEQ ID NO: 110) [OC125 VL]
DIELTQSPAILSVSPGERVSFSCRASQSIGTDMHWYQQRTNGSPRLLIKYASE
SISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSYSWPLTFGAGTKLEIK
(SEQ ID NO: 111) [AR9.6 VL]
EIVLTQSPATLSVSPGERATLSCRASESVDNYGISFMNWYQQKPGQAPRLLI YGASNQGSGIPARFSGSGSGTDFTLTISSLEPEDAAVYYCQQTKEVPWTFGGGTKV EIK
(SEQ ID NO: 112) [H1H8794 VL]
DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAAS SLQGGVPSRFSGGGSGTDFTLTITSLQPEDFATFYCQQSYSSPPITFGQGTRLEIK
(SEQ ID NO: 113) [B43.13 VL vl]
Figure imgf000066_0001
Figure imgf000067_0001
QGDSLRSYYAS
(SEQ ID NO: 125) [Lexatumumab LC CDR2] GKNNRPS
(SEQ ID NO: 126) [Lexatumumab LC CDR3] NSRDSSGNHVV
(SEQ ID NO: 127) [Conatumumab CDR-H1] SGDYFWS
(SEQ ID NO: 128) [Conatumumab CDR-H2]
HIHNSGTTYYNPSLKS
(SEQ ID NO: 129) [Conatumumab CDR-H3] DRGGDYYYGMDV
(SEQ ID NO: 130) [Conatumumab CDR-L1] RASQGISRSYLA
(SEQ ID NO: 131) [Conatumumab CDR-L2] GASSRAT
(SEQ ID NO: 132) [Conatumumab CDR-L3] QQFGSSPWT
(SEQ ID NO: 133) [Drozitumab CDR-H1]
DYAMS
(SEQ ID NO: 134) [Drozitumab CDR-H2]
GINWQGGSTGYADSVKG
(SEQ ID NO: 135) [Drozitumab CDR-H3] ILGAGRGWYFDY
(SEQ ID NO: 136) [Drozitumab CDR-L1]
SGDSLRSYYAS
(SEQ ID NO: 137) [Drozitumab CDR-L2]
GANNRPS
(SEQ ID NO: 138) [Drozitumab CDR-L3] NSADSSGNHVV
(SEQ ID NO: 139) [Tigatuzumab CDR-H1] SYVMS
(SEQ ID NO: 140) [Tigatuzumab CDR-H2] TISSGGSYTYYPDSVKG
(SEQ ID NO: 141) [Tigatuzumab CDR-H3] RGDSMITTDY
(SEQ ID NO: 142) [Tigatuzumab CDR-L1] KASQDVGTAVA
(SEQ ID NO: 143) [Tigatuzumab CDR-L2] WASTRHT
(SEQ ID NO: 144) [Tigatuzumab CDR-L3] QQYSSYRT
(SEQ ID NO: 145) [DS-8273a CDR-H1]
GYFMN
(SEQ ID NO: 146) [DS-8273a CDR-H2]
RFNPYNEDTFYNQKFKG
(SEQ ID NO: 147) [DS-8273a CDR-H3] SAYYFDSGGYFDY
(SEQ ID NO: 148) [DS-8273a CDR-L1]
RSSQSLVHSNKNTYLH
(SEQ ID NO: 149) [DS-8273a CDR-L2]
KVSNRFS
(SEQ ID NO: 150) [DS-8273a CDR-L3]
SQSTHVPWT
(SEQ ID NO: 151) [LBY135 CDR-H1]
DYTIH
(SEQ ID NO: 152) [LBY135 CDR-H2]
WFYPGGGYIKYNEKFKD
(SEQ ID NO: 153) [LBY135 CDR-H3]
HEEGIYFDY
(SEQ ID NO: 154) [LBY135 CDR-L1]
KASQDVNTAIA
(SEQ ID NO: 155) [LBY135 CDR-L2]
WASTRHT
(SEQ ID NO: 156) [LBY135 CDR-L3 vl]
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
( Q ) [ ]
Figure imgf000085_0001
(SEQ ID NO:218) [Lexatumumab VH variant 1 - G112C mutation]
EVQLVQSGGGVERPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVS GINWNGGSTGYADSVKGRVTISRDNAKNSLYLQMNSLRAEDTAVYYCAKILGAG RGWYFDLWCKGTTVTVSS
(SEQ ID NO:219) [Lexatumumab VH variant 2 - K113C mutation]
EVQLVQSGGGVERPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVS GINWNGGSTGYADSVKGRVTISRDNAKNSLYLQMNSLRAEDTAVYYCAKILGAG RGWYFDLWGCGTTVTVSS
(SEQ ID NO:220) [Lexatumumab VH variant 3 - G44C mutation]
EVQLVQSGGGVERPGGSLRLSCAASGFTFDDYGMSWVRQAPGKCLEWVSG INWNGGSTGYADSVKGRVTISRDNAKNSLYLQMNSLRAEDTAVYYCAKILGAGR GWYFDLWGKGTTVTVSS
EXAMPLES
The invention is now described by way of the following non-limiting examples, which explain the methods used to generate the bispecific molecule that binds MUC16 and DR5 as well as variations in their format, and the biological activity of these molecules.
Example 1: Design of binding molecules recognizing MUC16 and DR5
The present inventors have developed binding molecules that bind both MUC16 and DR5 and that induce cell death in cancer cells expressing both MUC16 and DR5. The molecular design used in these experiments has an IgG antibody that has specificity for MUC16, with a scFv that has specificity for DR5 coupled to the C terminus of the heavy chain of the IgG through a small peptide linker. A schematic representation of the design is shown in FIG. 1.
To test feasibility of this concept, four bispecific antigen binding molecules based on the format depicted in FIG. 1 were prepared (IMV-18, MCLX-SE, IMV-AA, and MC- AA) using methods well known to the skilled in the art. Each of these bispecific antigen binding molecules had the same anti-MUC16 CDRs and anti-DR5 CDRs. The anti-MUC 16 CDRs were: CDR-H1 - SEQ ID NO:1; CDR-H2 - SEQ ID NO:2; CDR-H3 - SEQ ID NO:6; CDR-L1 - SEQ ID NO:62; CDR-L2 - SEQ ID NO:63; and CDR-L3 - SEQ ID NO:65. The anti-DR5 CDRs were: CDR-H1 - SEQ ID NO: 121; CDR2-H2 - SEQ ID NO: 122; CDR-H3 - SEQ ID NO: 123; CDR-L1 -SEQ ID NO: 124; CDR-L2 - SEQ ID
NO: 125; and CDR-L3 - SEQ ID NO: 126.
The SEQ ID NOs for the light chain and the heavy chain fused to the scFv of these molecules, as well as additional sequence information are provided below in Table 9.
Table 9. Sequence NOs. of the heavy chain fusion and the light chain of exemplary bispecific antigen binding molecules
Figure imgf000087_0001
Figure imgf000088_0001
We also designed other bispecific molecules that targeted DR5 and a cell surface antigen different from MUC16, as well as monospecific antibodies to compare activities. See Table 10. All of these other bispecific molecules comprised the same anti-DR5 scFv.
Table 10. Comparator Bispecific Molecules and Monospecific Antibodies
Figure imgf000088_0002
Figure imgf000089_0001
Example 2. Expression and purification of bispecific tetravalent molecules recognizing human MUC16, or another antigen, and human DR5
Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies or immunoglobulin chains, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, Calif) and following the manufacturer's instructions. Alternatively, antibodies, immunoglobulin chains and antibody -like binding proteins of the invention can be synthesized by recombinant DNA techniques as is well- known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.
The expression vectors were constructed, and transfection of cells and antibody production and purification were carried out by methods well known in the art. Genes for both the heavy chain-scFv fusion and the light chain of the bispecific binding compounds set forth above were designed and optimized for expression in CHO cells, and the sequences were introduced into a protein expression vector of BioIntron, Building 5, No.388 Galileo Road, Zhangjiang High-Tech Park, Shanghai, China. CHO cells were transiently transfected with the expression vector and cultured for 5-7 days. Cultures were then harvested, and a two-step purification was performed. Recombinant binding molecules or antibodies were purified from culture supernatant by Protein A affinity chromatography, and then additionally purified by preparative size exclusion chromatography (SEC) by methods well known in the art. Upon purification, the antibodies were analyzed for monomer purity and for the presence of fragments or degradation, by methods well known in the art, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with protein staining, and analytical SEC-HPLC, and their concentration was determined by UV spectroscopy. The extinction coefficient was calculated as in Pace et al, Protein Sci. 1995;4:2411-23. All purified proteins contained no fragments or other impurities and were 97.5-100% monomeric. An example of this analysis is depicted in FIG. 2.
For IMV-M and its variants (IMV-Mvl, IMV-Mv2, IMV-Mv3 and IMY-Mv4) in which the scFv portion of the heavy chain fusion was stabilized by a disulfide bond between two cysteines, one each introduced into specific positions in the VL and VH of the scFv (see Table 9), some were purified using only Protein A affinity chromatography without the need for the additional SEC step. The final yields of each of these bispecific antibodies as determined is set below.
Figure imgf000090_0001
Example 3. Cytotoxicity of the bispecific molecules recognizing human MUC16 and human DR5.
To evaluate the cytotoxic effects of antibodies of invention, the following cell lines obtained at the American Type Culture Collection (ATCC), www.ATCC.org, have been used: NCI-H292 lung carcinoma (CRL-1848™), an adherent cell line, CAOV-3 ovarian adenocarcinoma (HTB-75), an adherent cell line, NIH:0VCAR-3, also known as OVCAR- 3 ovarian adenocarcinoma (HTB-161), an adherent cell line, MM. IS multiple myeloma (CRL-2974™), a suspension cell line, SU-DHL-8 large cell lymphoma (CRL-2961™), a suspension cell line, Ramos Burkitt’s lymphoma (CRL-1596™), a suspension cell line, RPMI-8226 plasmacytoma (CCL-155™), a suspension cell line, and HP AC pancreatic adenocarcinoma (CRL-2119™), an adherent cell line, and a suspension cell line obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ), https://www.dsmz.de, MOLP-8 multiple myeloma (ACC 569). All these cell lines express DR5 transcripts (Table 11). NCI-H292, CAOV-3, OVCAR-3, PK-59, HCC827, NCI-H1975, NCC-StC-K140, HDQ-P1 and HP AC express MUC16 transcripts and MUC16 protein. All these cell lines for which data for MUC16 protein expression are available, express MUC16 protein. Proteomics data reporting that MUC16 protein is expressed by OVCAR-3, NCI-H292, HCC827, NCI-H1975 and NCC-StC-K140 are shown in Table 11, and, in addition, flow cytometric or Western blot data demonstrating MUC16 protein expression by 0VCAR3, HP AC and CAOV-3 cells are reported in Chen et al., Cancer Res. 2007, 67, 4924-5998; Haridas et al, PLoS ONE 2011; 6: e26839; and Kline et al., Oncotarget 2017; 8, 52045-5260. MM. IS, SU-DHL-8, Ramos, RPMI-8226, and Molp-8 express CD38 transcripts (Table 11) and CD38 protein [Deckert et al., Clin. Cancer Res. 2014, 20, 4574-4583; Moreno et al, Clin Cancer Res. 2019, 25, 3176-3187], MM.1S, SU- DHL-8, Ramos, RPMI-8226, and Molp-8 cell lines express LIV-1 transcripts (Table 11).
Table 11. Expression of molecules by cells.
Figure imgf000091_0001
PK 59 6.97 NA 0.04 6.43
Figure imgf000092_0001
TR - transcripts; PE - protein expression (proteomics); NA - not available; PE for LIV-1 and for DR5 were not available for these cell lines.
Broad Institute DepMap, https://depmap.org/portal/depmap
The TR data are presented as log2(transcripts per million +1); the PE data are presented as log2 (relative data), as described in Nusinow et al., 2020, Cell 180, 387-402. a) Adherent cells. Cells were plated in a 96-well tissue culture plate at 2.5 x 103 cells/well and were allowed to adhere. The next day, the test-reagents were added, and cells were incubated for two additional days with the test antibodies, and CellTiter-Glo® (Promega) assay was performed in accordance with the manufacturer’s protocol. Each condition was in triplicate. Untreated cells (negative control) were exposed to culture medium only. The values were normalized to untreated control. b) Suspension cells. On the day of the assay, cells were plated in a 96 well plate at 4 x 103 cells/well and the test-antibodies were added. After two days of incubation with the test antibodies, CellTiter-Glo® (Promega) assay was performed in accordance with the manufacturer’s protocol. Each condition was in triplicate. Untreated cells (negative control) were exposed to culture medium only. The values were normalized to untreated control.
We examined the cytotoxicity of the anti-MUC16/anti-DR5 bispecific molecule 3A5/LEX (IMV-18; Table 9) and the parental anti-DR5 antibody lexatumumab (Table 10) towards three cell lines, NCI-H292, CAOV-3 and OVCAR-3. These three cell lines express both MUC16 and DR5 transcripts above the low-expression threshold level of 2.0 (Table 11). The results of the cytotoxicity experiments are displayed in FIG. 3 A. 3A5/LEX anti- MUC16/anti-DR5 produced robust cytotoxic effect in all three cell lines at a concentration 0.3 nM, and this cytotoxic effect was progressively enhanced with the concentration increase to 1 nM and 3 nM. At 3 nM, the surviving fractions of NCI-H292, Caov-3 OVCAR3 cells were 0.48, 0.20 and 0.21, respectively. The parental anti-DR5 antibody lexatumumab was markedly less potent in killing these three cell lines, and most cells survived exposure to the concentration of lexatumumab as high as 10 nM with the surviving fractions of 0.94, 0.95, and 0.81, respectively. Thus, binding of the monospecific antibody to DR5 without additional targeting of MUC16 was not sufficient to induce a strong DR5- mediated cytotoxic effect, while the anti-MUC16/anti-DR5 bispecific antibody had high cytotoxic potency.
We also made and tested the ability of two other bispecific molecules that targeted DR5 and an antigen other than MUC16 (IMV-15 and IMV-20; Table 10) to induce a cytotoxic effect in five cell lines, MM. IS, SU-DHL-8, Ramos, RP MI-8226, and MOLP-8. The results are shown in FIG. 3B. Neither the anti-CD38/anti-DR5 antibody IMV-15, nor the anti-LIV-l/anti-DR5 antibody IMV-20 were able to kill any of these cell lines, demonstrating that not all cell surface antigens are suitable for co-targeting DR5 with a bispecific antibody targeting DR5. This, together with the previously reported result that an anti-CD44v6/anti-DR5 bispecific did not potentiate the agonistic effect of DR5 in CD44v6- expressing cells (US Patent No. 10,858,438), further demonstrates that the cytotoxicity of the bispecific antigen-binding molecules disclosed herein could not be predicted. To further evaluate the cytotoxic effects of the bispecific antigen-binding molecules disclosed herein, cell lines, all adherent, are obtained at the American Type Culture Collection (ATCC), www.ATCC.org, RIKEN BioResource Research Center (RIKEN), https://cell.brc.riken.jp/en, or Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ), https://www.dsmz.de. The following cell lines are used: HP AC pancreatic adenocarcinoma (CRL-2119™; ATCC), PK-59 pancreatic carcinoma (RCB1901; RIKEN); HCC827 lung adenocarcinoma (CRL-2868; ATCC), NCI- H1975 lung adenocarcinoma (CRL-5908; ATCC); NIH:OVCAR-3, also known as OVCAR-3 ovarian adenocarcinoma (HTB-161; ATCC); NCC-StC-K140 gastric carcinoma (RCB2224; RIKEN); and HDQ-P1 breast ductal carcinoma (ACC 494; DSMZ).
We also examined the cytotoxicity of the anti-MUC16/anti-DR5 bispecific molecule 3A5/LEX (MCLX-SE; Table 9); a comparator bispecific antibody which only targeted DR5 on the cell surface, FLX-SE (Table 10); and the parental monospecific anti-MUC16 antibody 3A5 (MC-SE, Table 10). We compared the cytotoxicity of these three antibodies towards six cell lines, HP AC, PK-59, NCI-H1975, OVCAR-3, HDQ-P1 and NCC-StC- K140. The protocol for adherent cells, described above, was used except 3 x 103 HDQ-P1 cells, or 8 x 103 NCC-StC-K140 were plated per well. These six cell lines express both MUC16 and DR5 transcripts above the low-expression threshold level of 2.0 (Table 11). The results of the cytotoxicity experiments are displayed in FIG 3C. The anti-MUC16/anti- DR5 bispecific antibody MCLX-SE produced robust cytotoxic effect in these six cell lines at concentrations below 1 nM, while the anti-fluorescein/anti-DR5 bispecific antibody FLX-SE produced only a minor if any cytotoxic effect on these cell lines even at 10 nM, which was the highest tested concentration. The monospecific anti-MUC16 antibody was not cytotoxic for these cell lines in this concentration range. Thus, neither binding of the antibody FLX-SE to DR5 without additional targeting of MUC16, nor binding of the antibody MC-SE to MUC16 without additional targeting of DR5 was sufficient to induce a strong cytotoxic effect, while the anti-MUC16/anti-DR5 bispecific antibody had high cytotoxic potency.
Cells are plated in a 96-well tissue culture plate at 2.5 x 103 cells/well and are allowed to adhere. The next day, the test-reagents (IMV-18 (Table 9), MCLX-SE (Table 9), 11D10X (Table 10) and 11D10-SE (Table 10), parental anti-MUC16 antibody 3A5 (SEQ ID NOs:115 and 119), parental anti-MUC16 antibody MC-SE (SEQ ID NOs:116 and 120), the non-targeting bispecific antibody FLX-SE (Table 10), or IMV-15) are added to different culture plates, and cells are incubated for two additional days. CellTiter-Glo® (Promega) assays are performed in accordance with the manufacturer’s protocol. Each condition is assayed in triplicate. Untreated cells (negative control) are exposed to culture medium only. The values are normalized to untreated control.
We compare the cytotoxicity of the anti-MUC16/anti-DR5 bispecific antibodies 3A5/anti-DR5 and 1 lD10/anti-DR5. The KD values of the parental antibodies 3A5 and 11D10 reported in US7989595 and Chen et al. Cancer Res. 2007:67, 4924-4932 towards MUC16 on the surface of OVCAR-3 cells is 360 pM and 52 pM, respectively, i.e., the affinity of 11D10 is 7-fold higher than that of 3A5. Example 4. Evaluation of the anti-tumor activity of IMV-18 in a HPAC xenograft model
The anti-tumor activity of IMV-18, a bispecific antibody targeting MUC16 and DR5, was examined in a xenograft model of HP AC pancreatic adenocarcinoma cell line (CRL-2119™, American Type Culture Collection, www.atcc.org) in immunodeficient mice. HPAC cells express both MUC16 and DR5 (Table 11). For comparison, some mice were treated with IMV-21, a bispecific antibody employing the same anti-DR5 scFv, but targeting a different antigen, CD74. BALB/c nude female mice, 6 -8-week-old, 18 to 21 g were used in this study. Prior to inoculation into mice, HPAC cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 in F12/D MEM medium supplemented with 5% fetal bovine serum, 0.5 mM sodium pyruvate, 0.002 mg/ml insulin, 5 microgram'ml transferrin, 40 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor. The tumor cells were routinely sub-cultured before reaching confluence by trypsin-EDTA treatment, not to exceed 4-5 passages. For tumor inoculation, the cells growing in an exponential growth phase were harvested and counted. Each mouse was inoculated subcutaneously on the right flank with inoculated subcutaneously with 5 x 106 HPAC cells in the presence of Corning® Matrigel® Matrix in accordance with the manufacturer’s protocol. Treatment of tumorbearing mice with the test agents were started when the mean tumor volume reached about 145 mmf Based on the tumor volume, mice were randomly assigned to respective groups, five mice per group, such that the average starting tumor size was the same for each treatment group. The average body weight of mice in each group wns no less than 18g. Mice were injected intravenously once (day 1, FIG. 4) with either IMV-21 (5 mg/kg in phosphate buffered saline (PBS)), or IMV-18 (5 mg/kg in PBS), or vehicle only (PBS).
Body weights of all animals were measured daily throughout the study. The measurement of tumor size was conducted twice weekly with a caliper and the tumor volume (mm3) is estimated using the formula: tumor volume = a x b x b/2, where “a” and “b” are long and short diameters of a tumor, respectively. As shown in FIG. 4A, treatment with IMV-18 caused marked delay in average tumor growth in mice, compared with the vehicle control, while treatment with IMV-21 did not cause any delay in tumor growth. The body weight of mice did not decrease indicating absence of systemic toxicity of these molecules for the mice (FIG. 4B). Plots of tumor growth in individual mice (FIG. 4C) demonstrates that while in mice treated with vehicle only or with IMV-21, growth of HPAC xenografts was robust; in mice treated with IMV-21 there was no delay in tumor growth. Two out of five mice treated with IMV-18 experienced marked delay in tumor growth, and in the remaining three mice, tumors disappeared with no regrowth for the entire duration of the experiment. The fact that IMV-21, a bispecific antibody, composed of an IgG antibody targeting a distinct antigen (CD74), and of the anti-DR5 scFv identical to that in IMV-18, was inactive, demonstrates that the anti-tumor activity of IMV-18 was MUC16-dependent.
Example 5, Anti-proliferative effects and apoptosis in PK-59 ceils induced by the bispecific molecules recognizing human MUC16 and human DR5.
Cells were plated onto a tissue culture plate at the density of 1.5 x 103 cells per well. 24 h later, a test agent was added (each condition in triplicate) along with Incucy te® Caspase-3/7 Green Dye for Apoptosis, Catalog No. 4440, Sartorius (green fluorescence) and Incucyte® Nuclight Rapid Red Dye for nuclear labeling, Catalog No. 4717, Sartorius (red fluorescence) in accordance with the manufacturer’s protocol. Caspase-3/7 Green Dye is a fluorogenic substrate of caspase 3 and caspase 7, two caspases that become active during later stages of apoptosis [Elmore S., Toxicol Pathol, 2007; 35(4): 495-516], In the presence of this dye, healthy cells are not fluorescent, while cells that are undergoing apoptosis become green fluorescent. Exposure of cells to Nuclight Rapid Red Dye makes their nuclei red fluorescent. The green fluorescence (green) and red fluorescence (red) objects per well were counted even7 two hours using IncuCyte S3 (Sartorius AG, sartorius.com). Each condition was in triplicate. The anti -proliferative effect of the bispecific anti-MUC16/anti-DR5 antibody MCLX-SE is shown in FIG. 5A. Average numbers of cells with red fluorescent nucleus per well (average of three wells) (Y-axis in LoglO scale) were plotted versus time (X-axis; hours). In the absence of any test-agents (Medium) cells proliferated exponentially (linear semi-exponential plot) without slowing down, with the doubling time of 35 hours. In the presence of MCLX-SE, at the lowest tested concentration of 41.2 pM, proliferation of cells was drastically slowed down within the first 24 hours of exposure to a near complete arrest. In contrast, at this concentration the monospecific parental anti-MUC16 antibody MC-SE did not affect cell proliferation (doubling time remained 35 hours without slowing down), while the comparator bispecific antibody FLX-SE only partially slowed down cell proliferation. At higher concentrations, 123 pM, 370 pM, 1.11 nM, and 10 nM, exposure to MCLX-SE led to only one doubling of the number of cells, followed by a nearly complete arrest of proliferation, while in the presence of MC-SE cells continued to proliferate without slowing down with the doubling times between 35 to 40 hours, while FLX-SE only partially slowed down cell proliferation even at the highest concentration tested, 10 nM.
The induction of apoptosis in cells by the bispecific anti-MUC16/anti-DR5 antibody MCLX-SE is shown in FIG. 5B and FIG. 5C. As shown in FIG. 5B, upon exposure to 41.2 pM of bispecific anti-MLiC16/anti-DR5 antibody MCLX-SE, a majority’ of cells rapidly underwent apoptosis. By 18 hours of exposure about 1900 of cells contained activated caspase 3 and/or caspase 7. In contrast, only a small fraction of cells underwent apoptosis during their exposure to the comparator bispecific antibody FLX-SE, which targeted DR5 on the cell surface, but did not target MUC16. MC-SE, another comparator antibody, a mono-specific antibody targeting MUC16, did not induce any significant apoptosis of the cells. Exposure of cells to higher concentrations of MCLX-SE induced even a more pronounced apoptosis (FIG. 5C), while the comparator antibody FLX-SE induced apoptosis of only a minor fraction of cells even at the highest tested concentration of 10 nM, and the other comparator antibody, MC-SE, did not cause any significant apoptosis at any concentration (FIG. 5C).
Example 6. Evaluation of the anti-tumor activity of MCLX-SE in MUC16- positive/DRS positive xenograft models
The anti -tumor activity of MCLX-SE was examined in immunodeficient mice in the following xenograft models using MUC 16-positive cell lines that express both MUC16 and DR5 above the low-expression threshold (Table 11). The cell lines tested w'ere: HP AC pancreatic adenocarcinoma cell line (CRL-2119™), HCC827 (HCC827 lung adenocarcinoma (CRL-2868)), PK-59 cells (pancreatic adenocarcinoma), and NCI-H1975 cells (lung adenocarcinoma). For comparison, some mice were treated with either FLX-SE (Table 10), or with MC-SE. BALB/c nude female mice were used in this study. Treatment of tumor-bearing mice with the test agents was started when the mean tumor volume reached about 100-150 mm3. Based on the tumor volume, mice w'ere randomly assigned to respective groups, such that the average starting tumor size was the same for each treatment group. Mice were injected intravenously with one of these agents, or vehicle only (PBS). The measurement of tumor size was conducted with a caliper and the tumor volume (mm3) was estimated using the formula: tumor volume = a x b x b/2, where “a” and “b” are long and short diameters of a tumor, respectively. As shown in FIG. 6A - FIG. 6D, treatment with the anti-MUC16/anti/DR5 bispecific antibody MCLX-SE caused marked delay in average tumor growth in mice in all four xenograft models, compared with the vehicle control. In addition, in the studies of HP AC and HCC827 xenografts, two additional controls were included, MC-SE, a monospecific antibody targeting MUC16, and FLX-SE, a bispecific antibody targeting only DR5. As shown in FIG. 6A and FIG. 6B, treatment with either FLX-SE, or with MC-SE, did not cause any delay in tumor growth of HP AC or HCC827 xenograft tumors, demonstrating that targeting of both antigens was required for achieving anti-tumor activity at this dose, and that the anti-tumor activity of the anti- MUC16/antiDR5 antibody was both MUC16-dependent and DR5-dependent.
Expression of MUC16 protein in xenografts was examined by immunohistochemistry. The following reagents were used: rabbit monoclonal anti-MUC16 antibody, Abeam # 110640MXB (primary antibody); Polyclonal Goat anti-Rabbit Immunoglobulins, Dako (Agilent Technologies), cat# 4003 (secondary antibody); Dako antibody dilute solution, Dako#S2022; Wash buffer, Dako#K8007; Citrate 6.0 (MXB Biotechnologies, Fuzhou, Fujian, China #MVS-0066. Tumor tissues embedded in paraffin were cut with a microtome to the thickness of 4 pm, and then processed by a protocol well known in the arts. After heat-induced antigen unmasking in citrate pH 6.0, sections were immersed in 3% hydrogen peroxide solution for 5 min. To avoid nonspecific staining, the sections were then incubated in blocking serum for 30 min containing normal goat serum at room temperature, followed by an overnight incubation with a rabbit monoclonal anti- MUC16 antibody (Abeam #110640, 1:500 dilution). Then the sections were exposed to secondary antibody conjugated to HRP. The nuclei were stained with hematoxylin. Slides were then scanned using Aperio Scanner: Versa 8 (Leica), at 200X magnification. Images were then opened with HALO, using the pen tool to select an annotation layer. The necrotic areas were excluded in the annotation layer. The following xenografts were examined: Capan-1, PK-59, HCC827, NCI-H1975, HP AC; all in Balb/c nude mice, and OVCAR-3 in SCID mice. OVCAR-3 displayed strong MUC16 expression; PK-59, HCC827, and HP AC displayed medium MUC16 expression, NCI-H1975 displayed weak MUC16 expression, while Capan-1 displayed very weak to negative MUC16 expression. These data were in good agreement with Table 11. Example 7. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5 and FLX-SE to human recombinant DR5.
Human DR5 recombinant protein (TRAIL R2, catalog number TR2-H5229; amino acids 56-182) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86 400-682-2521, www.acrobiosystems.cn. A high-binding ELISA plate is coated with human DR5, 1 pg/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour. The blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed. The wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc-gamma polyclonal antibody, washed, and the bound polyclonal antibody is detected with 3, 3', 5,5'- tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm. Normal polyclonal human IgG is used to detect non-specific binding of human IgG to the plate coated with recombinant human DR5.
Example 8. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE to human recombinant MUC16.
Human recombinant MUC16 (catalog number CA5-H52H6, amino acids 13810- 14451) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86400-682-2521, www.acrobiosystems.cn. A high-binding ELISA plate is coated with human MUC16, 1 microgram/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour. The blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX- SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed. The wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc- gamma polyclonal antibody, washed and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm.
Example 9. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE to human recombinant CD74.
Human recombinant CD74 (catalog number CD4-H524c, amino acids 73-232) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86400-682-2521, www.acrobiosystems.cn. A high- binding ELISA plate is coated with human CD74, 1 microgram/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour. The blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti- MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed. The wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc-gamma polyclonal antibody, washed and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm.
Example 10. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE to human recombinant CD38.
Human CD38 (catalog number CD8-H5224, amino acids 43-300) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86400-682-2521, www.acrobiosystems.cn. A high-binding ELISA plate is coated with human CD38, 1 microgram/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour. The blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti- MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed. The wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc-gamma polyclonal antibody, washed and detected with 3.3'.5.5'-tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm.
Example 11. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16/anti-DR5, and FLX-SE to human recombinant LIV-1/SLC39A6.
Human LIV-1 (catalog number LV1-H5223, amino acids 29-325) is obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86400-682-2521, www.acrobiosystems.cn. A high-binding ELISA plate is coated with human LIV-1, 1 microgram/mL, or with blocking buffer (2% bovine serum albumin in PBS) overnight at 4° C. The solutions are aspirated, and blocking buffer is then added to all wells for 1 hour. The blocking buffer is aspirated, and then various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti- MUC16/anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). Plates are incubated at room temperature for 1 hour, the antibody is aspirated, and the wells are washed. The wells are then incubated with horse radish peroxidase-conjugated anti-human IgG Fc-gamma polyclonal antibody, washed and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by stop solution (ThermoFisher Scientific) and absorbance read at 450 nm.
Example 12. The anti-tumor activity of the anti-MUC16-anti-DR5 bispecific antibodies does not depend on its interaction with Fc receptors gamma in a pancreatic tumor xenograft model.
As described in Shivange et al. 2018; Cancer Cell 34, 331-345, the interaction of anti-DR5 antibodies with Fc gamma receptors, specifically, with Fc gamma receptor 2B found primarily on B cells, is critical for anti-tumor activity in xenograft models in mice. Antibodies with impeded ability to interact with these receptors did not demonstrate antitumor activity in these models. Surprisingly, the anti-MUC16-anti-DR5 bispecific antibodies disclosed herein do not require interaction with these receptors.
The anti -tumor activities of four variants of the anti-MUC16-anti-DR5 bispecific antibody (IMV-18, MCLX-SE, IMV-AA and MC-AA) against a pancreatic carcinoma xenograft model were compared. These variants (described in Table 9) were nearly identical except for several point mutations in the Fc region that affected the affinities of these variants towards Fc receptors gamma on immune cells of nude mice, as described in [Shivange et al. 2018; Cancer Cell 34, 331-345], IMV-18 contains a wild-type human IgGl Fc; MCLX-SE has enhanced affinity towards Fc gamma receptor 2B, IMV-AA has impeded affinity towards all Fc receptors gamma, and MC-AA has impeded affinity towards Fc receptors gamma except restored affinity towards Fc gamma receptor 2B. In some mice these antibodies were co-injected with an excess (30 mg/kg) of a non-targeting mouse IgGl antibody (muIgGl, Table 10), which contains a wild-type human IgGl Fc. This co-inj ection was done to allow muIgGl to compete with the anti-MUC16-anti-DR5 bispecific antibody for binding to Fc gamma receptors.
MUC16-positive/DR5 positive xenograft pancreatic carcinoma model PK-59 (RCB1901, Cell Bank, RIKEN BioResource Research Center (BRC) 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan) was used in this study. BALB/c nude were injected subcutaneously with the PK-59 cells and the resulting tumor-bearing mice were treated with one of the anti-MUC16-anti-DR5 bispecific antibodies (or an appropriate control) once the mean tumor volume reached about 100-150 mm3. Based on the tumor volume, mice were randomly assigned to respective groups, such that the average starting tumor size was the same for each treatment group. Mice were injected intravenously once on day 1 with one of the anti-MUC16-anti-DR5 bispecific antibodies (5 mg/kg) or vehicle (PBS), or with an anti-MUC16-anti-DR5 bispecific antibody (5 mg/kg) and muIgGl (30 mg/kg), four mice per group.
The measurement of tumor size was conducted with a caliper and the tumor volume (mm3) was estimated using the formula: tumor volume = a x b x b/2, where “a” and “b” are long and short diameters of a tumor, respectively. The data are presented as a mean + SEM (n = 4). At some time points the SEM is too small to be seen in the plot. As shown in FIG. 7, all anti-MUC16/anti/DR5 bispecific antibodies were equally active in their ability to delay the growth of PK-59 xenografts and irrespective of the presence or absence of an excess of mouse IgGl antibody.
Example 13. IVM-M displays dose-dependent anti-tumor activity in a pancreatic tumor xenograft model.
As described in Example 12, BALB/c nude were subcutaneously injected with PK- 59 pancreatic carcinoma cells. IVM-M treatment began when the mean tumor volume reached about 100-150 mm3. Based on the tumor volume, mice were randomly assigned to respective groups, such that the average starting tumor size was the same for each treatment group. Mice were injected intravenously once on day 1 with IMV-M at either 1 mg/kg, 2.5 mg/kg or 5 mg/kg, or vehicle only (PBS), four mice per group. The measurement of tumor size was conducted with a caliper and the tumor volume (mm3) was estimated using the formula: tumor volume = a x b x b/2, where “a” and “b” are long and short diameters of a tumor, respectively. The data are presented as a mean + SEM (n = 4). In some points the SEM is too small to be seen in the plot. As shown in FIG. 8, treatment with the anti- MUC16/anti/DR5 bispecific antibody IMV-M caused marked delay in average tumor growth in mice at all three doses and was dose-dependent.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web at tigr.org and/or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.
EQUIVALENTS
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

Claims
1. A bispecific binding molecule comprising: a. a first antigen-binding domain that specifically binds to the extracellular domain of human MUC16; and b. a second antigen-binding domain that specifically binds human DR5.
2. The bispecific binding molecule of claim 1, wherein the first antigen-binding domain binds to an epitope present in more than one tandem repeat/SEA segment within the extracellular domain of MUC16.
3. The bispecific binding molecule of claim 1, wherein the first antigen-binding domain is competed for MUC16 binding by one or more of the following antibodies: OC125, H185, Mil, OV197, 5E11, AR9.6, H1H8794, VK-8, B43.13, or 3A5.
4. The bispecific binding molecule of claim 1, wherein the first antigen-binding domain binds to a polypeptide or peptide consisting essentially of the amino acid sequence of at least one of SEQ ID NOs: 176-181, or to an extracellular fragment of MUC16 isolated from the cell culture media of OVCAR-3 cells.
5. The bispecific binding molecule of any one of claims 1-4, wherein the first antigenbinding domain comprises: a. a heavy chain variable region comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein:
CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1;
CRD-H2 comprises the amino acid sequence of any one of SEQ ID NOs:2- 5; and
CDR2-H3 comprises the amino acid sequence of any one of SEQ ID NOs:6- 61 (CDR3-H3); and b. a light chain variable region comprising three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:
CDR-L1 comprises the amino acid sequence of SEQ ID NO:62; CDR-L2 comprises the amino acid sequence of SEQ ID NO:63; and CDR-L3 comprises the amino acid sequence of SEQ ID NO65.
6. The bispecific binding molecule of claim 5, wherein:
CDR-H1 comprises the amino acid sequence of SEQ ID NO:1;
CDR-H2 comprises the amino acid sequence of SEQ ID NO:2;
CDR-H3 comprises the amino acid sequence of SEQ ID NO:6;
CDR-L1 comprises the amino acid sequence of SEQ ID NO:62;
CDR-L2 comprises the amino acid sequence of SEQ ID NO:63; and CDR-L3 comprises the amino acid sequence of SEQ ID NO:65.
7. The bispecific binding molecule of claim 5 or 6, wherein the first antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO:97.
8. The bispecific binding molecule of claim 7, wherein the first antigen-binding domain comprises a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 97.
9. The bispecific binding molecule of any one of claims 5-8, wherein the first antigenbinding domain comprises a light chain variable region that comprises the amino acid sequence of SEQ ID NOs.: 104 or 105, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to either SEQ ID NO: 104 or SEQ ID NO: 105.
10. The bispecific binding molecule of claim 9, wherein the first antigen-binding domain comprises a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 104 or SEQ ID NO: 105.
11. The bispecific binding molecule of any one of claims 7-10, wherein the first antigen-binding domain comprises: a. a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO:97, or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to SEQ ID NO:97; and b. a light chain variable region that comprises the amino acid sequences of either SEQ ID NO: 104 or 105, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to either SEQ ID NO: 104 or 105.
12. The bispecific binding molecule of claim 11, wherein the first antigen-binding domain comprises: a. a heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 97; and b. a light chain variable region that comprises the amino acid sequences of either SEQ ID NO: 104 or 105.
13. The bispecific binding molecule of any one of claims 5-12, wherein the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs. : 115-118, or a heavy chain comprising the same three heavy chain CDRs as, and an amino acid sequence having at least 90% sequence identity to, any one of SEQ ID NOs.:115-118.
14. The bispecific binding molecule of claim 13, wherein the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs.: 115-118.
15. The bispecific binding molecule of any one of claims 5-14, wherein the first antigen-binding domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 120; or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to, SEQ ID NO: 119 or SEQ ID NO: 120.
16. The bispecific binding molecule of claim 15, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 119 or the amino acid sequence of SEQ ID NO: 120.
17. The bispecific binding molecule of any one of claims 13-16, wherein the first antigen-binding domain comprises: a. a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs. : 115-118, or a heavy chain comprising the same three heavy chain CDRs as, and an amino acid sequence having at least 90% sequence identity to, any one of SEQ ID NOs.: 115-118; and b. a light chain comprising the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 120; or an amino acid sequence having the same three light chain CDRs as, and at least 90% sequence identity to, SEQ ID NO: 119 or SEQ ID NO: 120.
18. The bispecific binding molecule of claim 17, wherein the first antigen-binding domain comprises: a. a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs.:115-118; and b. a light chain comprising the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 120.
19. The bispecific binding molecule of any one of claims 1-18, wherein the first antigen-binding domain comprises two heavy chains and two light chains.
20. The bispecific binding molecule of claim 19, wherein each of the two heavy chains has an identical amino acid sequence and each of the two light chains has an identical amino acids sequence.
21. The bispecific binding molecule of any one of claims 1-20, wherein the second antigen-binding domain is competed for DR5 binding by one or more of the following antibodies: Conatumumab, Drozitumab, Lexatumumab, LBY135, Tigatuzumab, and DS- 8273a.
22. The bispecific binding molecule of any one of claims 1-21, wherein the second antigen-binding domain is competed for DR5 binding by TRAIL or a fragment of TRAIL that binds to DR5.
23. The bispecific binding molecule of any one of claims 1-22, wherein the second antigen-binding domain comprises a second heavy chain variable region comprising second heavy chain complementarity determining regions (CDRs) comprising the amino acid sequences SEQ ID NO: 121 (second CDR-H1); SEQ ID NO: 122 (second CDR2-H2); and SEQ ID NO: 123 (second CDR3-H3); and a second light chain variable region comprising second light chain complementarity determining regions comprising amino acid sequences SEQ ID NO: 124 (second CDR-L1); SEQ ID NO: 125 (second CDR-L2); and SEQ ID
NO: 126 (second CDR-L3).
24. The bispecific binding molecule of claim 23, wherein the second heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 158, SEQ ID NOs:218-220, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to any one of SEQ ID NO:158 or SEQ ID NOs:218-220.
25. The bispecific binding molecule of claim 24, wherein the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 158.
26. The bispecific binding molecule of claim 24, wherein the second heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NOs:218-220.
27. The bispecific binding molecule of any one of claims 23-26, wherein the second light chain variable region comprises the amino acid sequence of any one of SEQ ID NO: 165, SEQ ID NOs: 215-217, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to any one of SEQ ID NO: 165 or SEQ ID NOs: 215-217.
28. The bispecific binding molecule of claim 27, wherein the second light chain variable region comprises the amino acid sequence of SEQ ID NO: 165.
29. The bispecific binding molecule of claim 27, wherein the second light chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 215-217.
30. The bispecific binding molecule of any one of claims 24-29, wherein: a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO:158; and b. the second light chain variable region comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having the same three heavy chain CDRs as, and at least 90% sequence identity to SEQ ID NO:165.
31. The bispecific binding molecule of claim 30, wherein: a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 158; and b. the second light chain variable region comprises the amino acid sequence of SEQ ID NO: 165.
32. The bispecific binding molecule of any one of claims 24-29, wherein: a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO:218; and b. the second light chain variable region comprises the amino acid sequence of SEQ ID NO:215.
33. The bispecific binding molecule of any one of claims 24-29, wherein: a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO:219; and b. the second light chain variable region comprises the amino acid sequence of SEQ ID NO:215.
34. The bispecific binding molecule of any one of claims 24-29, wherein: a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO:219; and b. the second light chain variable region comprises the amino acid sequence of SEQ ID NO:216.
35. The bispecific binding molecule of any one of claims 24-29, wherein: a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO:220; and b. the second light chain variable region comprises the amino acid sequence of SEQ ID NO:217.
36. The bispecific binding molecule of any one of claims 1-35, wherein the second antigen-binding domain is a scFv fragment of an antibody.
37. The bispecific binding molecule of claim 36, wherein the scFv fragment comprises, in N- to C-terminal order, a light chain variable region, a peptide linker, and a heavy chain variable region.
38. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO: 172; or an amino acid sequence having the same three heavy chain CDRs and the same three light chain CDRs as, and at least 90% sequence identity to, SEQ ID NO: 172.
39. The bispecific binding molecule of claim 38, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO: 172.
40. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:210.
41. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:211.
42. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:212.
43. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:213.
44. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:214.
45. The bispecific binding molecule of any one of claims 36-44, wherein the N- terminus of the second antigen-binding domain is fused directly or through a peptide linker having a length of 4 to 20 amino acids to the C-terminus of one of the heavy chains of the first antigen-binding domain.
46. The bispecific binding molecule of claim 45, wherein the peptide linker has the amino acid sequence of SEQ ID NO: 173.
47. The bispecific binding molecule of any one of claims 36-46, comprising two scFv fragments that specifically bind DR5, wherein each of the two scFv fragments is bound to a different heavy chain of the first antigen-binding domain.
48. The bispecific binding molecule of claim 47, wherein each of the two scFv fragments have the identical amino acid sequence.
49. A bispecific binding molecule comprising: a. two antibody light chains, wherein each light chain has an amino acid sequence independently selected from SEQ ID NO: 119 and SEQ ID
NO: 120; and b. two antibody heavy chain fusions, wherein each heavy chain fusion has an amino acid sequence independently selected from the formula: X-L- Y, wherein:
X is the amino acid sequence of any one of SEQ ID NOs.: 115-118;
L is the amino acid sequence of SEQ ID NO: 173; and
Y is the amino acid sequence of any one of SEQ ID NO: 172 or SEQ ID NOs:210-214.
50. The bispecific binding molecule of claim 49, wherein each light chain has the identical amino acid sequence, and each heavy chain fusion has the identical amino acid sequence.
51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 174.
52. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 175.
53. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 186.
54. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 187.
- Il l -
55. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:210.
56. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:211.
57. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:212.
58. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:213.
59. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:214.
60. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 174.
61. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 175.
62. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 186.
63. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 187.
64. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:210.
65. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:211.
66. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:212.
67. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:213.
68. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:214.
69. A pharmaceutical composition comprising a bispecific binding molecule of any one of claims 1-68; and a pharmaceutically acceptable carrier.
70. An isolated nucleic acid sequence encoding a heavy chain fusion comprising an amino acid sequence of any one of SEQ ID NOs: 174, 175, 186, 187, or 210-214.
71. The isolated nucleic acid sequence of claim 70 comprising SEQ ID NO:182 or 183.
72. An expression vector comprising the nucleic acid sequence of claim 70 or 71.
73. The expression vector of claim 72, additionally comprising a nucleic acid sequence encoding a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119 or 120.
74. The expression vector of claim 73, wherein the nucleic acid sequence encoding the light chain comprises SEQ ID NO: 184 or 185.
75. A host cell harboring a first expression vector of claim 72; and a second expression vector comprising a nucleic acid sequence encoding a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119 or 120.
76. The host cell of claim 75, wherein nucleic acid sequence encoding the light chain comprises SEQ ID NO: 184 or 185.
77. A host cell harboring the expression vector of claim 73 or 74.
78. A method of manufacturing a bispecific binding molecule, comprising the steps of: a. culturing the host cell of any one of claims 75-77 under conditions allowing for expression of the nucleic acid sequence encoding the heavy chain fusion and expression of the nucleic acid sequence encoding the light chain, and association of the expressed heavy chain fusion and the expressed light chain into the bispecific binding molecule; and b. recovering the bispecific binding molecule from the culture.
79. A method of treating a MUC16-related fibrotic disease disorder, inflammatory disorder, immune disorder, or autoimmune disorder, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the bispecific binding molecule of any one of claims 1-68, or the pharmaceutical composition of claim 69.
80. The method of claim 79, wherein the MUC16-mediated fibrotic disorder is pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, cirrhosis, kidney fibrosis, glial scar, myocardial fibrosis, arterial stiffness, arthrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, or adhesive capsulitis.
81. A method of treating a cancer or malignancy characterized by over-expression of MUC16, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the bispecific binding molecule of any one of claims 1-68, or the pharmaceutical composition of claim 69.
82. The method of claim 81, wherein the cancer or malignancy is a gynecologic cancer (cancer of the female reproductive tract), including the cervix, endometrium, fallopian tubes, ovaries, uterus, and vagina; pancreatic cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, or lung cancer.
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