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WO2025011471A1 - Egfr/c-met bispecific binding protein and use thereof - Google Patents

Egfr/c-met bispecific binding protein and use thereof Download PDF

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Publication number
WO2025011471A1
WO2025011471A1 PCT/CN2024/103914 CN2024103914W WO2025011471A1 WO 2025011471 A1 WO2025011471 A1 WO 2025011471A1 CN 2024103914 W CN2024103914 W CN 2024103914W WO 2025011471 A1 WO2025011471 A1 WO 2025011471A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
peptide
bispecific antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/103914
Other languages
French (fr)
Inventor
Yong Zheng
Dongmei LEI
Yuehua ZHOU
Yanshu DI
Shuting YI
Hu LONG
Shuntao LUO
Xiaoxi YUAN
Mingxiong LI
Wei Shan
Senwu LI
Hongmei SONG
Miao TAN
Xiangyang TAN
Junyou GE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sichuan Kelun Biotech Biopharmaceutical Co Ltd
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Sichuan Kelun Biotech Biopharmaceutical Co Ltd
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Publication of WO2025011471A1 publication Critical patent/WO2025011471A1/en
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Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present disclosure relates to therapeutic bispecific antibodies, and more specifically, to bispecific antibodies against EGFR and c-MET and methods of use thereof.
  • EGFR epidermal growth factor receptor
  • HER1 epidermal growth factor receptor
  • HER2 erbB2, NEU
  • HER3 erbB3
  • HER4 erbB4
  • the HER family plays an important regulatory role in cellular physiological processes.
  • EGFR is a type I transmembrane glycoprotein composed of 1210 amino acids, with a molecular weight of about 170KD.
  • EGFR is highly expressed in various tumors such as colorectal cancer, head and neck cancer, and non-small cell lung cancer, and it is also expressed in normal epithelial tissues such as skin and lungs.
  • Tyrosine protein kinase Met also known as hepatocyte growth factor receptor (HGFR)
  • HGFR hepatocyte growth factor receptor
  • c-MET is a type I transmembrane glycoprotein composed of 1390 amino acids, with a molecular weight of about 190KD, and comprises two polypeptide chains connected by disulfide bonds through cleavage, namely the ⁇ chain (50 kDa) and the ⁇ chain (140 kDa) .
  • MET protein can be divided into SEMA domain (Semaphorin domain, SEMA) , PSI domain (Plexin-semaphorin-integrin domain, PSI) , four immunoglobulin-like repeat domains (Immunoglobulin-plexins-transcription domain, IPT) , a transmembrane region, a juxtamembrane region (JM) , a tyrosine kinase domain (TK) and a carboxy-terminal tail region (CT) .
  • the natural ligand for the c-MET receptor is hepatocyte growth factor (HGF) , an inactive protein that is converted to its active form by proteolytic cleavage.
  • HGF hepatocyte growth factor
  • c-MET Aberrant regulation of c-MET has been reported in a variety of cancers, including colorectal cancer, non-small cell lung cancer, gastric cancer, and breast cancer. High activation of c-MET and its downstream signaling pathways have been shown to trigger hyperproliferation, tumor invasion, and angiogenesis, and be associated with poor survival.
  • Anti-EGFR macromolecular antibody drugs such as cetuximab, panitumumab, nimotuzumab, and necitumumab have been approved for marketing, targeting colorectal cancer, non-small cell lung cancer, etc.
  • EGFR-tyrosine kinase inhibitors such as osimertinib, alimertinib, and avitinib, have become the standard treatment for advanced cancer patients positive for EGFR mutation.
  • c-MET-TKIs have also achieved good efficacy in non-small cell lung cancer.
  • the EGFR and c-MET signaling pathways have been clinically verified respectively.
  • the bispecific antibody targeting EGFR and c-MET at the same time is expected to simultaneously inhibit the signaling pathways of EGFR and c-MET, avoiding the occurrence and development of tumors caused by the activation of c-MET signaling pathway after EGFR inhibition, thereby improving the overall efficacy and safety.
  • Anti-c-MET and anti-EGFR antibodies are known in the art, for example, Telisotuzumab vedotin (Teliso-v or ABBV399) , which is disclosed in U. S. Patent No. 10603389, is anti-c-MET antibody ABT-700 conjugated to vc-MMAE (vedotin) , and MRG003 (ADC-3 or MYK-3) , which is disclosed in WO2023040941, is anti-EGFR antibody BA03 conjugated to vedotin.
  • Bispecific antibody BSAB01 disclosed in WO2010115551 comprises the EGFR binding VH/VL pair (Fab) of cetuximab and the c-Met binding VH/VL pair (Fab) of onartuzumab, which is currently in Phase III trials.
  • Amivantamab a bispecific antibody marketed under the tradename is disclosed in U.S. Patent No. 9593164.
  • Amivantamab comprises an Anti-EGFR Fab comprising the VH and VL of Zalutumumab (See, U.S. Patent No. 7247301 and U.S. Patent No.
  • the present invention provides a high-affinity EGFR/c-MET bispecific antibody, which can specifically recognize or bind EGFR and c-MET and simultaneously block the binding of EGF to EGFR and the binding of HGF to c-MET, thus, simultaneously blocking the downstream signaling pathways of both EGFR and c-MET, thereby increasing the inhibition of cell proliferation.
  • the present invention provides a bispecific antibody, comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH) , and the first VL and the first VH collectively form a domain capable of specifically binding to c-MET; and the second antigen-binding domain comprises a second VL and a second VH, and the second VL and the second VH together form a domain capable of specifically binding to EGFR.
  • the bispecific antibody binds to c-MET and EGFR with increased affinity relative to other known antibodies.
  • the first antigen-binding domain and the second antigen-binding domain are each independently selected from scFv, Fab, and scFab.
  • the bispecific antibody further comprises an Fc domain, wherein the Fc domain comprises a first Fc domain monomer and a second Fc domain monomer, and the first and second Fc domain monomers comprise one or more modifications that promote heterodimerization of the Fc domain monomers.
  • the Fc domain comprises a first Fc domain monomer comprising modifications that form a knob structure and a second Fc domain monomer comprising modifications that form a hole structure, wherein the hole structure can be paired with the knob structure to form a heterodimeric Fc domain.
  • the first Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 49 or 51
  • the second Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 50 or 52.
  • the first antigen-binding domain and the second antigen-binding domain are each linked to one of the first and second Fc domain monomers of the Fc domain.
  • the first antigen-binding domain is linked to the first Fc domain monomer, and the second antigen-binding domain is linked to the second Fc domain monomer; or the first antigen-binding domain is linked to the second Fc domain monomer, and the second antigen-binding domain is linked to the first Fc domain monomer.
  • the first VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 17 or 59; and/or the first VH comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 18 or 60.
  • the first VL comprises:
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 34
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 36
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; wherein the CDRs are defined by the Kabat numbering system;
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 34
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 36
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; wherein the CDRs are defined by the Chothia numbering system;
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 34
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 36
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; wherein the CDRs are defined by the Abm numbering system; or
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 35
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 37
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; wherein the CDRs are defined by the IMGT numbering system; and/or
  • the first VH comprises:
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 39
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 43
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47; wherein the CDRs are defined by the Kabat numbering system;
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 40
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 44
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47; wherein the CDRs are defined by the Chothia numbering system;
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 42
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 46
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47; wherein the CDRs are defined by the Abm numbering system; or
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 41
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 45
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 48; wherein the CDRs are defined by the IMGT numbering system.
  • the first VL comprises:
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 34
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 36
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38;
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 35
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 37
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38;
  • the first VH comprises:
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 39
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 43
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47;
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 40
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 44
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47;
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 42
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 46
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47;
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 41
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 45
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 48.
  • the first VL comprises the amino acid sequence as shown in SEQ ID NO: 17 or 59, and/or the first VH comprises the amino acid sequence as shown in SEQ ID NO: 18 or 60; or the first VL comprises the amino acid sequence as shown in SEQ ID NO: 17, the first VH comprises the amino acid sequence as shown in SEQ ID NO: 18; or the first VL comprises the amino acid sequence as shown in SEQ ID NO: 59, the first VH comprises the amino acid sequence as shown in SEQ ID NO: 60.
  • the second VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15; and/or the VH comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16.
  • the second VL comprises:
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 19
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 21
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; wherein the CDRs are defined by the Kabat numbering system;
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 19
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 21
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; wherein the CDRs are defined by the Chothia numbering system;
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 19
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 21
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; wherein the CDRs are defined by the Abm numbering system; or
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 20
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 22
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; wherein the CDRs are defined by the IMGT numbering system; and/or
  • the second VH comprises:
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 24, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32; wherein the CDRs are defined by the Kabat numbering system;
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32; wherein the CDRs are defined by the Chothia numbering system;
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 27, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 31, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32; wherein the CDRs are defined by the Abm numbering system; or
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 26
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 30,
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 33; wherein the CDRs are defined by the IMGT numbering system.
  • the second VL comprises:
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 19
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 21
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23;
  • LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 20
  • LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 22
  • LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23;
  • the second VH comprises:
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 24, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32;
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32;
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 27, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 31, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32; or
  • HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 26
  • HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 30
  • HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 33.
  • the second VL comprises the amino acid sequence as shown in SEQ ID NO: 15, and/or the second VH comprises the amino acid sequence as shown in SEQ ID NO: 16.
  • the first antigen-binding domain is a Fab
  • the second antigen-binding domain is an scFv.
  • the bispecific antibody comprises a peptide chain I-A, a peptide chain I-B and a peptide chain I-C; wherein the peptide chain I-A comprises the first VL and a light chain constant region; the peptide chain I-B comprises: the first VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain I-C comprises: the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) .
  • the peptide chain I-A comprises from N-terminus to C-terminus the first VL and the light chain constant region;
  • the peptide chain I-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ;
  • the peptide chain I-C comprises from N-terminus to C-terminus (i) the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the second VH, the second VL and the second Fc domain monomer (or the first Fc domain monomer) .
  • the adjacent domains of the peptide chain I-A are connected optionally with or without a linker
  • the adjacent domains of the peptide chain I-B are connected optionally with or without a linker
  • the adjacent domains of the peptide chain I-C are connected optionally with or without a linker.
  • the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) .
  • the peptide linkers are each independently selected from the group consisting of peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown as (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
  • the peptide chain I-A comprises the amino acid sequence as shown in SEQ ID NO: 1
  • the peptide chain I-B comprises the amino acid sequence as shown in SEQ ID NO: 2 or 9
  • the peptide chain I-C comprises the amino acid sequence as shown in SEQ ID NO: ID NO: 3 or 10.
  • the first antigen-binding domain is an scFv
  • the second antigen-binding domain is a Fab
  • the bispecific antibody comprises a peptide chain II-A, a peptide chain II-B and a peptide chain II-C; wherein the peptide chain II-A comprises the second VL and a light chain constant region; the peptide chain II-B comprises the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain II -C comprises: the first VL, the first VH and the second Fc domain monomer (or the first Fc domain monomer) .
  • the peptide chain II-A comprises from N-terminus to C-terminus the second VL and the light chain constant region; and the peptide chain II-B comprises from N-terminus to C-terminus the second VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain II-C comprises from N-terminus to C-terminus (i) the first VL, the first VH and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the first VH, the first VL and the second Fc domain monomer (or the first Fc domain monomer) .
  • the adjacent domains of the peptide chain II-A are connected optionally with or without a linker; the adjacent domains of the peptide chain II-B are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain II-C are connected optionally with or without a linker.
  • the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) .
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
  • the peptide chain II-A comprises the amino acid sequence as shown in SEQ ID NO: 4
  • the peptide chain II-B comprises the amino acid sequence as shown in SEQ ID NO: 5 or 7
  • the peptide chain II-C comprises the amino acid sequence as shown in SEQ ID NO: 6 or 8.
  • the first antigen-binding domain is a Fab
  • the second antigen-binding domain is a scFab
  • the bispecific antibody comprises a peptide chain III-A, a peptide chain III-B and a peptide chain III-C; wherein the peptide chain III-A comprises the first VL and a light chain constant region; the peptide chain III-B comprises: the first VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain III-C comprises: the second VL, a light chain constant region, the second VH, a heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) .
  • the peptide chain III-A comprises from N-terminus to C-terminus the first VL and the light chain constant region;
  • the peptide chain III-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ;
  • the peptide chain III-C comprises from N-terminus to C-terminus (i) the second VL, the light chain constant region, the second VH, the heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the second VH, the heavy chain CH1 region, the second VL, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer) .
  • the adjacent domains of the peptide chain III-A are connected optionally with or without a linker; the adjacent domains of the peptide chain III-B are connected optionally with or without a linker; and/or the adjacent domains of the peptide chain III-C are connected optionally with or without a linker.
  • the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) ; or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • G glycines
  • S serines
  • the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the CH1 region of the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 54.
  • the first antigen-binding domain is a scFab
  • the second antigen-binding domain is a Fab
  • the bispecific antibody comprises a peptide chain IV-A, a peptide chain IV-B and a peptide chain IV-C; wherein the peptide chain IV-A comprises the second VL and a light chain constant region; the peptide chain IV-B comprises: the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain IV-C comprises: the first VL, a light chain constant region, the first VH, a heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) .
  • the peptide chain IV-A comprises from N-terminus to C-terminus the second VL and the light chain constant region
  • the peptide chain IV-B comprises from N-terminus to C-terminus the second VH, the CH1 region of the heavy chain and the first Fc domain monomer (or the second Fc domain monomer)
  • the peptide chain IV-C comprises from N-terminus to C-terminus (i) the first VL, the light chain constant region, the first VH, the heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) ; or (ii) the first VH, the heavy chain CH1 region, the first VL, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer) .
  • the adjacent domains of the peptide chain IV-A are connected optionally with or without a linker, and the adjacent domains of the peptide chain IV-B are connected optionally with or without a linker; and/or the adjacent domains of the peptide chain IV-C are connected optionally with or without a linker.
  • the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) .
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68;
  • the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
  • the peptide chain IV-A comprises the amino acid sequence as shown in SEQ ID NO: 4
  • the peptide chain IV-B comprises the amino acid sequence as shown in SEQ ID NO: 7
  • the peptide chain IV-C comprises the amino acid sequence as shown in SEQ ID NO: 11.
  • the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the second antigen-binding domain comprises domain swaps in the form of CrossMab.
  • the bispecific antibody comprises a peptide chain V-A, a peptide chain V-B, a peptide chain V-C and a peptide chain V-D; wherein the peptide chain V-A comprises the first VL and a light chain constant region; the peptide chain V-B comprises: the first VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) , and the peptide chain V-C comprises: the second VH, a light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) , the peptide chain V-D comprises: the second VL and a heavy chain CH1 region.
  • the peptide chain V-A comprises from N-terminus to C-terminus the first VL and the light chain constant region;
  • the peptide chain V-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ;
  • the peptide chain V-C comprises from N-terminus to C-terminus the second VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer)
  • the peptide chain V-D comprises from N-terminus to C-terminus the second VL and the heavy chain CH1 region.
  • the adjacent domains of the peptide chain V-A are connected optionally with or without a linker
  • the adjacent domains of the peptide chain V-B are connected optionally with or without a linker
  • the adjacent domains of the peptide chain V-C are connected optionally with or without a linker
  • the adjacent domains of the peptide chain V-D are connected optionally with or without a linker.
  • the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) .
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54;
  • the peptide chain V-A comprises the amino acid sequence as shown in SEQ ID NO: 1
  • the peptide chain V-B comprises the amino acid sequence as shown in SEQ ID NO: 9
  • the peptide chain V-C comprises the amino acid sequence as shown in SEQ ID NO: 12
  • the sequence of the peptide chain V-D is shown in SEQ ID NO: 13.
  • the first antigen-binding domain and the second antigen-binding domain are Fab, and the Fab of the first antigen-binding domain comprises domain swaps in the form of CrossMab.
  • the bispecific antibody comprises a peptide chain VI-A, a peptide chain VI-B, a peptide chain VI-C and a peptide chain VI-D; wherein the peptide chain VI-A comprises the second VL and a light chain constant region; the peptide chain VI-B comprises: the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain VI-C comprises: the first VH, a light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) ; the peptide chain VI-D comprises: the first VL and a heavy chain CH1 region.
  • the peptide chain VI-A comprises from N-terminus to C-terminus the second VL and the light chain constant region
  • the peptide chain VI-B comprises from N-terminus to C-terminus the second VH, the CH1 region of the heavy chain, and the first Fc domain monomer (or the second Fc domain monomer)
  • the peptide chain VI-C comprises from N-terminus to C-terminus the first VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer)
  • the peptide chain VI-D comprises from N-terminus to C-terminus the first VL and the heavy chain CH1 region.
  • the adjacent domains of the peptide chain VI-A are connected optionally with or without a linker; the adjacent domains of the peptide chain VI-B are connected optionally with or without a linker; the adjacent domains of the peptide chain VI-C are connected optionally with or without a linker; and/or the adjacent domains of the peptide chain VI-D are connected optionally with or without a linker.
  • the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) .
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
  • both the first antigen-binding domain and the second antigen-binding domain are scFvs.
  • the bispecific antibody comprises a peptide chain VII-A and a peptide chain VII-B; wherein the peptide chain VII-A comprises: the first VL, the first VH and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain VII-B comprises: the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) .
  • the peptide chain VII-A comprises from N-terminus to C-terminus (i) the first VL, the first VH and the first Fc domain monomer (or the second Fc domain monomer) ; or (ii) the first VH, the first VL and the first Fc domain monomer (or the second Fc domain monomer ) ; and/or the peptide chain VII-B comprises from N-terminus to C-terminus (i) the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the second VH, the second VL and the second Fc domain monomer (or the first Fc domain monomer) .
  • the adjacent domains of the peptide chain VII-A are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain VII-B are connected optionally with or without a linker.
  • the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • G glycines
  • S serines
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the peptide chain VII-A comprises the amino acid sequence as shown in SEQ ID NO: 14, and/or the peptide chain VII-B comprises the amino acid sequence as shown in SEQ ID NO: 8.
  • the bispecific antibody comprises:
  • peptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • peptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 2
  • peptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 3;
  • peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5
  • peptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6;
  • peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7
  • peptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8;
  • peptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • peptide IV-B comprising the amino acid sequence shown in SEQ ID NO: 7
  • peptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11;
  • peptide chain V-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • peptide chain V-B comprising the amino acid sequence shown in SEQ ID NO: 9
  • peptide chain V-C comprising the amino acid sequence shown in SEQ ID NO: 12
  • peptide chain V-D comprising the amino acid sequence as shown in SEQ ID NO: 13;
  • peptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14
  • peptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.
  • the bispecific antibody comprises an enhanced tumor suppressive effect relative to a monospecific anti-c-MET antibody and/or a monospecific anti-EGFR antibody; wherein the amino acid sequences of the CDRs of the monospecific anti-c-MET antibody are identical to the amino acid sequences of the CDRs of the first antigen-binding domain, and the amino acid sequences of the CDRs of the monospecific anti-EGFR antibody are identical to the amino acid sequences of the CDRs of the second antigen-binding domain.
  • the tumor suppression effect comprises: inhibition of EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the present invention further provides an isolated nucleic acid molecule or set of nucleic acid molecules, comprising a nucleotide sequence encoding the bispecific antibody disclosed herein.
  • the present invention provides one or more vectors, comprising the isolated nucleic acid molecule or set of nucleic acid molecules encoding the bispecific antibody disclosed herein.
  • the nucleotide sequences encoding the different peptide chains of the bispecific antibody disclosed herein are located in different vector molecules.
  • the one or more vectors is a cloning vector or an expression vector.
  • the present invention further provides a host cell comprising the isolated nucleic acid molecule or set of nucleic acid molecules disclosed herein encoding the bispecific antibody disclosed herein, or the one or more vectors disclosed herein encoding the bispecific antibody disclosed herein.
  • the host cell expresses the bispecific antibody disclosed herein.
  • the present invention further provides a method for preparing the bispecific antibody disclosed herein, comprising culturing the host cell comprising the isolated nucleic acid molecule or set of nucleic acid molecules disclosed herein encoding the bispecific antibody disclosed herein, or the one or more vectors disclosed herein encoding the bispecific antibody disclosed herein under conditions allowing the expression of the bispecific antibody, and recovering the bispecific antibody from the culture of the host cell.
  • the present invention further provides a conjugate comprising the bispecific antibody disclosed herein and a conjugation moiety linked thereto.
  • the conjugation moiety is selected from a therapeutic agent (such as a cytotoxic agent, cytokine, toxin, or radionuclide) .
  • the present invention further provides a pharmaceutical composition comprising the bispecific antibody disclosed herein, or the isolated nucleic acid molecule or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition further comprises an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug with antitumor activity.
  • the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.
  • the bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as mixed components.
  • the present invention further provides use of the bispecific antibody disclosed herein, or the isolated nucleic acid molecule or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell disclosed herein, or the conjugate disclosed herein, or the pharmaceutical composition disclosed herein, in the preparation of a medicament, wherein the medicament is used for preventing and/or treating and/or acting as an adjuvant in treating diseases related to c-MET and/or EGFR in a subject, and/or for inhibiting c-MET and/or EGFR activity in vitro or in a subject.
  • the disease related to c-MET and/or EGFR is cancer.
  • the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification.
  • cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cavity cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.
  • the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, one or more vectors, host cell, conjugate, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, such as administered simultaneously, separately or sequentially.
  • the another pharmaceutically active agent is a drug with an anti-tumor activity.
  • the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.
  • the subject is resistant to erlotinib, gefitinib, afatinib, CO-1686, AZD9192 or cetuximab treatment.
  • the present invention further provides a method for inhibiting the activity of c-MET and/or EGFR in a cell, comprising contacting the cell with the bispecific antibody disclosed herein, or the isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein, or the pharmaceutical composition comprising the bispecific antibody disclosed herein, or the isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein.
  • the cells are cells expressing c-MET and/or EGFR, such as tumor cells.
  • the present invention further provides a method for preventing and/or treating and/or acting as an adjuvant in treating diseases associated with c-MET and/or EGFR in a subject, wherein the method comprises administering to the subject in need thereof an effective amount of the bispecific antibody disclosed herein, or the isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein, or the pharmaceutical composition comprising the bispecific antibody disclosed herein, or the isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein.
  • the disease associated with c-MET and/or EGFR is cancer.
  • the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification.
  • the cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cavity cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.
  • the method further comprises administering to the subject a second therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof; optionally, the second therapy may be applied simultaneously, separately or sequentially.
  • the subject is resistant to a treatment using erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
  • Fig. 1A Schematic diagram of the bispecific antibody structure.
  • Fig. 1B Schematic diagram of the bispecific antibody structure.
  • Fig. 1C Schematic diagram of the bispecific antibody structure.
  • Fig. 1D Schematic diagram of the bispecific antibody structure.
  • Fig. 2A Determination of the binding activity of bispecific antibodies to human non-small cell lung cancer cells HCC827.
  • Fig. 2B Determination of the binding activity of bispecific antibodies to human non-small cell lung cancer cells HCC827.
  • Fig. 3A Determination of the binding activity of bispecific antibodies to human gastric cancer cell line MKN45.
  • Fig. 3B Determination of the binding activity of bispecific antibodies to human gastric cancer cell line MKN45.
  • Fig. 4A Determination of the binding activity of bispecific antibodies to human lung adenocarcinoma NCI-H1975 cells.
  • Fig. 4B Determination of the binding activity of bispecific antibodies to human lung adenocarcinoma NCI-H1975 cells.
  • the application provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR.
  • the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH) , and the first VL and the first VH collectively form a domain capable of specifically binding c-MET.
  • the second antigen-binding domain comprises a second VL and a second VH, and the second VL and the second VH together form a domain capable of specifically binding EGFR.
  • said first antigen-binding domain and said second antigen-binding domain are each independently selected from an scFv, a Fab, and an scFab.
  • the Fab domains may be arranged according to their native orientation or comprise domain substitutions or swaps that promote correct VH and VL pairing (e.g., domain swaps in the Crossmab format) .
  • the domain swap of the CrossMab format is selected from:
  • CrossMab Fab In Fab, the antibody light chain CL-VL and the antibody heavy chain CH1-VH are swapped;
  • CrossMab CH1-CL In Fab, the CH and CL of the antibody are swapped.
  • CrossMab is a method for preventing mispairing of light and heavy chains, see US patent US9266967B2, which is incorporated herein by reference in its entirety.
  • This method mainly utilizes the principle that the same parts of an antibody repel each other, that is, VH and VH repel each other, CH1 and CH1 repel each other, CL and CL repel each other, and VL and VL repel each other.
  • the purpose of preventing light chain mismatch is achieved by regional interchange of the heavy and light chains of one of the Fab in bispecific antibodies (e.g., light chain CL-VL is exchanged with heavy chain CH1-VH, VL and VH are interchanged, or CH and CL are interchanged) .
  • the bispecific antibody further comprises an Fc domain.
  • the Fc domain comprises first and second Fc domain monomers.
  • the first and second Fc domain monomers comprise one or more modifications that promote heterodimerization of the Fc domain monomers.
  • the Fc domain comprises a first Fc domain monomer comprising modifications that form a knob structure and a second Fc domain monomer comprising modifications that form a hole structure, wherein the hole structure can be paired with the knob structure to form a heterodimeric Fc domain.
  • hole structure can be paired with the knob structure to form a “knob in hole structure” , which can be used to reduce heavy chain mismatch in the bispecific antibody.
  • a “knob” structure and a “hole” structure can be introduced by mutation in the corresponding positions of the CH3 domains of two Fc domain monomers to form a specific interaction interface between two Fc domain monomers (see Ridgway et al., Protein Eng., 9: 617-621 (1996) ; WO 2006/028936; incorporated herein by reference in its entirety) .
  • Amino acid substitutions that can be used to form a “knob” structure include, but are not limited to, S354C and T366W, wherein the numbering is according to the Eu numbering scheme.
  • Amino acid substitutions that can be used to form a “hole” structure include, but are not limited to, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the Eu numbering scheme. Due to mutual attraction between the “knob” structure and “hole” structure, as well as mutual repulsion between the “knob” structure and the “knob” structure, the “knob in hole structure” can effectively reduce the heavy chain mismatch in the bispecific antibody.
  • the Fc domain monomer is derived from the Fc domain of a human immunoglobulin, and comprises modifications that form a knob structure or a hole structure.
  • the human immunoglobulin is IgG1, IgG2, IgG3 or IgG4.
  • the Fc domain monomer comprises one or more modifications that form a knob structure, wherein the one or more modifications comprise S354C and/or T366W amino acid substitutions, wherein the numbering is according to the Eu numbering scheme.
  • the Fc domain monomer comprises S354 and T366W amino acid substitutions, wherein the numbering is according to the Eu numbering scheme.
  • the Fc domain monomer comprises one or more modifications that form a hole structure, wherein the one or more modifications comprise Y349C, T366S, L368A, and/or Y407V amino acid substitutions, wherein the numbering is according to the Eu numbering scheme. In certain embodiments, the Fc domain monomer comprises Y349C, T366S, L368A, and Y407V amino acid substitutions, wherein the numbering is according to the Eu numbering scheme.
  • the Fc domain further has altered effector function (e.g., enhanced ADCC activity) compared to a wild-type Fc region.
  • altered effector functions may be introduced by, for example, mutation or chemical modification.
  • the first Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 49 or 51
  • the second Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 50 or 52.
  • said first antigen-binding domain and said second antigen-binding domain are each linked to one of the first and second Fc domain monomers.
  • the first antigen-binding domain is linked to the first Fc domain monomer, and the second antigen-binding domain is linked to the second Fc domain monomer. In certain embodiments, the first antigen-binding domain is linked to the second Fc domain monomer, and the second antigen-binding domain is linked to the first Fc domain monomer.
  • the individual CDRs of an antigen-binding domain disclosed herein can be determined according to any CDR numbering scheme known in the art.
  • an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the Kabat numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the Kabat numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the Chothia numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the Chothia numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the MacCallum numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the MacCallum numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the IMGT numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the IMGT numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the AbM numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the AbM numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the AHo numbering scheme.
  • an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the AHo numbering scheme.
  • the individual CDRs of an antigen-binding domain of an antibody provided herein are each independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the antigen-binding domain, wherein the structural analysis identifies residues in the variable region (s) predicted to make contact with an epitope region of c-MET or EGFR.
  • the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 17 or 59, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 18 or 60 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat
  • the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 17, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 18 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat, Chothia, Mac
  • the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 59, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 60 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat, Chothia
  • the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 17, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 60 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat, Chothia, Mac
  • the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 59, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 18 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat, Chothia
  • the first VL comprises:
  • LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34
  • LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36
  • LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; wherein the CDRs are defined by the Kabat numbering system;
  • LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34
  • LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36
  • LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; wherein the CDRs are defined by the Chothia numbering system;
  • LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34
  • LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36
  • LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; wherein the CDRs are defined by the Abm numbering system; or
  • LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35
  • LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37
  • LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; wherein said CDRs are defined by the IMGT numbering system; and/or
  • the first VH comprises:
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 39
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 43
  • HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47; wherein the CDRs are defined by the Kabat numbering system;
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: in 44
  • HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47; wherein the CDRs are defined by the Chothia numbering system;
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 42
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46
  • HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47; wherein the CDRs are defined by the Abm numbering system; or
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45
  • HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48; where the CDRs are defined by the IMGT numbering system.
  • the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 17 or 59.
  • the first VL comprises an amino acid sequence set forth in SEQ ID NO: 17 or 59.
  • the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 or 59.
  • the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 18 or 60.
  • the first VH comprises an amino acid sequence set forth in SEQ ID NO: 18 or 60.
  • the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18 or 60.
  • the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 17 or 59 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100%(e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 18 or 60.
  • the first VL comprises an amino acid sequence set forth in SEQ ID NO: 17 or 59 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 18 or 60.
  • the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 or 59 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18 or 60.
  • the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 17 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 18.
  • the first VL comprises an amino acid sequence set forth in SEQ ID NO: 17 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18.
  • the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 59 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 60.
  • the first VL comprises an amino acid sequence set forth in SEQ ID NO: 59 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 59 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 60.
  • the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 17 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 60.
  • the first VL comprises an amino acid sequence set forth in SEQ ID NO: 17 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 60.
  • the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 59 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 18.
  • the first VL comprises an amino acid sequence set forth in SEQ ID NO: 59 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 59 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18.
  • the second VL comprises:
  • LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19
  • LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21
  • LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; wherein the CDRs are defined by the Kabat numbering system;
  • LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19
  • LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21
  • LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; wherein the CDRs are defined by the Chothia numbering system;
  • LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19
  • LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21
  • LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; wherein the CDRs are defined by the Abm numbering system; or
  • LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20
  • LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22
  • LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; wherein said CDRs are defined by the IMGT numbering system; and/or
  • the second VH comprises:
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; wherein the CDRs are defined by the Kabat numbering system;
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29,
  • HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; wherein the CDRs are defined by the Chothia numbering system;
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; wherein the CDRs are defined by the Abm numbering system; or
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30,
  • HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; where the CDRs are defined by the IMGT numbering system.
  • the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 15.
  • the first VL comprises an amino acid sequence set forth in SEQ ID NO: 15.
  • the first VL consists of the amino acid sequence set forth in SEQ ID NO: 15.
  • the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 16.
  • the first VH comprises an amino acid sequence set forth in SEQ ID NO: 16.
  • the first VH consists of the amino acid sequence set forth in SEQ ID NO: 16.
  • the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 15 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 16.
  • the first VL comprises an amino acid sequence set forth in SEQ ID NO: 15 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 15 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 16.
  • the instant disclosure provides a bispecific antibody that cross-competes for binding to c-MET and/or EGFR with any of the antibodies described herein. In certain embodiments, the instant disclosure provides a bispecific antibody that binds to the same or an overlapping epitope of c-MET and/or EGFR as an antibody described herein.
  • the epitope of an antibody can be determined by, e.g., NMR spectroscopy, surface plasmon resonance X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry) , array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping) .
  • NMR spectroscopy surface plasmon resonance X-ray diffraction crystallography studies
  • ELISA assays hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry)
  • array-based oligo-peptide scanning assays e.g., site-directed mutagenesis mapping
  • crystallization may be accomplished using any of the known methods in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4) : 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are herein incorporated by reference in their entireties) .
  • Antibody antigen crystals may be studied using well known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.
  • the epitope of an antibody is determined using alanine scanning mutagenesis studies.
  • bispecific antibodies that recognize and bind to the same or overlapping epitopes of c-MET and/or EGFR can be identified using routine techniques such as an immunoassay, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competition binding assays also can be used to determine whether two antibodies have similar binding specificity for an epitope.
  • such an assay involves the use of purified antigen (e.g., c-MET or EGFR) bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin.
  • a test immunoglobulin is present in excess.
  • a competing antibody is present in excess, it will inhibit specific binding of a reference or antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more.
  • a competition binding assay can be configured in a large number of different formats using either labeled antigen or labeled antibody.
  • the antigen is immobilized on a 96-well plate.
  • the ability of unlabeled antibodies to block the binding of labeled antibodies to the antigen is then measured using radioactive or enzyme labels.
  • one, two, or more mutations are introduced into an Fc region (e.g., a CH2 domain (residues 231-340 of human IgG1) ) and/or a CH3 domain (residues 341-447 of human IgG1, numbered according to the EU numbering system) and/or a hinge region (residues 216-230, numbered according to the EU numbering system) , to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • Fc region e.g., a CH2 domain (residues 231-340 of human IgG1)
  • CH3 domain residues 341-447 of human IgG1, numbered according to the EU numbering system
  • a hinge region residues 216-230, numbered according to the EU numbering system
  • one, two, or more mutations are introduced into an Fc region to, e.g., reduce or ablate effector function of the Fc region.
  • the antibodies comprise an Fc region comprising: (i) E233A and L235A (EALA) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (ii) L234A and L235A (LALA) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (iii) L234A L235A D265S (LALADS) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (iv) L234A L235A P329G (LALAPG) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (v) L234A L235A P329A (LALAPA) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (vi)
  • any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody described herein having two heavy chain constant regions.
  • polypeptides comprising one or more LCDR, HCDR, VL, and/or VH amino acid sequences as described above.
  • the polypeptide comprises the LCDR1, LCDR2, and/or LCDR3 of the VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59, as determined by any of the methods discussed above.
  • the polypeptide comprises the LCDR1, LCDR2, and LCDR3 of the VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59.
  • the polypeptide comprises the LCDR1, LCDR2, and/or LCDR3 amino acid sequences set forth in SEQ ID NOs: 34, 36, and 38; 35, 37, and 38; 19, 21, and 23; or 20, 22, and 23, respectively.
  • the polypeptide comprises the HCDR1, HCDR2, and/or HCDR3 of the VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60, as determined by any of the methods discussed above. In certain embodiments, the polypeptide comprises the HCDR1, HCDR2, and HCDR3 of the VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60.
  • the polypeptide comprises the HCDR1, HCDR2, and/or HCDR3 amino acid sequences set forth in SEQ ID NOs: 39, 43, and 47; 40, 44, and 47; 42, 46, and 47; 41, 45, and 48; 24, 28, and 32; 25, 29, and 32; 27, 31, and 32; or 26, 30, and 33, respectively.
  • the polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17, and 59.
  • the polypeptide comprises the VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17, and 59.
  • the polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18, and 60.
  • the polypeptide comprises the VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18, and 60.
  • the first antigen-binding domain is a Fab and the second antigen-binding domain is an scFv.
  • the bispecific antibody comprises polypeptide chain I-A, polypeptide chain I-B, and polypeptide chain I-C; wherein (a) the elements comprised in polypeptide chain I-A comprise a first VL and a first CL; the elements comprised in polypeptide chain I-B comprise a first VH, a first heavy chain CH1 region, and a first Fc monomer; and the elements comprised in polypeptide chain I-C comprise a second VL, a second VH, a second heavy chain CH1 hinge region, and a second Fc monomer; or (b) the elements comprised in polypeptide chain I-A comprise a first VL and a CL; the elements comprised in polypeptide chain I-B comprise a first VH, a first heavy chain CH1 region, and a second Fc monomer; and the elements comprised in polypeptide chain I-C comprise a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc monomer.
  • the elements comprised in polypeptide chain I-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain I-B from the N-terminal end to the C-terminal end comprises the first VH, the first heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain I-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, the second heavy chain CH1 hinge region, and the second Fc monomer, or (ii) the second VH, the second VL, the second heavy chain CH1 hinge region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain I-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain I-B from the N-terminal end to the C-terminal end comprise the first VH
  • the adjacent elements comprising the polypeptide chain I-A are optionally connected to each other by peptide linkers or not; the adjacent elements comprising the polypeptide chain I-B are optionally connected to each other by peptide linkers or not; and/or the adjacent elements comprising the polypeptide chain I-C are optionally connected by a peptide linker or not.
  • the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) .
  • each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues.
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
  • each peptide linker may independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues.
  • each peptide linker may independently comprise the peptide GGGG (SEQ ID NO: 61) .
  • the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprises the amino acid sequence shown in SEQ ID NOs: 55-58, 61 and 63-68.
  • the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53.
  • the first heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54.
  • the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 17
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
  • polypeptide chain I-A comprises the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain I-B comprises the amino acid sequence shown in SEQ ID NO: 2 or 9
  • polypeptide chain I-C comprises the amino acid sequence shown in SEQ ID NO: 3 or 10.
  • the first antigen-binding domain is an scFv and the second antigen-binding domain is an Fab.
  • the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein (a) the elements comprised in polypeptide chain II-A comprise a second VL and a second CL; the elements comprised in polypeptide chain II-B comprise a second VH, a second heavy chain CH1 region, and a first Fc monomer; the elements comprised in polypeptide chain II-C comprise a first VL, a first VH, a first heavy chain CH1 hinge region, and a second Fc monomer, or (b) the elements comprised in polypeptide chain II-A comprise a second VL and a second CL; the elements comprised in polypeptide chain II-B comprise a second VH, a second heavy chain CH1 region, and a second Fc monomer; and the elements comprised in polypeptide chain II-C comprise a first VL, a first VH, a first heavy chain CH1 hinge region, and a first Fc monomer.
  • the elements comprised in the polypeptide chain II-A from the N-terminal end to the C-terminal end comprise the second VL and the second CL; the elements comprised in polypeptide chain II-B from the N-terminal end to the C-terminal end comprise the second VH, the second heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain II-C from the N-terminal end to the C-terminal end comprise: (i) the first VL, the first VH, the first heavy chain CH1 hinge region, and the second Fc monomer, or (ii) the first VH, the first VL, the first heavy chain CH1 hinge region, and the second Fc monomer, or (b) the elements comprised in polypeptide chain II-A from the N-terminal end to the C-terminal end comprise the second VL and the second CL; the elements comprised in polypeptide chain II-B from the N-terminal end to the C-terminal end comprise the second VL and the second CL;
  • the adjacent elements comprised in the polypeptide chain II-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain II-B are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in the polypeptide chain II-C are optionally connected to each other by a peptide linker or not.
  • the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) .
  • each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues.
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
  • the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53
  • the first and second heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54
  • the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 17
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
  • the peptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4
  • the peptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 5 or 7
  • the peptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 6 or 8.
  • the first antigen-binding domain is a Fab and the second antigen-binding domain is a scFab (single chain Fab) .
  • the bispecific antibody comprises polypeptide chain III-A, polypeptide chain III-B, and polypeptide chain III-C; wherein (a) the elements comprised in polypeptide chain III-A comprise the first VL and the first CL; the elements comprised in polypeptide chain III-B comprise the first VH, the first heavy chain CH1 region, and the first Fc monomer; and the elements comprised in polypeptide chain III-C comprise the second VL, the second CL, the second VH, the second heavy chain CH1 region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain III-A comprise the first VL and the first CL; the elements comprised in polypeptide chain III-B comprise the first VH, the first heavy chain CH1 region, and the second Fc monomer; and the elements comprised in polypeptide chain III-C comprise the second VL, the second CL, the second VH, the second heavy chain CH1 region, and the first Fc monomer.
  • the elements comprised in polypeptide chain III-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain III-B from the N-terminal end to the C-terminal end comprise the first VH, the first heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain III-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second CL, the second VH, the second heavy chain CH1 region, and the second Fc monomer; or (ii) the second VH, the second heavy chain CH1 region, the second VL, the second CL, and the second Fc monomer; or (b) the elements comprised in polypeptide chain III-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain III-B from the N-terminal end to the C-terminal
  • the adjacent elements comprising the polypeptide chain III-A are optionally connected to each other by a peptide linker or not, and the adjacent elements comprising the polypeptide chain III-B are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprising the polypeptide chain III-C are optionally connected to each other by a peptide linker or not.
  • the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) .
  • each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues.
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
  • the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53
  • the first and second heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54
  • the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 17
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
  • the first antigen-binding domain is a scFab and the second antigen-binding domain is a Fab.
  • the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B; and polypeptide chain IV-C, wherein (a) the elements comprised in polypeptide chain IV-A comprise the second VL and second CL; the elements comprised in polypeptide chain IV-B comprise the second VH, the second heavy chain CH1 region, and the first Fc monomer; and the elements comprised in polypeptide chain IV-C comprise the first VL, the first CL, the first VH, the first heavy chain CH1 region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain IV-A comprise the second VL and second CL; the elements comprised in polypeptide chain IV-B comprise the second VH, the second heavy chain CH1 region, and the second Fc monomer; and the elements comprised in polypeptide chain IV-C comprise the first VL, the first CL, the first VH, the first heavy chain CH1 region, and the first Fc monomer.
  • the elements comprised in polypeptide chain IV-A from the N-terminal end to the C-terminal end comprise the second VL and the second CL; the elements comprised in polypeptide chain IV-B from the N-terminal end to the C-terminal end comprise the second VH, the second heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain IV-C from the N-terminal end to the C-terminal end comprise the first VL, the first CL, the first VH, the first heavy chain CH1 region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain IV-A from the N-terminal end to the C-terminal end comprise the second VL and the second CL; the elements comprised in polypeptide chain IV-B from the N-terminal end to the C-terminal end comprise the second VL and the second CL, the second VH, the second heavy chain CH1 region, and the second Fc monomer; and/or the elements comprise
  • the adjacent elements comprising the polypeptide chain IV-A are optionally connected to each other by a peptide linker or not; the adjacent elements of the polypeptide chain IV-B are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprising the polypeptide chain IV-C are optionally connected to each other by a peptide linker or not.
  • the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) .
  • each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues.
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
  • the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53
  • the first and second heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54
  • the first heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 59 and the first VH comprises the amino acid sequence shown in SEQ ID NO: 60.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 17
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
  • the peptide chain IV-A comprises the amino acid sequence shown in SEQ ID NO: 4
  • the peptide chain IV-B comprises the amino acid sequence shown in SEQ ID NO: 7
  • the peptide chain IV-C comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the first antigen-binding domain and the second antigen-binding domain are Fabs
  • the Fab of the second antigen-binding domain comprises a domain swap in the form of a CrossMab Fab .
  • the Fab of the second antigen-binding domain comprises a domain swap of the VH and VL in the form of CrossMab VH-VL .
  • the Fab of the second antigen-binding domain comprises a domain swap of the CH1 and CL in the form of CrossMab CH1-CL .
  • the bispecific antibody in the form of CrossMab CH1-CL comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D; wherein (a) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a first Fc monomer; the elements comprised in polypeptide chain V-C comprise a second VH, a second CL, a second heavy chain CH1 hinge region, and a second Fc monomer; and the polypeptide chain V-D comprise a second VL and a second heavy chain CH1 region; or (b) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise the first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised
  • the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the first CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain V-C comprise from N-terminus to C-terminus the second VH, the second CL, the second heavy chain CH1 hinge region, and the second Fc monomer, and/or the elements comprised in polypeptide chain V-D comprise from N-terminus to C-terminus the second VL and second heavy chain CH1 region; or (b) the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in
  • the adjacent elements comprised in the polypeptide chain V-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-B are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-C are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in the polypeptide chain V-D are optionally connected to each other by a peptide linker or not.
  • the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) .
  • each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues.
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
  • the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53
  • the first and second heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54
  • the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 17
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
  • polypeptide chain V-A comprises the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain V-B comprises the amino acid sequence shown in SEQ ID NO: 9
  • polypeptide chain V-C comprises the amino acid sequence shown in SEQ ID NO: 12
  • polypeptide chain V-D comprises the amino acid sequence shown in SEQ ID NO: 13.
  • the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the first antigen-binding domain comprises a domain swap in the form of a CrossMab.
  • the Fab of the first antigen-binding domain comprises a domain swap in the form of CrossMab CH1-CL .
  • the bispecific antibody comprises polypeptide chain VI-A, polypeptide chain VI-B, polypeptide chain VI-C, and polypeptide chain VI-D; wherein (a) the elements comprised in polypeptide chain VI-A comprise the second VL and the CL; the elements comprised in polypeptide chain VI-B comprise the second VH, the heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain VI-C comprise the first VH, the CL, first heavy chain CH1 hinge region, and the second Fc monomer; and the elements comprised in polypeptide chain VI-D comprise the first VL and the heavy chain CH1 region; or (b) the elements comprised in polypeptide chain VI-A comprise the second VL and the CL; the elements comprised in polypeptide chain VI-B comprise the second VH, the heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain VI-C comprise the first VH, the CL, the first heavy chain CH1 hinge region, and the
  • the elements comprised in polypeptide chain VI-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain VI-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain VI-C from the N-terminal end to the C-terminal end comprise the first VH, the CL, the first heavy chain CH1 hinge region, and the second Fc monomer; and/or the elements comprised in polypeptide chain VI-D from the N-terminal end to the C-terminal end comprise the first VL and the heavy chain CH1 region; or (b) the elements comprised in polypeptide chain VI-A from the N-terminal end to the C-terminal end comprises the second VL and the CL; the peptide chain VI-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH
  • the adjacent elements comprised in the polypeptide chain VI-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain VI-B are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain VI-C are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in the polypeptide chain VI-D are optionally connected to each other by a peptide linker or not.
  • the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) .
  • each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues.
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) .
  • the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprises the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the CL comprises the amino acid sequence shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54, and/or the first heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 17
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
  • the bispecific antibody in the form of CrossMab VH-VL comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D; wherein (a) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a first Fc monomer; the elements comprised in polypeptide chain V-C comprise a second VL, a second heavy chain CH1 hinge region, and a second Fc monomer; and the polypeptide chain V-D comprise a second VH and second CL; or (b) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-C comprise the second
  • the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the first CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain V-C comprise from N-terminus to C-terminus the second VL, the second heavy chain CH1 hinge region, and the second Fc monomer, and/or the elements comprised in polypeptide chain V-D comprise from N-terminus to C-terminus the second VH and second CL; or (b) the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the first CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-C
  • the adjacent elements comprised in the polypeptide chain V-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-B are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-C are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in the polypeptide chain V-D are optionally connected to each other by a peptide linker or not.
  • the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) .
  • each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues.
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
  • the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the first and second CL comprise the amino acid sequence shown in SEQ ID NO: 53
  • the first and second heavy chain CH1 region comprise the amino acid sequence shown in SEQ ID NO: 54
  • the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 17
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
  • the bispecific antibody in the form of CrossMab Fab-Fab comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D; wherein (a) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a first Fc monomer; the elements comprised in polypeptide chain V-C comprise a second VL, a second CL, a second heavy chain CH1 hinge region, and a second Fc monomer; and the polypeptide chain V-D comprise a second VH and second heavy chain CH1 region; or (b) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-
  • the elements comprised in polypeptide chain V-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain V-C comprise from N-terminus to C-terminus the second VL, the second CL, the second heavy chain CH1 hinge region, and the second Fc monomer, and/or the elements comprised in polypeptide chain V-D comprise from N-terminus to C-terminus the second VH and second heavy chain CH1 region; or (b) the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the first CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the second Fc monomer;
  • the adjacent elements comprised in the polypeptide chain V-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-B are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-C are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in the polypeptide chain V-D are optionally connected to each other by a peptide linker or not.
  • the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) .
  • each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues.
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
  • the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  • the first and second CL comprise the amino acid sequence shown in SEQ ID NO: 53
  • the first and second heavy chain CH1 region comprise the amino acid sequence shown in SEQ ID NO: 54
  • the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 17
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
  • the first antigen-binding domain and the second antigen-binding domain are both scFv.
  • the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein (a) the elements comprised in polypeptide chain VII-A comprise the first VL, the first VH, the first heavy chain CH1 hinge region, and the first Fc monomer; and the elements comprised in polypeptide chain VII-B comprise the second VL, the second VH, the second heavy chain CH1 hinge region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain VII-A comprise the first VL, the first VH, the first heavy chain CH1 hinge region and the second Fc monomer; and the elements comprised in polypeptide chain VII-B comprise the second VL, the second VH, the second heavy chain CH1 hinge region, and the first Fc monomer.
  • the elements comprised in polypeptide chain VII-A from the N-terminal end to the C-terminal end comprise (i) the first VL, the first VH, the first heavy chain CH1 hinge region, and the first Fc monomer, or (ii) the first VH, the first VL, the first heavy chain CH1 hinge region, and the first Fc monomer; and/or the elements comprised in polypeptide chain VII-B from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, the second heavy chain CH1 hinge region, and the second Fc monomer, or (ii) the second VH, the second VL, the second heavy chain CH1 hinge region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain VII-A from the N-terminal end to the C-terminal end comprise (i) the first VL, the first VH, the first heavy chain CH1 hinge region, and the second Fc monomer, or (ii)
  • the adjacent elements comprising the polypeptide chain VII-A are optionally connected to each other by a peptide linker or not, and/or the adjacent elements comprising the polypeptide chain VII-B are optionally connected to each other by a peptide linker or not.
  • the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) .
  • each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues.
  • the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
  • the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n , wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  • the peptide linkers each independently comprises the amino acid sequence shown in SEQ ID NO: 55-58, 61, or 63-68.
  • the first and second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first VH comprises the amino acid sequence shown in SEQ ID NO: 18
  • the first VL comprises the amino acid sequence shown in SEQ ID NO: 17
  • the second VH comprises the amino acid sequence shown in SEQ ID NO: 16
  • the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
  • polypeptide chain VII-A comprises the amino acid sequence shown in SEQ ID NO: 14
  • polypeptide chain VII-B comprises the amino acid sequence shown in SEQ ID NO: 8.
  • bispecific antibody comprising the ADC comprises:
  • polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 2
  • polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 3;
  • polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 9
  • polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 10;
  • polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5
  • polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6;
  • polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7
  • polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8;
  • polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7
  • polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11;
  • polypeptide chain V-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain V-B comprising the amino acid sequence shown in SEQ ID NO: 9
  • polypeptide chain V-C comprising the amino acid sequence shown in SEQ ID NO: 12
  • polypeptide chain V-D comprising the amino acid sequence shown in SEQ ID NO: 13;
  • polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14
  • polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.
  • the bispecific antibody has an enhanced tumor suppressive effect relative to the monospecific anti-c-MET antibody and/or the monospecific anti- EGFR antibody; wherein the CDR amino acid sequences of CDRs of the monospecific anti-c-MET are identical to the amino acid sequences of CDRs sequence of the first antigen-binding domain, and the amino acid sequences of CDRs sequence of the monospecific anti-EGFR antibody are identical to the CDR amino acid sequences of CDRs of the second antigen-binding domain.
  • the tumor suppressive effects include: Inhibition of EGFR and c-MET signaling, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the application provides an isolated nucleic acid molecule or set of nucleic acid molecules comprising a nucleotide sequence encoding a bispecific antibody as described above.
  • the nucleotide sequence can be replaced according to the codon degeneracy.
  • the nucleotide sequence is codon-optimized.
  • the present application provides a vector comprising an isolated nucleic acid molecule or set of nucleic acid molecules as described above.
  • vectors of the invention are, for example, plasmids, cosmids, phages, lentiviruses, and the like.
  • the vector is capable of expressing a bispecific antibody of the invention in a subject (e.g., a mammal, e.g., a human) .
  • the vector comprises a first nucleotide sequence encoding the first peptide chain of the bispecific antibody of the present invention and a second nucleotide sequence encoding the second peptide chain of the bispecific antibody of the present invention, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different vectors.
  • the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.
  • the vector comprises a first nucleotide sequence encoding the first peptide chain of the bispecific antibody of the present invention, a second nucleotide sequence encoding the second peptide chain of the bispecific antibody of the present invention and a third nucleotide sequence encoding the third peptide chain of the bispecific antibody of the present invention, wherein the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence exist in the same or on different vectors.
  • the vector of the present invention comprises a first vector comprising the first nucleotide sequence, a second vector comprising the second nucleotide sequence, and a third vector comprising the third nucleotide sequence.
  • the vector comprises a first nucleotide sequence encoding the first peptide chain of the bispecific antibody of the present invention, a second nucleotide sequence encoding the second peptide chain of the bispecific antibody of the present invention, a third nucleotide sequence encoding the third peptide chain of the bispecific antibody of the present invention and a fourth nucleotide sequence encoding the fourth peptide chain of the bispecific antibody of the present invention, wherein the first nucleotide sequence, the second nucleotide sequence, said third nucleotide sequence and said fourth nucleotide sequence are present on the same or different vectors from each other.
  • the vector of the present invention comprises a first vector comprising the first nucleotide sequence, a second vector comprising the second nucleotide sequence, a third vector comprising the third nucleotide sequence, and a fourth vector comprising the fourth nucleotide sequence.
  • the nucleotide sequences encoding the different peptide chains of the bispecific antibody are located on different vector molecules.
  • the vector is a cloning vector or an expression vector.
  • the application provides a host cell comprising an isolated nucleic acid molecule or set of nucleic acid molecules as described above, or a vector as described above.
  • Host cells can be eukaryotic cells (e.g., mammalian cells, insect cells, yeast cells) or prokaryotic cells (e.g., E. coli) .
  • Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.
  • Suitable insect cells include, but are not limited to, Sf9 cells.
  • the host cell of the invention is a mammalian cell, such as a CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44) .
  • the bispecific antibody of the present invention can be prepared by various methods known in the art, for example, by genetic engineering and recombination techniques. For example, a DNA molecule encoding the peptide chain of the bispecific antibody of the present invention is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, which is then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions, and express the bispecific antibody of the present invention.
  • the present application provides a method for preparing the above-mentioned bispecific antibody, which comprises, under conditions that allow the expression of the bispecific antibody, culturing the above-mentioned host cell, and recovering the bispecific antibody from the culture of the host cell.
  • a bispecific antibody of the invention may be derivatized, e.g. linked to another molecule (e.g. another polypeptide or protein) .
  • another molecule e.g. another polypeptide or protein
  • the bispecific antibodies of the invention are also intended to include such derivatized forms.
  • a bispecific antibody of the invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage or otherwise) to one or more other molecular moieties, such as another antibody, a detection reagent, a pharmaceutical reagent, and/or proteins or polypeptides (e.g., avidin or polyhistidine tags) capable of mediating the binding of the bispecific antibody to another molecule.
  • the present application provides a conjugate comprising the bispecific antibody as described above and a conjugation moiety linked thereto.
  • the conjugation moiety is selected from a therapeutic agent (e.g., a cytotoxic agent, cytokine, toxin, or radionuclide) .
  • a therapeutic agent e.g., a cytotoxic agent, cytokine, toxin, or radionuclide
  • the conjugation moiety is selected from substances capable of improving the biological properties of the bispecific antibody (such as increasing the serum half-life) , such as chemical groups such as polyethylene glycol (PEG) , formazan group or ethyl group, or sugar group.
  • substances capable of improving the biological properties of the bispecific antibody such as increasing the serum half-life
  • chemical groups such as polyethylene glycol (PEG) , formazan group or ethyl group, or sugar group.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody as described above, or the isolated nucleic acid molecule or set of nucleic acid molecules as described above, or the vector as described above, or the host cell as described above, or the conjugate as described above, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical compositions further comprise an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug having antineoplastic activity.
  • the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
  • the bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as mixed components.
  • the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate in the pharmaceutical composition of the invention is sufficient (e.g., in a subject) to exert a tumor suppressive effect (e.g., a tumor suppressive effect superior to a monospecific anti-c-MET antibody and/or a monospecific anti-EGFR antibody, wherein the amino acid sequences of CDRs of the monospecific anti-c-MET antibody are identical to the amino acid sequences of CDRs of the first antigen-binding domain, and the amino acid sequences of CDRs of the monospecific anti-EGFR antibody are identical to the amino acid sequences of CDRs of the second antigen-binding domain) .
  • a tumor suppressive effect e.g., a tumor suppressive effect superior to a monospecific anti-c-MET antibody and/or a monospecific anti-EGFR antibody, wherein the amino acid sequences of CDRs of the monospecific anti-c-MET antibody are identical to the amino acid sequences of
  • the tumor suppressive effects include: Inhibition of EGFR and c-MET signaling, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the application provides use of the bispecific antibody as described above, or the isolated nucleic acid molecule or set of nucleic acid molecules as described above, or the vector as described above, or the host cell as described above, or the conjugate as described above, or the pharmaceutical composition as described above in the preparation of medicine, wherein the medicine is used for preventing and/or treating and/or acting as an adjuvant in treating diseases related to c-MET and/or EGFR, and/or for inhibiting the activity of c-MET and/or EGFR in vitro or in a subject.
  • the disease related to c-MET and/or EGFR is cancer.
  • the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification.
  • the cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.
  • the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, such as administered simultaneously, separately, or sequentially.
  • the additional pharmaceutically active agent is a drug having antineoplastic activity.
  • the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
  • the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
  • the present application provides a method for inhibiting the activity of c-MET and/or EGFR in a cell, comprising contacting the cell with the bispecific antibody as described above, or the isolated nucleic acid molecule or set of nucleic acid molecules as described above, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned conjugate, or the above-mentioned pharmaceutical composition.
  • the cells are cells expressing c-MET and/or EGFR, such as tumor cells.
  • the present application provides a method for preventing and/or treating and/or acting as an adjuvant in treating a disease related to c-MET and/or EGFR in a subject, wherein the method comprises administering to the subject in need thereof an effective amount of the above-mentioned bispecific antibody, or the above-mentioned isolated nucleic acid molecule or set of nucleic acid molecules, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned conjugate, or pharmaceutical composition as described above.
  • the disease related to c-MET and/or EGFR is cancer.
  • the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification.
  • the cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.
  • the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and any combination thereof.
  • the second therapy may be applied simultaneously, separately or sequentially to the methods described above.
  • the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
  • the bispecific antibody of the present invention and the pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablet, pill, suspension, emulsion, solution, gel, capsule, powder, granule, elixir, lozenge, suppository, injection (including solution for injection, sterile powder for injection and concentrated solution for injection) , inhalant, spray, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • Pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage.
  • a preferred dosage form is injection. Such injection can be a sterile solution for injection.
  • the sterile solution for injection can be prepared by incorporating in an appropriate solvent a necessary dose of the bispecific antibody or the pharmaceutical composition of the present invention, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusting agent, surfactant, adjuvant, ionic strength enhancer, isotonic agent, preservative, diluent, or any combination thereof) , followed by filter sterilization.
  • a sterile solution for injection can be prepared as sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) for ease of storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as sterile pyrogen-free water.
  • bispecific antibodies of the present invention may be presented in pharmaceutical compositions in unit dosage form for ease of administration.
  • the bispecific antibody and the pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, groin, intravesical, topical (e.g., powder, ointment, or drops) , or nasal route.
  • the preferred route/mode of administration is parenteral (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection) .
  • the bispecific antibody and the pharmaceutical composition of the present invention are administered by intravenous injection or injection.
  • the pharmaceutical composition of the present invention may comprise a “therapeutically effective amount” or “prophylactically effective amount” of the bispecific antibody of the present invention.
  • the “prophylactically effective amount” means an amount sufficient to prevent, arrest, or delay the occurrence of a disease.
  • the “therapeutically effective amount” refers to an amount sufficient to cure, or at least partially prevent the disease and its complications in a patient already suffering from the disease.
  • the therapeutically effective amount of the bispecific antibody of the invention may vary according to the following factors: The severity of the disease to be treated, the general state of the patient's immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered at the same time, etc.
  • dosing regimens can be adjusted to obtain the optimum desired response (e.g., a therapeutic or prophylactic response) .
  • a single dose can be administered, multiple doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the subject may be a mammal, such as a human.
  • a bispecific antibody described herein can also be used to assay c-MET and/or EGFR protein levels in a biological sample using classical immunohistological methods known to those of skill in the art, including immunoassays, such as the enzyme linked immunosorbent assay (ELISA) , immunoprecipitation, or Western blotting.
  • immunoassays such as the enzyme linked immunosorbent assay (ELISA) , immunoprecipitation, or Western blotting.
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I) , carbon (14C) , sulfur (35S) , tritium (3H) , indium (121In) , and technetium (99Tc) ; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase
  • radioisotopes such as iodine (125I, 121I) , carbon (14C) , sulfur (35S) , tritium (3H) , indium (121In) , and technetium (99Tc)
  • luminescent labels such as luminol
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • Such labels can be used to label a bispecific antibody described herein.
  • the present disclosure relates to the use of a bispecific antibody of the present disclosure for in vitro detection of c-MET and/or EGFR protein in a biological sample.
  • the present disclosure relates to the use of a bispecific antibody of the disclosure, for assaying and/or detecting c-MET and/or EGFR protein levels in a biological sample in vitro, optionally wherein the bispecific antibody is conjugated to a radionuclide or detectable label, and/or carries a label described herein, and/or wherein an immunohistological method is used.
  • Assaying for the expression level of c-MET and/or EGFR protein is intended to include qualitatively or quantitatively measuring or estimating the level of c-MET and/or EGFR protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing to the disease associated protein level in a second biological sample) .
  • c-MET and/or EGFR polypeptide expression level in the first biological sample can be measured or estimated and compared to a standard c-MET and/or EGFR protein level, the standard being taken, for example, from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder.
  • the present disclosure relates to an in vitro method for assaying and/or detecting c-MET and/or EGFR protein levels in a biological sample, comprising qualitatively or quantitatively measuring or estimating the level of c-MET and/or EGFR protein in a biological sample, by an immunohistological method.
  • biological sample refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing c-MET and/or EGFR.
  • Methods for obtaining tissue biopsies and body fluids from animals e.g., humans or cynomolgus monkeys are well known in the art.
  • Biological samples include peripheral blood mononuclear cells (PBMCs) .
  • PBMCs peripheral blood mononuclear cells
  • a bispecific antibody described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description.
  • Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro assessment and evaluation of cancer (e.g., tumorigenesis) or other disorders may be utilized to predict, diagnose, and monitor to evaluate patient samples, including those known to have or suspected of having cancer or other disorders, or with regard to an anticipated or desired therapeutic response.
  • the assessment and evaluation of cancer or other disorders are also useful in determining the suitability of a patient for a clinical trial of a drug or for the administration of a particular chemotherapeutic agent, a radiotherapeutic agent, or an antibody, including combinations thereof, versus a different agent or antibody.
  • This type of prognostic and diagnostic monitoring and assessment is already in practice utilizing antibodies against the HER2 protein in breast cancer (HercepTestTM, Dako) where the assay is also used to evaluate patients for antibody therapy using In vivo applications include directed cell therapy and immune system modulation and radio imaging of immune responses. Therefore, in certain embodiments, the present disclosure relates to a bispecific antibody and/or pharmaceutical composition of the present disclosure for use as a diagnostic.
  • the present disclosure relates to a bispecific antibody and/or pharmaceutical composition of the present disclosure for use in a method for the prediction, diagnosis, and/or monitoring of a subject having or suspected to have cancer or another disorder, and/or with regard to an anticipated or desired therapeutic response.
  • the present disclosure relates to the use of a bispecific antibody of the disclosure, for predicting, diagnosing, and/or monitoring of a subject having or suspected to have cancer or another disorder, and/or with regard to an anticipated or desired therapeutic response by assaying and/or detecting c-MET and/or EGFR protein levels in a biological sample of the subject in vitro.
  • a bispecific antibody of the present disclosure can be used in immunohistochemistry of biopsy samples.
  • the method is an in vitro method.
  • a bispecific antibody of the present disclosure can be used to detect levels of c-MET and/or EGFR, or levels of cells which contain c-MET and/or EGFR on their membrane surface, the levels of which can then be linked to certain disease symptoms.
  • Bispecific antibodies described herein may carry a detectable or functional label and/or may be conjugated to a radionuclide or detectable label. When fluorescence labels are used, currently available microscopy and fluorescence-activated cell sorter analysis (FACS) or combination of both methods procedures known in the art may be utilized to identify and to quantitate the specific binding members.
  • FACS fluorescence-activated cell sorter analysis
  • Bispecific antibodies described herein may carry or may be conjugated to a fluorescence label.
  • fluorescence labels include, for example, reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes, and DyLight dyes.
  • the Bispecific antibody of the present disclosure may carry or may be conjugated to a radioactive label or radionuclide, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 67Cu, 90Y, 99Tc, 111In, 117Lu, 121I, 124I, 125I, 131I, 198Au, 211At, 213Bi, 225Ac, and 186Re.
  • radioactive labels currently available counting procedures known in the art may be utilized to identify and quantitate the specific binding of a bispecific antibody to c-MET and/or EGFR.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques as known in the art. This can be achieved by contacting a sample or a control sample with bispecific antibody under conditions that allow for the formation of a complex between the bispecific antibody and c-MET and/or EGFR. Any complexes formed between the bispecific antibody and c-MET and/or EGFR are detected and compared in the sample and the control. In light of the specific binding of the bispecific antibodies described herein for c-MET and EGFR, the bispecific antibodies can be used to specifically detect c-MET and/or EGFR.
  • the bispecific antibodies described herein can also be used to purify c-MET and/or EGFR via immunoaffinity purification.
  • an assay system which may be prepared in the form of a test kit, kit, or kit-of-parts for the quantitative analysis of the extent of the presence of, for instance, c-MET and/or EGFR ligand complexes.
  • the system, test kit, kit, or kit-of-parts may comprise a labeled component, e.g., a labeled antibody, and one or more additional immunochemical reagents.
  • kits comprising one or more bispecific antibodies described herein, or pharmaceutical compositions or conjugates thereof.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more bispecific antibodies provided herein.
  • the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein.
  • the kits may contain a T cell mitogen, such as, e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA) , or a TCR complex stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody.
  • PHA phytohaemagglutinin
  • PMA phorbol myristate acetate
  • TCR complex stimulating antibody such as an anti-CD3 antibody and anti-CD28 antibody.
  • Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical
  • kits that can be used in the above methods.
  • a kit comprises a bispecific antibody described herein, preferably purified antibody, in one or more containers.
  • kits described herein contain a substantially isolated c-MET and/or EGFR antigen as a control.
  • the kits described herein further comprise a control antibody which does not react with c-MET or EGFR antigen.
  • kits described herein contain one or more elements for detecting the binding of an antibody to a c-MET and/or EGFR antigen (e.g., the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody can be conjugated to a detectable substrate) .
  • a kit provided herein can include a recombinantly produced or chemically synthesized c-MET and/or EGFR antigen.
  • the antigen provided in the kit can also be attached to a solid support.
  • the detecting means of the above-described kit includes a solid support to which a c-MET and/or EGFR antigen is attached.
  • a kit can also include a non-attached reporter-labeled anti-human antibody or anti-mouse/rat antibody.
  • binding of the bispecific antibody to the antigen can be detected by binding of the said reporter-labeled antibody.
  • the present disclosure relates to the use of a kit of the present disclosure for in vitro assaying and/or detecting c-MET and/or EGFR antigen in a biological sample.
  • Embodiment 1 A bispecific antibody or bispecific antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR, optionally wherein the bispecific antibody binds to c-MET and EGFR with increased affinity relative to other known antibodies.
  • Embodiment 2 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 1, wherein the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH) , the first VL and the first VH together form a domain capable of specifically binding c-MET; the second antigen-binding domain includes a second VL and a second VH, which together form a specific domain that binds EGFR;
  • VL light chain variable region
  • VH first heavy chain variable region
  • first VL comprises:
  • CDR complementarity determining region
  • CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34
  • CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36
  • CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDRs are defined by the Chothia numbering system
  • CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34
  • CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36
  • CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDRs are defined by the Abm numbering system; or
  • a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 35
  • a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 37
  • a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDRs are defined by the IMGT numbering system
  • the first VH contains:
  • CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 39
  • CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 43
  • CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47, wherein the CDRs are defined by the Kabat numbering system
  • CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 40
  • CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 44
  • CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47, wherein the CDRs are defined by the Chothia numbering system
  • CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 42
  • CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 46
  • CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47, wherein the CDRs are defined by the Abm numbering system; or
  • a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 41
  • a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 45
  • a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 48, where the CDRs are defined by the IMGT numbering system.
  • Embodiment 3 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 2, wherein the second VL comprises:
  • CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19
  • CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21
  • CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDRs are defined by the Kabat numbering system
  • CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19
  • CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21
  • CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDRs are defined by the Chothia numbering system
  • CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19
  • CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21
  • CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDRs are defined by the Abm numbering system; or
  • a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 22, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDRs are defined by the IMGT numbering system; and/or
  • the second VH contains:
  • CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 24, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 28, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32, wherein the CDRs are defined by the Kabat numbering system;
  • CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 25
  • CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 29
  • CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32, wherein the CDRs are defined by the Chothia numbering system
  • a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 27, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 31, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32, wherein the CDRs are defined by the Abm numbering system; or
  • a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 26
  • a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 30
  • a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 33, where the CDRs are defined by the IMGT numbering system.
  • Embodiment 4 The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 2-3, wherein (i) the first VL comprises the amino acid sequence shown in SEQ ID NO: 17 and the first VH comprises the amino acid sequence shown in SEQ ID NO: 18; or (ii) the first VL comprises the amino acid sequence shown in SEQ ID NO: 59 and the first VH comprises the amino acid sequence shown in SEQ ID NO: 60.
  • Embodiment 5 The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 2-4, wherein the second VL comprises the amino acid sequence shown in SEQ ID NO: 15, and/or the second VH comprises the amino acid sequence shown in SEQ ID NO: 16.
  • Embodiment 6 The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 5, wherein the bispecific antibody or its antigen-binding fragment further comprises an Fc dimer comprising first and second Fc monomers, and the first and second Fc monomers each independently comprise a modification of one or more amino acids that promotes heterodimerization of the first and second Fc monomers.
  • Embodiment 7 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 6, wherein the Fc comprises a first Fc monomer containing amino acid modifications capable of forming a knob structure and a second Fc monomer containing amino acid modifications capable of forming a hole structure, wherein the hole structure is capable of pairing with the knob structure to form a heterodimeric Fc dimer.
  • Embodiment 8 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 6 or 7, wherein (a) the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50; or (b) the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
  • Embodiment 9 The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 6-8, wherein the first antigen-binding domain and the first Fc monomer are connected and the second antigen-binding domain is connected to the second Fc monomer; or the first antigen-binding domain and the second Fc monomer are connected and the second antigen-binding domain is connected to the first Fc monomer.
  • Embodiment 10 The bispecific antibody or bispecific antigen-binding fragment thereof described in any one of embodiments 1-9, wherein the first antigen-binding domain is a Fab and the second antigen-binding domain is an scFv.
  • Embodiment 11 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 10, wherein the bispecific antibody comprises polypeptide chain I-A, polypeptide chain I-B, and polypeptide chain I-C; wherein
  • the elements comprised in polypeptide chain I-A from the N-terminal end to the C-terminal end comprise the first VL and CL; the elements comprised in polypeptide chain I-B from the N-terminal end to the C-terminal end comprise the first VH, the heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain I-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, and the second Fc monomer; or (ii) the second VH, the second VL, and the second Fc monomer; or
  • the elements comprised in polypeptide chain I-A from the N-terminal end to the C-terminal end comprise the first VL and CL; the elements comprised in polypeptide chain I-B from the N-terminal end to the C-terminal end comprise the first VH, the heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain I-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, and the first Fc monomer; or (ii) the second VH, the second VL, and the first Fc monomer.
  • Embodiment 12 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 10 or 11, wherein (a) the polypeptide chain I-A comprises the amino acid sequence shown in SEQ ID NO: 1; the polypeptide chain I-B comprises the amino acid sequence shown in SEQ ID NO: 2; and/or the polypeptide chain I-C comprises the amino acid sequence shown in SEQ ID NO: 3; or (b) the polypeptide chain I-A comprises the amino acid sequence shown in SEQ ID NO: 1; the polypeptide chain I-B comprises the amino acid sequence shown in SEQ ID NO: 9; and/or the polypeptide chain I-C comprises the amino acid sequence shown in SEQ ID NO: 10.
  • Embodiment 13 The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 9, wherein the first antigen-binding domain is an scFv, and the second antigen-binding domain is a Fab.
  • Embodiment 14 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 13, wherein
  • the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein the elements comprised in polypeptide chain II-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain II-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain II-C from N-terminal end to C-terminal end comprise:
  • the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein the elements comprised in polypeptide chain II-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain II-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain II-C from the N-terminal end to the C-terminal end comprise:
  • Embodiment 15 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 13 or 14, wherein (a) the polypeptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4; the polypeptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 5; and/or the polypeptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 6; or (b) the polypeptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4; the polypeptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 7; and/or the polypeptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 8.
  • Embodiment 16 The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-9, wherein the first antigen-binding domain is an scFab, and the second antigen-binding domain is a Fab.
  • Embodiment 17 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 16, wherein
  • the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C; wherein the elements comprised in polypeptide chain IV-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain IV-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain IV-C from the N-terminal end to the C-terminal end comprise:
  • the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C; wherein the elements comprised in polypeptide chain IV-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain IV-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain IV-C from the N-terminal end to the C-terminal end comprise:
  • the first VH, the heavy chain CH1 region, the first VL, the CL, and the first Fc monomer are the first VH, the heavy chain CH1 region, the first VL, the CL, and the first Fc monomer.
  • Embodiment 18 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 16 or 17, wherein the polypeptide chain IV-A comprises the amino acid sequence shown in SEQ ID NO: 4; the polypeptide chain IV-B comprises the amino acid sequence shown in SEQ ID NO: 7; and/or the polypeptide chain IV-C comprises the amino acid sequence shown in SEQ ID NO: 11.
  • Embodiment 19 The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-9, wherein the first antigen-binding domain and the second antigen-binding domain are each a Fab, and wherein the second antigen-binding domain Fab comprises a CrossMab form of domain swapping.
  • Embodiment 20 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 19, wherein
  • the bispecific antibody comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D;
  • the elements comprised in polypeptide chain V-A from the N-terminal end to the C-terminal end comprise the first VL and CL; the elements comprised in polypeptide chain V-B from the N-terminal end to the C-terminal end comprise the first VH, the heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain V-C from the N-terminal end to the C-terminal end comprise the second VH, the CL, and the second Fc monomer; and/or the elements comprised in polypeptide chain V-D from the N-terminal end to the C-terminal end comprise the second VL and heavy chain CH1 region; or
  • the bispecific antibody comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D;
  • the elements comprised in polypeptide chain V-A from the N-terminal end to the C-terminal end comprise the first VL and the CL; the elements comprised in polypeptide chain V-B from the N-terminal end to the C-terminal end comprise the first VH, the heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-C from the N-terminal end to the C-terminal end comprise the second VH, the CL, and the first Fc monomer; and/or the elements comprised in polypeptide chain V-D from the N-terminal end to the C-terminal end comprise the second VL and heavy chain CH1 region.
  • Embodiment 21 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 19 or 20, wherein the polypeptide chain V-A comprises the amino acid sequence shown in SEQ ID NO: 1; the polypeptide chain V-B comprises the amino acid sequence shown in SEQ ID NO: 9; the polypeptide chain V-C comprises the amino acid sequence shown in SEQ ID NO: 12; and/or the polypeptide chain V-D comprises the amino acid sequence shown in SEQ ID NO: 13.
  • Embodiment 22 The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-9, wherein both the first antigen-binding domain and the second antigen-binding domain are scFv.
  • Embodiment 23 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 22, wherein
  • the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B;wherein the elements comprised in polypeptide chain VII-A from the N-terminal end to the C-terminal end comprise:
  • the elements comprised in polypeptide chain VII-B rom the N-terminal end to the C-terminal end comprise:
  • the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein the polypeptide chain VII-A from the N-terminal end to the C-terminal end comprise:
  • the elements comprised in polypeptide chain VII-B contains from the N-terminal end to the C-terminal end comprise:
  • Embodiment 24 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 22 or 23, wherein the polypeptide chain VII-A comprises the amino acid sequence shown in SEQ ID NO: 14, and/or the polypeptide chain VII-B comprises the amino acid sequence shown in SEQ ID NO: 8.
  • Embodiment 25 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 11, 14, 17, 20 or 23, wherein adjacent elements comprising each of the polypeptide chains are optionally connected through a peptide linker or not.
  • Embodiment 26 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 25, wherein each peptide linker is independently the same peptide linker or a different peptide linker, wherein each peptide linker selected from the group consisting of a rigid peptide linker and a flexible peptide linker.
  • Embodiment 27 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 26, wherein each peptide linker is independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
  • Embodiment 28 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 27, wherein each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of a peptide linker subunit comprising the amino acid sequence GGGGS (SEQ ID NO: 57) .
  • Embodiment 29 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 28, wherein each peptide linker independently comprises the amino acid sequence shown in SEQ ID NO: 55-58, or 63-68.
  • Embodiment 30 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 11, 14, 17, 20 or 23, wherein the CL comprises the amino acid sequence shown in SEQ ID NO: 53; and/or the heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54.
  • Embodiment 31 The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 30, wherein the bispecific antibody or its antigen-binding fragment comprises:
  • polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 2 or 9
  • polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 3 or 10;
  • polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5 or 7
  • polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6 or 8;
  • polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7
  • polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11;
  • polypeptide chain V-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain V-B comprising the amino acid sequence shown in SEQ ID NO: 9
  • polypeptide chain V-C comprising the amino acid sequence shown in SEQ ID NO: 12
  • polypeptide chain V-D comprising the amino acid sequence shown in SEQ ID NO: 13;
  • polypeptide chain VII-A comprises the amino acid sequence shown in SEQ ID NO: 14, and/or polypeptide chain VII-B comprising the amino acid sequence of SEQ ID NO: 8.
  • Embodiment 32 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 28, wherein the bispecific antibody or antigen-binding fragment thereof comprises:
  • polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 2
  • polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 3;
  • polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 9
  • polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 10;
  • polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5
  • polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6;
  • polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7
  • polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8;
  • polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4
  • polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7
  • polypeptide chain IV-C with the amino acid sequence shown in SEQ ID NO: 11;
  • polypeptide chain V-A comprising the amino acid sequence shown in SEQ ID NO: 1
  • polypeptide chain V-B comprising the amino acid sequence shown in SEQ ID NO: 9
  • polypeptide chain V-C comprising the amino acid sequence shown in SEQ ID NO: 12
  • polypeptide chain V-D comprising the amino acid sequence shown in SEQ ID NO: 13;
  • polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14
  • polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.
  • Embodiment 33 The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 30, wherein the bispecific antibody or its antigen-binding fragment has an enhanced tumor inhibitory effect compared to the monospecific anti-c-MET antibody and/or the monospecific anti-EGFR antibody; and wherein
  • amino acid sequences of the CDRs of the monospecific anti-c-MET antibody are the same as the amino acid sequences of the CDRs of the first antigen-binding domain and the amino acid sequence of the CDRs of the monospecific anti-EGFR antibody are the same as the amino acid sequences of the CDRs of the second antigen-binding domain, or
  • amino acid sequences of the CDRs of the monospecific anti-c-MET antibody are the same as the amino acid sequences of the CDRs of the second antigen-binding domain and the amino acid sequence of the CDRs of the monospecific anti-EGFR antibody are the same as the amino acid sequences of the CDRs of the first antigen-binding domain.
  • Embodiment 34 The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 33, wherein the tumor inhibitory effect includes inhibition of the EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or complement-dependent cytotoxicity (CDC) activity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Embodiment 35 An isolated nucleic acid molecule or set of nucleic acid molecules, comprising a nucleotide sequence encoding the bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-34.
  • Embodiment 36 A vector comprising the isolated nucleic acid molecule or set of nucleic acid molecules of embodiment 35.
  • Embodiment 37 The vector according to embodiment 36, wherein the nucleotide sequences encoding the different peptide chains of the bispecific antibody are located in different vector molecules.
  • Embodiment 38 The vector according to embodiment 36 or 37, wherein the vector is a cloning vector or an expression vector.
  • Embodiment 39 A host cell comprising the isolated nucleic acid molecule or set of nucleic acid molecules of embodiment 35, or the vector of any one of embodiments 36-38.
  • Embodiment 40 A method for preparing the bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1-34, comprising culturing the host cell of embodiment 39 under conditions allowing the expression of the bispecific antibody or bispecific antigen-binding fragment thereof, and recovering the bispecific antibody from the culture of the host cell.
  • Embodiment 41 A pharmaceutical composition containing one or more bispecific antibodies or bispecific antigen-binding fragment thereof according to any one of embodiments 1-34 and one or more pharmaceutically acceptable carriers and/or excipients.
  • Embodiment 42 The pharmaceutical composition of embodiment 41, wherein the pharmaceutical composition further comprises one or more additional pharmaceutically active agents selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
  • additional pharmaceutically active agents selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
  • Embodiment 43 Use of the bispecific antibody according to any one of embodiments 1 to 34 or the pharmaceutical composition according to embodiment 41 or 42 in preparation of a medicament, wherein the medicament is used to prevent and/or treat and/or assist in the treatment of diseases related to c-MET and/or EGFR in a subject, and/or for inhibiting c-MET cells or activity of c-MET and/or EGFR in vitro or in the body of a subject; wherein the diseases related to c-MET and/or EGFR are EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations and/or c-MET cancers associated with MET gene amplification.
  • Embodiment 44 The use of according to embodiment 43, wherein the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, liver cell carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, and head and neck cancer.
  • the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue
  • Embodiment 45 The use of according to embodiment 43 or 44, wherein said bispecific antibody or bispecific antigen-binding fragment thereof or pharmaceutical composition thereof and another pharmaceutically active agent are administered in combination, either simultaneously, separately or sequentially; wherein the another pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c- MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
  • the another pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c- MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
  • Embodiment 46 The use of any one of embodiments 43-45, wherein the medicament is used in a subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192 or cetuximab.
  • Embodiment 47 A method for inhibiting the activity of c-MET and/or EGFR in cells, comprising combining the cells with the bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-34 or the composition or the pharmaceutical composition of embodiment 41 or 42; wherein the cells are cells that express c-MET and/or EGFR.
  • Embodiment 48 A method for preventing and/or treating and/or adjunctive treatment of a disease associated with c-MET and/or EGFR in a subject, the method comprising administering an effective amount to a subject in need thereof the bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 34 or the pharmaceutical composition according to embodiment 41 or 42, wherein the disease associated with c-MET and/or EGFR is a cancer comprising EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and/or c-MET gene amplification.
  • Embodiment 49 The method of embodiment 48, wherein the cancer is selected from the group consisting of epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, and prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.
  • the cancer is selected from the group consisting of epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, and prostate cancer, bladder cancer, pharyngeal cancer
  • Embodiment 50 The method of embodiment 48 or 49, wherein the method further comprises administering a second therapy to the subject, the second therapy being selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof; optionally, the second therapy can be applied simultaneously, separately or sequentially.
  • the second therapy being selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof; optionally, the second therapy can be applied simultaneously, separately or sequentially.
  • Embodiment 51 The method of any one of embodiments 47-50, wherein the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
  • Embodiment 52 A bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 34 or the pharmaceutical composition according to embodiment 41 or 42, for preventing and/or treating and/or adjunctive treatment of a disease associated with c-MET and/or EGFR in a subject, wherein the disease associated with c-MET and/or EGFR is a disease comprising EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and/or c-MET gene amplification cancer.
  • Embodiment 53 The bispecific antibody or bispecific antigen-binding fragment thereof or the pharmaceutical composition of embodiment 52, wherein the cancer is selected from the group consisting of epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, and prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.
  • the cancer is selected from the group consisting of epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal
  • Embodiment 54 The bispecific antibody or bispecific antigen-binding fragment thereof or the pharmaceutical composition of embodiment 52 or 53, wherein the method further comprises administering a second therapy to the subject, the second therapy being selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof; optionally, the second therapy can be applied simultaneously, separately or sequentially.
  • the second therapy being selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof; optionally, the second therapy can be applied simultaneously, separately or sequentially.
  • Embodiment 55 The bispecific antibody or bispecific antigen-binding fragment thereof or the pharmaceutical composition of any one of embodiments 52-54, wherein the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
  • Antibody framework region Amino acid residues in antibody variable regions other than CDR residues
  • IMGT Based on the numbering system of The international ImMunoGeneTics information (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003.
  • Kabat Alignment and numbering system for immunoglobulins proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) .
  • Chothia The immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia &Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) .
  • HCDR1 Complementarity-determining region 1 in the variable region of an immunoglobulin heavy chain
  • HCDR2 Complementarity-determining region 2 in the variable region of an immunoglobulin heavy chain
  • HCDR3 Complementarity-determining region 3 in the variable region of an immunoglobulin heavy chain
  • LCDR1 Complementarity-determining region 1 in the variable region of an immunoglobulin light chain
  • LCDR2 Complementarity-determining region 2 in the variable region of an immunoglobulin light chain
  • LCDR3 Complementarity-determining region 3 in the variable region of an immunoglobulin light Chains
  • an immunoglobulin molecule may be composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC) .
  • Antibody light chains can be classified as kappa ( ⁇ ) and lambda ( ⁇ ) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also comprising a “D” region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH) .
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3) .
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL) .
  • the light chain constant region consists of one domain, CL.
  • the constant domains are not directly involved in antibody-antigen binding, but exhibit a variety of effector functions, such as mediating the binding of immunoglobulin with host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q) .
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) , interspersed with more conserved regions called framework regions (FRs) .
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged from the amino terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antigen-binding site, respectively.
  • the allocation of amino acids in each region or domain can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991) ) , or Chothia &Lesk (1987) J. Mol. Biol. 196: 901-917 and Chothia et al. (1989) Nature 342: 878-883.
  • antibody further includes embodiments in which heavy chain constant domains may comprise a C-terminal lysine or lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide.
  • the term further includes embodiments in which the N-terminal amino acid of the antibody variable domains has undergone cyclization to pyroglutamate.
  • various species of the antibodies therein may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and/or comprise an N-terminal glutamine or glutamic acid or cyclization of the N-terminal amino acid to pyroglutamate.
  • antibody includes not only whole antibodies but also antigen-binding fragments of antibodies.
  • CDR complementarity determining region
  • the term “complementarity determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding.
  • CDR complementarity determining region
  • the precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example according to the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268 –9272) , the MacCallum numbering system (MacCallum et al., (1996) J Mol Biol 262: 732-745, see also, e.g., Martin A.
  • the CDRs comprised in the antibody of the present invention or an antigen-binding fragment thereof can be determined according to various numbering systems known in the art.
  • the antibodies or antigen-binding fragments thereof of the invention comprise CDRs identified by the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering system.
  • the entire amino acid sequence of the V H is commonly numbered according to Kabat while the three CDRs within the variable region may be defined according to any one of the aforementioned numbering schemes.
  • the numbering of the amino acid positions in the V H may be sequential beginning with amino acid position 1 and continuing sequentially to the end of the sequence or according to Kabat.
  • the amino acid positions in the V H and V L herein are defined according to sequential numbering.
  • the numbering of the amino acid positions in the heavy chain constant domain may be sequential beginning with amino acid position 1 and continuing sequentially to the end of the sequence or according to Eu numbering.
  • the IgG1 heavy chain constant domain amino acid sequence has 330 amino acids sequentially numbered 1 to 330.
  • the corresponding sequence numbered according to Eu begins with position number 118 and ends with position number 447. Unless specified otherwise, the amino acid positions in the heavy and light chains herein are defined according to sequential numbering.
  • framework region or “FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes) , IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG e.g., IgG1, IgG2, IgG3 or IgG4 subtypes
  • IgA1, IgA2, IgD, IgE or IgM antibodies e.g., IgA1, IgA2, IgD, IgE or IgM antibodies.
  • bispecific antibody refers to an antibody with binding specificities for two different antigens (or epitopes) , which comprises two antibodies with binding specificities for different antigens (or epitopes) .
  • Antigen-binding domains thereby being able to bind two different binding sites and/or target molecules.
  • Each antigen-binding domain comprised in the bispecific antibody can be independently selected from a full-length antibody (such as an IgG antibody) or an antigen-binding fragment thereof (such as Fv, Fab, scFab or scFv) .
  • the individual antigen-binding domains are linked by a peptide linker.
  • the term “Fv fragment” means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • the Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site.
  • the six CDRs are generally believed to confer antigen-binding specificity to an antibody.
  • a variable region such as the Fd fragment, which comprises only three CDRs specific for an antigen is capable of recognizing and binding antigens, although perhaps with a lower affinity than the full binding site.
  • Fc fragment means a fragment formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain antibody fragments.
  • the Fc fragment of an antibody has several different functions but is not involved in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker.
  • Such scFv molecules can have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art peptide linkers consist of the repeated GGGGS amino acid sequence (SEQ ID NO: 57) or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 (SEQ ID NO: 58) can be used, but variants thereof can also be used.
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains, usually consisting of one peptide chain comprising VL and CL and another peptide chain comprising VH and CH1.
  • the Fab domains may be arranged according to the natural orientation described above, but may also comprise domain substitutions or exchanges that promote correct VH and VL pairing (such as a domain exchange in the form of a CrossMabs) ;
  • the term “scFab” refers to, a single polypeptide chain comprising VL, VH, CL and CH1 domains, wherein the adjacent domains are connected optionally by a linker.
  • the single polypeptide chain comprised in scFab starts from the N-terminal to the C-terminal comprises: (1) VL, CL, VH and CH1, where CL and VH are usually linked by a peptide linker (e.g., a flexible peptide linker) , or (2) VH, CH1, VL and CL, where CH1 and VL usually connected by a peptide linker (e.g., a flexible peptide linker) .
  • a peptide linker e.g., a flexible peptide linker
  • the terms “monoclonal antibody” , and “mAb” have the same meaning and are used interchangeably to refer to one antibody molecule from a group of highly homogeneous antibody molecules or a fragment of an antibody, that is, a population of antibody molecules that are identical except for natural mutations that may arise spontaneously. mAbs are highly specific for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least 2 or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is characterized as being obtained from a highly homogeneous population of antibodies and is not to be construed as requiring that the antibody be prepared by any particular method.
  • bispecific antibody or “BsAb” refers to an antibody having two distinct binding domains that enable the biantibody to bind to two different antigens or two different epitopes of the same antigen simultaneously.
  • CrossMab refers to a method in the construction of bispecific antibodies that enables the correct association of the light chains and their cognate heavy chains by exchange of heavy-chain and light-chain domains within the antigen-binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur.
  • Fab antigen-binding fragment
  • CrossMab Fab which refers to crossover or exchange in position of the complete VH-CH1 and VL-CL domains of one half of the bispecific antibody
  • CrossMab VH-VL which refers to the crossover or exchange in position of only the VH and VL domains of one half of the bispecific antibody
  • CrossMab CH1-CL which refers to the crossover or exchange in position of the CH1 and CL domains within the Fab region of one half of the bispecific antibody.
  • CrossMab antibodies have been described or claimed in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010136172, WO2010145792 and WO2013026831.
  • the term "CrossMab” antibody is generally recognized in the art; e.g., see Brinkmannand Kontennann, MAbs 9 (2) : 182-212 (2017) ; Kontermannand Brinkmann, Drug Discovery Today 20 (7) : 838-846 (2015) ; Schaefer et al., PNAS, 108 11187-1191 (2011) ; Kleinet al., MAbs 8 (6) : 1010-1020 (2016) ; and Klein et al., MAbs 4 (6) : 653-663 (2012) .
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.
  • the strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) or the half-maximal effect concentration (EC 50 ) of the interaction.
  • the specific binding properties between two molecules can be determined using methods well known in the art.
  • One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • Both the “association rate constant” (k a or k on ) and the “dissociation rate constant” (k dis or k off ) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187) .
  • the ratio k dis /k on is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473) .
  • KD, k on and k dis values can be measured by any effective method.
  • dissociation constants can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method) .
  • bioluminescence interferometry e.g., the ForteBio Octet method
  • surface plasmon resonance techniques e.g., Biacore
  • Kinexa can be used to measure dissociation constants.
  • the term “vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: Plasmid; Phagemid; Cosmid; Artificial chromosome, such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) ; bacteriophage such as lambda phage or M13 phage and animal virus, etc.
  • YAC yeast artificial chromosome
  • BAC bacterial artificial chromosome
  • PAC P1-derived artificial chromosome
  • bacteriophage such as lambda phage or M13 phage and animal virus, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses) , adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus) , poxviruses, baculoviruses, papillomaviruses, Polyoma vacuolar virus (e.g., SV40) .
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses poxviruses
  • baculoviruses papillomaviruses
  • Polyoma vacuolar virus e.g., SV40
  • a vector can comprise a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also comprise an origin of replication.
  • Expression and cloning vectors comprise nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes origins of replication or autonomously replicating sequences.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors comprise sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) , and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) .
  • the term “host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or such as fibroblasts, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells (such as CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cells ) , ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells and other animal cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • fibroblasts NS0 cells
  • Vero cells Hela cells
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by an adenine, or both a position in each of the polypeptides is occupied by lysine)
  • Percent identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of compared positions multiplied by 100. For example, two sequences are 60%identical if 6 out of 10 positions match.
  • the DNA sequences CTGACT and CAGGTT share 50%identity (3 out of a total of 6 positions match) .
  • comparisons are made when two sequences are aligned for maximum identity.
  • Such alignments can be achieved using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453 which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc. ) .
  • Align program DNAstar, Inc.
  • the algorithm of Appl Biosci., 4: 11-17 (1988) ) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine the percent identity between two amino acid sequences by using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.
  • the algorithm of Needleman and Wunsch J MoI Biol. 48: 444-453 (1970) ) that has been integrated into the GAP program of the GCG software package (available at www. gcg. com) can be used to determine the percent identity between two amino acid sequences by using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.
  • the term “pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) , and include but are not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearates and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline) , alcohols and polyols (e.g., glycerol) , and the like.
  • Stabilizer has the meaning generally understood by those skilled in the art, and it can stabilize the desired activity of the active ingredient in the medicine, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose) , amino acids (such as glutamic acid, glycine) , proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate) , etc.
  • prevention refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a tumor) in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical outcome.
  • a beneficial or desired clinical outcome includes, but is not limited to, relief of symptoms, reduction of the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delay or slowing of the progression, amelioration or palliation of the disease status, and relief of symptoms (whether partial or total) , whether detectable or not.
  • treatment can also refer to prolonging survival as compared to expected survival if not receiving treatment.
  • the term “subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject e.g., human
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (for example, a tumor)
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent an existing disease or complications. Determining such an effective amount is well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly, etc.
  • effector function refers to those biological activities that can be attributed to the antibody Fc region (native sequence Fc region or amino acid sequence variant Fc region) , and which are associated with the antibody varies with antibody isotype.
  • antibody effector functions include, but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC) , complement-dependent cytotoxicity (CDC) , antibody-dependent cellular phagocytosis (ADCP) , cell surface receptors such as B cell receptor, B cell activation, cytokine secretion, half-life/clearance rate of antibodies and antigen-antibody complexes, etc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • cell surface receptors such as B cell receptor, B cell activation, cytokine secretion, half-life/clearance rate of antibodies and antigen-antibody complexes, etc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells such as natural killer (NK) cells, neutrophils, or macrophages, allowing these cytotoxic effector cells to specifically bind to the antigen-attached target cells, and then kill the target cells by secreting cytotoxins.
  • the term “combination therapy” comprises combining a bispecific antibody or pharmaceutical composition of the invention with one or more additional active therapeutic agents (e.g. chemotherapeutic agents) of a second therapy or other prophylactic or therapeutic modalities (e.g. radiotherapy) .
  • additional active therapeutic agents e.g. chemotherapeutic agents
  • a second therapy or other prophylactic or therapeutic modalities e.g. radiotherapy
  • Combination therapy includes therapeutic agents that affect the immune response (e.g., enhance or activate the response) and therapeutic agents that affect (e.g., inhibit or kill) tumor/cancer cells. Combination therapy reduces the likelihood of drug-resistant cancer cells developing. Combination therapy may allow dose reduction of one or more of the agents to reduce or eliminate adverse effects associated with one or more of the agents.
  • Such combination therapies may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder or condition.
  • “combination” includes therapies that may be administered separately, e.g., formulated separately for separate administration (e.g., may be provided in a kit) , as well as therapies that may be administered together in a single formulation (i.e., a “co-formulation” ) .
  • the bispecific antibodies of the invention may be administered sequentially. In other embodiments, the bispecific antibodies can be administered simultaneously.
  • the bispecific antibodies of the invention may be used in any combination with at least one other (active) agent.
  • cancer and “tumor” are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant sites in the body through the lymphatic system or bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Laboratory Guide to Molecular Biology, 3rd Edition, John Wiley &Sons, Inc., 1995.
  • the Fab-scFv-Fc (KIH) bispecific antibodies comprise the Fab-scFv-Fc (KIH) bispecific antibody structure.
  • the knob-in-hole (KIH) structure is used between the heavy chains to prevent mismatching.
  • the schematic diagram of the structure is shown in Fig. 1A.
  • the amino acid sequences comprising the anti-EGFR and anti-c-MET bispecific Fab-scFv-Fc (KIH) bispecific antibodies are shown in Table 1.
  • the above amino acid sequences were sent to Genscript Biotechnology Co., Ltd. for codon optimization and DNA synthesis, and the light and heavy chains of the bispecific antibody were cloned into a pKL GS expression vector (containing two expression cassettes; designed by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. and gene synthesis by GenScript) .
  • the promoter of the first expression cassette is the mouse CMV promoter and the polyA signal sequence is the sv40 polyA signal sequence.
  • the promoter of the second expression cassette is the mouse CMV promoter and the polyA signal sequence is the thymidine kinase (TK) polyA signal sequence.
  • the scFv-Fc of each bispecific antibody was separately cloned into a pKL5 Vector (containing 1 expression cassette; designed by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. and gene synthesis by GenScript; the promoter is the mouse CMV promoter and the polyA signal sequence is the CMV polyA signal sequence) or pKL8 expression vector (containing 1 expression cassette; the promoter is mouse CMV promoter and the polyA signal sequence is the CMV polyA signal sequence) .
  • the plasmid pair was co-transfected into CHO-K1 cells (obtained from American Type Culture Collection (ATCC) , Manasas, VA) and the cells were cultured in a cell culture medium for expression of the bispecific antibodies. After allowing expression of the bispecific antibodies for a period of time, the bispecific antibodies were harvested from the culture medium and Protein A (MabSelect TM SuRe, GE) chromatography was used to capture the bispecific antibodies. The bispecific antibodies were then purified using cation chromatography ( CPX, Merck KGaA) to provide bispecific antibodies 07B, 10B, 38B, and 49B.
  • ATCC American Type Culture Collection
  • VA Manasas, VA
  • the Fab-scFab-Fc (KIH) bispecific antibodies comprise the Fab-scFab-Fc (KIH) bispecific antibody structure, and the KIH structure is used between the heavy chains to prevent mismatching.
  • a pair of disulfide bonds were introduced into the scFab-Fc chain, that is, the Q at position 106 of scFab-Fc was mutated to C, and the G at position 304 of scFab-Fc was mutated to C.
  • FIG. 1B A schematic diagram of the structure is shown in Fig. 1B.
  • the amino acid sequences comprising the anti-EGFR and anti-c-MET Fab-scFab-Fc (KIH) bispecific antibodies are shown in Table 2.
  • the above amino acid sequences were sent to Genscript Biotechnology Co., Ltd. for codon optimization and DNA synthesis.
  • the light and heavy chains of the bispecific antibody were cloned into the pKLGS expression vector of Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd., and the scFab-Fc of the bispecific antibody was cloned into the pKL5 expression vector of Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. .
  • the plasmid pair of the plasmid expressing the light and heavy chains of the bispecific antibody and the plasmid expressing the scFab-Fc of the bispecific antibody were co-transfected into CHO-K1 cells (ATCC) and the cells were cultured in cell culture medium for expression of the bispecific antibody 41B. After allowing expression of the bispecific antibodies for a period of time, the bispecific antibodies were harvested from the culture medium and captured by Protein A (MabSelect TM SuRe LX, GE) affinity chromatography. The bispecific antibodies were then purified using cation chromatography ( CPX, Merck KGaA) to provide bispecific antibody 41B.
  • the bispecific antibody comprises the CrossMab CH1-CL bispecific antibody structure, and the KIH structure is used between the heavy chains to prevent mismatching.
  • the structural diagram is shown in Fig. 1C.
  • the amino acid sequences comprising the anti-EGFR and anti-c-MET CrossMab CH1-CL bispecific antibodies are shown in Table 3.
  • the above amino acid sequences were sent to Genscript Biotechnology Co., Ltd. for codon optimization and DNA synthesis.
  • the light and heavy chains of bispecific antibody 55B were cloned into the pKLGS expression vector of Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd, and the VL-CH1 and VH-CL-Fc of the bispecific antibody were cloned into another pKLGS expression vector.
  • the two pKLGS plasmids were co-transfected into CHO-K1 cells (ATCC) and the cells were cultured in cell culture medium for expression of the bispecific antibody 55B.
  • bispecific antibodies After allowing expression of the bispecific antibodies for a period of time, the bispecific antibodies were harvested from the culture medium and Protein A (MabSelect TM SuRe, GE) affinity chromatography was used to capture the bispecific antibodies. The bispecific antibodies were then purified using cation chromatography ( CPX, Merck KGaA) to provide bispecific antibody 55B.
  • Protein A MobSelect TM SuRe, GE affinity chromatography
  • the scFv-Fc (KIH) bispecific antibodies comprise the scFv-Fc (KIH) bispecific antibody structure, and the KIH structure is used between the heavy chains to prevent mismatching.
  • the schematic diagram of the structure is shown in Fig. 1D.
  • the amino acid sequences comprising the anti-EGFR and anti-c-MET scFv-Fc (KIH) bispecific antibodies are shown in Table 4.
  • the above amino acid sequences were sent to Genscript Biotechnology Co., Ltd. for codon optimization and DNA synthesis.
  • the scFv-Fc (knob) and scFv-Fc (hole) of the bispecific antibody were cloned into the pKLGS expression vector of Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd.
  • the plasmid expressing the bispecific antibody was was transfected into CHO-K1 cells (ATCC) and the cells were cultured in cell culture medium for expression of bispecific antibody 56B.
  • bispecific antibodies After allowing expression of the bispecific antibodies for a period of time, the bispecific antibodies were harvested from the culture medium and Protein A (MabSelect TM SuRe LX, GE) affinity Chromatography was used to capture the bispecific antibody. The bispecific antibodies were then purified using cation ( CPX, Merck KGaA) chromatography to provide bispecific antibody 56B.
  • the amino acid sequences of monospecific anti-EGFR monoclonal antibodies Cetuximab, Zalutumumab, Necitumumab, and Nimotuzumab are from the Immunogenetics website, wherein the International Nonproprietary Name (INN) number for Cetuximab is 7906, the INN number for Zalutumumab is 8605, the INN number for Necitumumab is 9083, and the INN number for Nimotuzumab is 8545.
  • Zalutumumab is a parental monoclonal antibody of the bispecific antibody provided by the present invention.
  • the amino acid sequence of the variable region of Zalutumumab is the same as that of the anti-EGFR portion of the bispecific antibody provided by the present invention.
  • Onartuzumab is a parental monoclonal antibody of the bispecific antibody provided by the present invention.
  • the amino acid sequence of the variable region of Onartuzumab is the same as that of the anti-c-MET portion of the bispecific antibody provided by the present invention.
  • MRG003 is an ADC based on an anti-EGFR monoclonal antibody in clinical phase II and this ADC has shown good efficacy in patients with nasopharyngeal carcinoma and head and neck cancer.
  • BA03 is the antibody of MRG003.
  • the amino acid sequence of BA03 monoclonal antibody is from Chinese patent CN106999606B; the amino acid sequences of monospecific anti-c-MET monoclonal antibodies Onartuzumab and Telisotuzumab are from the website, wherein the INN number for Onartuzumab is 9368, the INN number for Telisotuzumab is 10366, and the sequence of CE355621 monoclonal antibody sequence is from Chinese patent CN107207564A.
  • expression plasmids were constructed that encoded the heavy and light chains of the monospecific antibodies.
  • the expression plasmids were separately transfected into CHO-K1 cells and the transfected cells were cultivated in culture medium under suitable conditions for expression of the monospecific antibodies into the culture medium. After allowing expression of the monospecific antibodies for a period of time, the monospecific antibodies were harvested from the culture medium and Protein A (MabSelect TM SuRe LX, GE) affinity Chromatography was used to capture the monospecific antibodies. The monospecific antibodies were then purified using cation ( CPX, Merck KGaA) chromatography to provide the monospecific antibodies.
  • the antibody Tagitanlimab (See WO2017148424) was used as a hydrophilic control and the antibody Sacituzumab (See WO2019114666) was used as a hydrophobic control.
  • Each of the control antibodies (produced by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. ) were diluted with diluent to make a solution of 1.0 mg/mL as the system suitability solution.
  • About 40 ⁇ g of sample was injected and analyzed in gradient elution: 0-3 minutes, maintaining mobile phase A of 95%and mobile phase B of 5%; 3-40 min, mobile phase B from 5%to 100%; 40-45 minutes, maintaining mobile phase A of 95%and mobile phase B of 5%.
  • the hydrophobicity value of the test sample was calculated according to the control sample with the formula: (retention time of the test sample -retention time of the hydrophilic control) / (retention time of the hydrophobic control -retention time of the hydrophilic control) , and the smaller the retention time and hydrophobicity value, the better the hydrophilicity of the antibody.
  • the results are shown in Table 5.
  • the bispecific antibodies all displayed good hydrophilicity. Good hydrophilicity is expected to be beneficial for the production of antibodies, quality control, or conjugation with small molecules, etc., and is expected to enhance the in vivo drug efficacy, improve the pharmacokinetic properties and the medication safety.
  • Human EGFR-His protein was diluted to 1 ⁇ g/mL with Carbonate-Bicarbonate Buffer (CBS) coating solution and added to the wells of a 96-well ELISA microtiter plate at 100 ng/well. The plate was incubated overnight at 4°C. The next day, the 96-well ELISA microtiter plate was washed once with PBST (0.05%Tween-20) and 100 ⁇ L of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37°C for 2 hours.
  • CBS Carbonate-Bicarbonate Buffer
  • the purified bispecific antibody and Nimotuzumab control antibody were serially diluted with 2%BSA, starting at 10 ⁇ g/mL, 3-fold gradient, 11 concentration points.
  • the blocking solution was then removed from the wells and the serially diluted antibody solutions added to the wells of the ELISA microtiter plate.
  • the plate was incubated at 37°C for 2 hours. Afterwards, the plate was washed 3 times with PBST (0.05%Tween-20) at 320 ⁇ L per well.
  • the plate was dried and then 100 ⁇ L HRP conjugated Goat anti-human IgG was added to each well, and the plate was incubated at 37 °C for 1 hour.
  • the plate was washed 5 times with PBST (0.05%Tween-20) at 320 ⁇ L per well. After drying the plate, 100 ⁇ L 3, 3', 5, 5'-tetramethylbenzidine (TMB) (Huzhou Yingchuang Biotechnology Co., Ltd. ) chromogenic substrate was added to each well and allowed to react at room temperature for 10 minutes. The reaction was stopped by adding 50 ⁇ L 2 M H 2 SO 4 . The plate was read at OD450nm absorbance with a microplate reader. The raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting, and the EC 50 of the binding of bispecific antibody and control antibody to EGFR-His was calculated.
  • TMB trimethylbenzidine
  • c-Met protein was added to the wells of a 96-well ELISA microtiter The plate was incubated overnight at 4°C. The next day, the 96-well ELISA microtiter plate was washed once with PBST (0.05%Tween-20) and 100 ⁇ L of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37°C for 2 hours. The purified bispecific antibody and Telisotuzumabcontrol antibody were serially diluted with 2%BSA, starting at 10 ⁇ g/mL, 3-fold gradient, 11 concentration points. The blocking solution was removed and the diluted antibody solution was added to the ELISA microtiter plate.
  • the plate was incubated at37°C for 2 hours. Afterwards, the plate was washed 3 times with PBST (0.05%Tween-20) at 320 ⁇ L per well. The plates were dried and then 100 ⁇ L HRP conjugated Goat anti-human IgG was added to each well, and the plate was incubated at 37 °C for 1 hour. Afterwards, the plate was washed 5 times with PBST (0.05%Tween-20) at 320 ⁇ L per well. After drying the plate, 100 ⁇ L 3, 3', 5, 5'-tetramethylbenzidine (TMB) (Huzhou Yingchuang Biotechnology Co., Ltd. ) chromogenic substrate was added to each well and allowed to react at room temperature for 10 minutes.
  • TMB 3, 3', 5, 5'-tetramethylbenzidine
  • the reaction was stopped by adding 50 ⁇ L 2 M H 2 SO 4 .
  • the plate was read at OD450nm absorbance with a microplate reader.
  • the raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting, and the EC 50 of the binding of bispecific antibody and control antibody to c-MET-His was calculated. All of the tested bispecific antibodies were able to normally bind to c-MET-His protein. The specific results are shown in Table 7.
  • Flow cytometry (Beckman, model Cytoflex) was used to detect the binding activity of the bispecific antibodies to human non-small cell lung cancer cell HCC827 (ATCC) displaying high expression of EGFR and low expression of c-MET, human gastric cancer cell MKN45 displaying high expression of c-MET and low expression of EGFR (Nanjing Kebai) and human lung adenocarcinoma cell NCI-H1975 (Nanjing Kebai) displaying low expression of EGFR and c-MET.
  • ATCC827 human non-small cell lung cancer cell HCC827
  • human gastric cancer cell MKN45 displaying high expression of c-MET and low expression of EGFR
  • human lung adenocarcinoma cell NCI-H1975 Najing Kebai
  • the adherent cells were digested with Trypsin-EDTA (0.25%, Shanghai Yuanpei) solution, and the cell density adjusted to 4.0 ⁇ 10 6 /mL.
  • the cells were washed twice with 1%BSA and resuspended in 1%BSA solution. Then 50 ⁇ L of cell suspension was added to each well of a 96-well sharp-bottomed plate (the number of cells was 2 ⁇ 10 5 per well) ;
  • the bispecific antibody was diluted with 1%BSA, starting from a final concentration of 10 ⁇ g/mL, under 3-fold serial dilution, with a total of 11 concentration points.
  • the diluted antibody was added to the wells of the conical bottom plate and the plate was incubated at 4°C for 60 minutes.
  • the cells were washed twice with 1%BSA and then 50 ⁇ L of diluted secondary antibody was added to each well, mixed well, and the plate was allowed to incubate at 4°C for 30 minutes. Afterwards, the cells were washed twice with 1%BSA and then resuspended in 200 ⁇ L 1%BSA for flow cytometry detection.
  • the Median PE value was exported into GraphPad Prism 6 software for calculating the EC 50 .
  • the results are shown in Figs. 2A, 2B, 3A, 3B, 4A, and 4B and Table 8 (antibody hlgG1 is a negative control antibody) .
  • the results showed that the bispecific antibodies could effectively bind to EGFR and c-MET regardless of their expression levels.
  • the maximum signal values of bispecific antibodies were stronger than those of EGFR and c-MET monoclonal antibodies, that is, tumor cells could bind more bispecific antibodies than monoclonal antibodies, which may produce stronger tumor suppressor effects.
  • Flow cytometry (Beckman, model Cytoflex) was used to detect the internalizing activity of the bispecific antibodies on human non-small cell lung cancer cell HCC827 displaying high expression of EGFR and low expression of c-MET, human gastric cancer cell MKN45 displaying high expression of c-MET and low expression of EGFR, and human lung adenocarcinoma cell NCI-H1975 displaying low expression of EGFR and c-MET.
  • the adherent cells were digested with Tryple TM Express (Thermo) solution and counted.
  • the cell density was adjusted to 4 ⁇ 10 6 cells/mL with 1%BSA and 50 ⁇ L of cell suspension was added to the wells of a 96-well microtiter plate (the number of cells per well was 2 ⁇ 10 5 cells/well) .
  • the bispecific antibody was diluted with 1%BSA starting from 5 ⁇ g/mL, under 4-fold serial dilution, with a total of 8 concentration points. Then 50 ⁇ L of diluted bispecific antibody per well was added to the wells of the 96-well microtiter plate and the plate was incubated at 4°C for 40 minutes.
  • the wells were washed twice with 1%BSA and the secondary antibody (anti-human F (ab') 2 Alexa Fluor 660) diluted 1: 200 with 1%BSA was added at 50 ⁇ L per well.
  • the plates were incubated at 4°C for 20 minutes, then the wells were washed twice with 1%BSA and the cells therein were resuspended in 150 ⁇ L of complete medium.
  • the resuspended cells were incubated at 4°C and 3°C for 4 hours after resuspension.
  • the cells were centrifuged at 500g for 3 minutes to remove the medium, and 100 ⁇ L of pH 2.0 acid solution was added per well and the plate was incubated for 2 minutes. Next, 60 ⁇ L of pH 13 base solution was added for neutralization followed by centrifugation to remove the supernatant fraction. Then, the cells were resuspended in 1%BSA for FACS analysis.
  • Flow cytometry (Beckman, model Cytoflex) was used to detect the internalizing activity of bispecific antibodies on human non-small cell lung cancer cell HCC827 displaying high expression of EGFR and low expression of c-MET, human gastric cancer cell MKN45 displaying high expression of c-MET and low expression of EGFR, and human lung adenocarcinoma cell NCI-H1975 displaying low expression of EGFR and c-MET.
  • the adherent cells were digested with Trypsin-EDTA (0.25%) (Shanghai Yuanpei) solution, the cells were counted, and the cell density was adjusted to 1 ⁇ 10 5 cells/mL with complete culture medium. Then 100 ⁇ L cell suspension was added to each well of a 96-well plate (the number of cells is 1 ⁇ 10 4 cells per well) and incubated at 37°C, CO 2 for 24 hours.
  • the serially diluted bispecific antibodies to be tested were combined with the diluted PHrodo reagent to provide a final reagent ratio of 1: 1 (30 ⁇ L: 30 ⁇ L) and incubated at room temperature for 30 minutes in the dark. Then, 50 ⁇ L of the bispecific antibody to be tested and reagent mixture was added to the wells of a 96-well plate and the 96-well plate was incubated at 37°C, 5%CO 2 for 24 hours. Afterwards, the culture medium was removed, the wells were washed once with sterile PBS, and then 100 ⁇ L Trypsin-EDTA (0.25%) was added to each well to release the cells from the wells. Next, 100 ⁇ L complete culture medium was added to neutralize the trypsin and the cells were dispersed by pipetting and testing by FACS.
  • Human EGFR-His protein was diluted to 1 ⁇ g/mL with Carbonate-Bicarbonate Buffer (CBS) coating solution and added to the wells of a 96-well ELISA microtiter plate at 100 ng/well. The plate was incubated overnight at 4°C. The next day, the 96-well ELISA microtiter plate was washed once with PBST (0.05%Tween-20) and 100 ⁇ L of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37°C for 2 hours. The purified bispecific antibodies were serially diluted with 2%BSA, starting at 10 ⁇ g/mL, 3-fold gradient, 11 concentration points.
  • CBS Carbonate-Bicarbonate Buffer
  • the blocking solution was removed from the wells and the serially diluted antibody solutions were added to the wells of the ELISA microtiter plate.
  • the plate was incubated at 37°C for 2 hours. Afterwards, the plate was washed 3 times with PBST (0.05%Tween-20) at 320 ⁇ L per well.
  • PBST 0.05%Tween-20
  • the plate was dried and then 100 ⁇ L of 1 ⁇ g/mL human c-MET-His-biotin protein (Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd) was added to each well of the ELISA microtiter plate and the plate was incubated at 37°C for 2 hours.
  • the wells of the plate were washed 3 times with 320 ⁇ L per well of PBST (0.05%Tween-20) . After drying the plate, 100 ⁇ L peroxidase-conjugated streptavidin secondary antibody was added to each well of the ELISA microtiter plate and the plate was incubated at 37°Cfor 1 hour. Afterwards, the wells were washed 5 times with 320 ⁇ L per well of PBST (0.05%Tween-20) . The wells were pat dried and 100 ⁇ L TMB (Huzhou Yingchuang Biotechnology Co., Ltd. ) chromogenic substrate was added to each well.
  • TMB Human Yingchuang Biotechnology Co., Ltd.
  • bispecific antibody molecules can bind c-MET and EGFR simultaneously, showing that binding to one protein does not affect the binding of the second protein, and that the bispecific antibodies can efficiently bind to two proteins simultaneously.
  • the bispecific antibodies can inhibit the signaling pathways of tumor cell growth mediated by c-MET and EGFR at the same time.
  • Bispecific antibody blocks the binding of EGF to EGFR.
  • Human EGF-His protein was diluted to 1 ⁇ g/mL with Carbonate-Bicarbonate Buffer (CBS) coating solution and added to the wells of a 96-well ELISA microtiter plate at 200 ng/well. The plate was incubated overnight at 4°C. The next day, the 96-well ELISA microtiter plate was washed once with PBST (0.05%Tween-20) and 100 ⁇ L of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37°C for 2 hours.
  • CBS Carbonate-Bicarbonate Buffer
  • the purified bispecific antibodies were serially diluted with 2%BSA, starting at 500 ⁇ g/mL, 3-fold gradient, 11 concentration points or starting from 100 ⁇ g/mL, 3-fold gradient, 11 concentration points.
  • EGFR-hFc-biotin Kactus Biosystems
  • the blocking solution was removed from the wells and 50 ⁇ L aliquots of the serially diluted bispecific antibody solutions and EGFR-hFc-biotin solution were added to the wells of the ELISA microtiter plate. The plate was incubated at 37°C for 2 hours.
  • the plate was washed 3 times with PBST (0.05%Tween-20) at 320 ⁇ L per well.
  • the plate was dried and then 100 ⁇ L of peroxidase-conjugated anti-streptavidin antibody was added to each well of the ELISA microtiter plate and the plate was incubated at 37°C for 1 hour.
  • the wells of the plate were washed 5 times with 320 ⁇ L per well of PBST (0.05%Tween-20) .
  • 100 ⁇ L TMB Humanzhou Yingchuang Biotechnology Co., Ltd.
  • chromogenic substrate was added to each well.
  • 50 ⁇ L of 2 M H2SO4 was added to the wells to terminate the reaction, and the plate was read at OD450 nm absorbance with a microplate reader.
  • the raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting, and the IC 50 of bispecific antibodies blocking the binding of EGF to EGFR was calculated.
  • the experimental results are shown in Table 12.
  • the bispecific antibody molecules can block the binding of EGF to EGFR.
  • the bispecific antibody blocks the binding of HGF to c-MET.
  • Human c-MET-hFc protein (Kactus Biosystems) was diluted to 1 ⁇ g/mL with Carbonate-Bicarbonate Buffer (CBS) coating solution and added to the wells of a 96-well ELISA microtiter plate at 100 ng/well. The plate was incubated overnight at 4°C. The next day, the 96-well ELISA microtiter plate was washed once with PBST (0.05%Tween-20) and 100 ⁇ L of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37°C for 2 hours.
  • CBS Carbonate-Bicarbonate Buffer
  • the purified bispecific antibodies were serially diluted with 2%BSA, starting at 500 ⁇ g/mL, 3-fold gradient, 11 concentration points.
  • HGF-his solution was diluted to 600 ng/mL with 2%BSA (the final concentration of HGF-his was 300 ng/mL) .
  • the blocking solution was removed from the wells and 50 ⁇ L aliquots of the serially diluted antibody solutions and EGFR-hFc-biotin solution were added to the wells of the ELISA microtiter plate. The plate was incubated at 37°C for 2 hours. Afterwards, the plate was washed 3 times with PBST (0.05%Tween-20) at 320 ⁇ L per well.
  • PBST 0.05%Tween-20
  • the plate was dried and then 100 ⁇ L of HRP-conjugated anti-his secondary antibody was added to each well of the ELISA microtiter plate and the plate was incubated at 37°C for 1 hour. Afterwards, the wells of the plate were washed 5 times with 320 ⁇ L per well of PBST (0.05%Tween-20) . After drying the plate, 100 ⁇ L TMB (Huzhou Yingchuang Biotechnology Co., Ltd. ) chromogenic substrate was added to each well. After 10 minutes at room temperature, 50 ⁇ L of 2 M H2SO4 was added to the wells to terminate the reaction, and the plate was read at OD450 nm absorbance with a microplate reader.
  • TMB Human Yingchuang Biotechnology Co., Ltd.
  • the raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting, and the IC 50 of the bispecific antibody blocking the binding of HGF to c-MET was calculated.
  • the experimental results are shown in Table 13.
  • the bispecific antibody molecules can block the binding of HGF to c-MET.
  • NCI-H292 cells Najing Kebai
  • human bronchoalveolar carcinoma cells NCI-H322M cells
  • Trypsin-EDTA 0.25%, Shanghai Yuanpei
  • the cell density was adjusted to 5 ⁇ 10 3 and 1 ⁇ 10 4 cells/mL, respectively.
  • 100 ⁇ L of cell suspension was added to a 96-well microtiter plate (final density 500 cells/well for NCI-H292 cells, 1000 cells/well for NCI-H322M cells) and the 96-well microtiter plate was incubated at 37°C in a CO 2 incubator for 24 hours.
  • the bispecific antibody to be tested and the control antibody were diluted with complete medium, starting at a final concentration of 500 ⁇ g/mL, 3-fold gradient, with a total of 11 concentration points.
  • 100 ⁇ l of the diluted bispecific and control antibody was added to the 96-well microtiter plate and incubated at 37°C, 5%CO 2 for 8 days.
  • 20 ⁇ L of Cell Counting-Lite reagent (Nanjing Novizan Biotechnology Co., Ltd. ) was added to each well of the 96-well microtiter plate and the plate was shaken at 600 rpm for 5 minutes at room temperature.
  • a microplate reader was used to detect the luminescence signal value.
  • the maximum proliferation inhibitory rate of bispecific antibodies on tumor cells is shown in Table 15. All the tested bispecific antibodies had obvious proliferation inhibitory activity on NCI-H292 and NCI-H322M cells, and the maximum proliferation inhibitory rates were higher than that of Onartuzumab.
  • Table 15 The maximum inhibition rate of tumor cell proliferation by bispecific antibody molecules

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Abstract

Related to the field of disease treatment, and provided are bispecific antibodies against EGFR and c-MET, nucleic acid molecules encoding them, and methods for preparing them. Said anti-EGFR and c-MET bispecific antibody has high specificity and high affinity for EGFR and c-MET. Also provided is the use of said bispecific antibody in the treatment and diagnosis of diseases.

Description

EGFR/C-MET BISPECIFIC BINDING PROTEIN AND USE THEREOF FIELD OF THE INVENTION
The present disclosure relates to therapeutic bispecific antibodies, and more specifically, to bispecific antibodies against EGFR and c-MET and methods of use thereof.
DESCRIPTION OF RELATED ART
EGFR (epidermal growth factor receptor, abbreviated as EGFR, ErbB-1 or HER1) is a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR) , HER2 (erbB2, NEU) , HER3 (erbB3) and HER4 (erbB4) . The HER family plays an important regulatory role in cellular physiological processes. EGFR is a type I transmembrane glycoprotein composed of 1210 amino acids, with a molecular weight of about 170KD. Its main structure includes a ligand-binding extracellular domain, a hydrophobic transmembrane region, and an intracellular region comprising tyrosine kinase. EGFR is highly expressed in various tumors such as colorectal cancer, head and neck cancer, and non-small cell lung cancer, and it is also expressed in normal epithelial tissues such as skin and lungs. Highly expressed EGFR in tumor tissue undergoes homologous or heterologous dimerization through the binding of ligands such as EGF or TGFα, and the dimerization leads to the activation of tyrosine kinases and protein phosphorylation in tumor cells, mediating gene transcription and activation of various cell signaling pathways in cell cycle progression, which plays an important role in promoting the survival, proliferation and migration of tumor cells and tumor angiogenesis.
Tyrosine protein kinase Met (c-MET) , also known as hepatocyte growth factor receptor (HGFR) , is a heterodimeric transmembrane tyrosine kinase receptor encoded by the Met proto-oncogene. c-MET is a type I transmembrane glycoprotein composed of 1390 amino acids, with a molecular weight of about 190KD, and comprises two polypeptide chains connected by disulfide bonds through cleavage, namely the α chain (50 kDa) and the β chain (140 kDa) . MET protein can be divided into SEMA domain (Semaphorin domain, SEMA) , PSI domain (Plexin-semaphorin-integrin domain, PSI) , four immunoglobulin-like repeat domains (Immunoglobulin-plexins-transcription domain, IPT) , a transmembrane region, a juxtamembrane region (JM) , a tyrosine kinase domain (TK) and a carboxy-terminal tail region (CT) . The natural ligand for the c-MET receptor is hepatocyte growth factor (HGF) , an inactive protein that is converted to its active form by proteolytic cleavage. Aberrant regulation of c-MET has been reported in a variety of cancers, including colorectal cancer, non-small cell lung cancer, gastric cancer, and breast  cancer. High activation of c-MET and its downstream signaling pathways have been shown to trigger hyperproliferation, tumor invasion, and angiogenesis, and be associated with poor survival.
Anti-EGFR macromolecular antibody drugs, such as cetuximab, panitumumab, nimotuzumab, and necitumumab have been approved for marketing, targeting colorectal cancer, non-small cell lung cancer, etc. EGFR-tyrosine kinase inhibitors (TKIs) , such as osimertinib, alimertinib, and avitinib, have become the standard treatment for advanced cancer patients positive for EGFR mutation. c-MET-TKIs have also achieved good efficacy in non-small cell lung cancer. The EGFR and c-MET signaling pathways have been clinically verified respectively.
Nearly 60%of tumors resistant to EGFR-TKIs had increased c-MET expression, amplified c-MET, or increased c-MET ligand HGF (Turke et al., Cancer Cell, 17: 77-88, 2010) , suggesting that c-MET signaling compensates for EGFR signaling, in the tumor cell. Both EGFR and c-MET signal through the same survival and anti-apoptotic pathways (ERK and AKT) . The bispecific antibody targeting EGFR and c-MET at the same time is expected to simultaneously inhibit the signaling pathways of EGFR and c-MET, avoiding the occurrence and development of tumors caused by the activation of c-MET signaling pathway after EGFR inhibition, thereby improving the overall efficacy and safety.
Anti-c-MET and anti-EGFR antibodies are known in the art, for example, Telisotuzumab vedotin (Teliso-v or ABBV399) , which is disclosed in U. S. Patent No. 10603389, is anti-c-MET antibody ABT-700 conjugated to vc-MMAE (vedotin) , and MRG003 (ADC-3 or MYK-3) , which is disclosed in WO2023040941, is anti-EGFR antibody BA03 conjugated to vedotin.
Several EGFR/c-Met bispecific antibodies have been disclosed in the art. Bispecific antibody BSAB01 disclosed in WO2010115551 comprises the EGFR binding VH/VL pair (Fab) of cetuximab and the c-Met binding VH/VL pair (Fab) of onartuzumab, which is currently in Phase III trials. Amivantamab, a bispecific antibody marketed under the tradenameis disclosed in U.S. Patent No. 9593164. Amivantamab comprises an Anti-EGFR Fab comprising the VH and VL of Zalutumumab (See, U.S. Patent No. 7247301 and U.S. Patent No. 7595378) and an anti-cMET Fab comprising the VH and VL of antibody 069 disclosed in U.S. Patent No. 9068011. There is a need in the art for EGFR/c-MET bispecific antibodies that bind both EGFR and c-MET and/or block the downstream signaling pathways of EGFR and c-MET by, e.g., simultaneously blocking the binding of EGF to EGFR and of HGF to c-MET.
SUMMARY OF THE INVENTION
The present invention provides a high-affinity EGFR/c-MET bispecific antibody, which can specifically recognize or bind EGFR and c-MET and simultaneously block the binding of EGF to EGFR and the binding of HGF to c-MET, thus, simultaneously blocking the downstream signaling pathways of both EGFR and c-MET, thereby increasing the inhibition of cell proliferation.
The present invention provides a bispecific antibody, comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH) , and the first VL and the first VH collectively form a domain capable of specifically binding to c-MET; and the second antigen-binding domain comprises a second VL and a second VH, and the second VL and the second VH together form a domain capable of specifically binding to EGFR. In a further embodiment, the bispecific antibody binds to c-MET and EGFR with increased affinity relative to other known antibodies.
In a further embodiment of the bispecific antibody, the first antigen-binding domain and the second antigen-binding domain are each independently selected from scFv, Fab, and scFab.
In a further embodiment of the bispecific antibody, the bispecific antibody further comprises an Fc domain, wherein the Fc domain comprises a first Fc domain monomer and a second Fc domain monomer, and the first and second Fc domain monomers comprise one or more modifications that promote heterodimerization of the Fc domain monomers.
In a further embodiment of the bispecific antibody, the Fc domain comprises a first Fc domain monomer comprising modifications that form a knob structure and a second Fc domain monomer comprising modifications that form a hole structure, wherein the hole structure can be paired with the knob structure to form a heterodimeric Fc domain.
In a further embodiment of the bispecific antibody, the first Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 49 or 51, and the second Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 50 or 52.
In a further embodiment of the bispecific antibody, the first antigen-binding domain and the second antigen-binding domain are each linked to one of the first and second Fc domain monomers of the Fc domain.
In a further embodiment of the bispecific antibody, the first antigen-binding  domain is linked to the first Fc domain monomer, and the second antigen-binding domain is linked to the second Fc domain monomer; or the first antigen-binding domain is linked to the second Fc domain monomer, and the second antigen-binding domain is linked to the first Fc domain monomer.
In a further embodiment of the bispecific antibody, the first VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 17 or 59; and/or the first VH comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 18 or 60.
In a further embodiment of the bispecific antibody, the first VL comprises:
(i) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 34, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 36, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; wherein the CDRs are defined by the Kabat numbering system;
(ii) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 34, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 36, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; wherein the CDRs are defined by the Chothia numbering system;
(iii) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 34, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 36, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; wherein the CDRs are defined by the Abm numbering system; or
(iv) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 35, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; wherein the CDRs are defined by the IMGT numbering system; and/or
the first VH comprises:
(v) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 39, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 43, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47; wherein the CDRs are defined by the Kabat numbering system;
(vi) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 40, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47; wherein the CDRs are defined by the Chothia numbering system;
(vii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 42, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 46, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47; wherein the CDRs are defined by the Abm numbering system; or
(viii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 45, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 48; wherein the CDRs are defined by the IMGT numbering system.
In a further embodiment of the bispecific antibody, the first VL comprises:
(ix) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 34, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 36, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; or
(x) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 35, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38;
and/or
the first VH comprises:
(xi) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 39, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 43, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47;
(xii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 40, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47;
(xiii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 42, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 46, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47; or
(xiv) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 45, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 48.
In a further embodiment of the bispecific antibody, the first VL comprises the amino acid sequence as shown in SEQ ID NO: 17 or 59, and/or the first VH comprises the amino acid sequence as shown in SEQ ID NO: 18 or 60; or the first VL comprises the amino acid sequence as shown in SEQ ID NO: 17, the first VH comprises the amino acid sequence as shown in SEQ ID NO: 18; or the first VL comprises the amino acid sequence as shown in  SEQ ID NO: 59, the first VH comprises the amino acid sequence as shown in SEQ ID NO: 60.
In a further embodiment of the bispecific antibody the second VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15; and/or the VH comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16.
In a further embodiment of the bispecific antibody, the second VL comprises:
(xv) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; wherein the CDRs are defined by the Kabat numbering system;
(xvi) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; wherein the CDRs are defined by the Chothia numbering system;
(xvii) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; wherein the CDRs are defined by the Abm numbering system; or
(xviii) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; wherein the CDRs are defined by the IMGT numbering system; and/or
the second VH comprises:
(xix) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 24, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32; wherein the CDRs are defined by the Kabat numbering system;
(xx) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32; wherein the CDRs are defined by the Chothia numbering system;
(xxi) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 27, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 31, and HCDR3  comprising the amino acid sequence as shown in SEQ ID NO: 32; wherein the CDRs are defined by the Abm numbering system; or
(xxii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 26, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 30, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 33; wherein the CDRs are defined by the IMGT numbering system.
In a further embodiment of the bispecific antibody, the second VL comprises:
(xxiii) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; or
(xxiv) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; and/or
the second VH comprises:
(xxv) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 24, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32;
(xxvi) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32;
(xxvii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 27, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 31, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32; or
(xxviii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 26, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 30, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 33.
In a further embodiment of the bispecific antibody, the second VL comprises the amino acid sequence as shown in SEQ ID NO: 15, and/or the second VH comprises the amino acid sequence as shown in SEQ ID NO: 16.
In a further embodiment of the bispecific antibody, the first antigen-binding domain is a Fab, and the second antigen-binding domain is an scFv.
In a further embodiment of the bispecific antibody, the bispecific antibody comprises a peptide chain I-A, a peptide chain I-B and a peptide chain I-C; wherein the peptide chain I-A comprises the first VL and a light chain constant region; the peptide chain I-B  comprises: the first VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain I-C comprises: the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the peptide chain I-A comprises from N-terminus to C-terminus the first VL and the light chain constant region; the peptide chain I-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain I-C comprises from N-terminus to C-terminus (i) the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the second VH, the second VL and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the adjacent domains of the peptide chain I-A are connected optionally with or without a linker, and the adjacent domains of the peptide chain I-B are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain I-C are connected optionally with or without a linker.
In a further embodiment of the bispecific antibody, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) . In a further embodiment of the bispecific antibody, the peptide linkers are each independently selected from the group consisting of peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown as (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In a further embodiment of the bispecific antibody, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In a further embodiment of the bispecific antibody, the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
In a further embodiment of the bispecific antibody, the peptide chain I-A comprises the amino acid sequence as shown in SEQ ID NO: 1, the peptide chain I-B comprises the amino acid sequence as shown in SEQ ID NO: 2 or 9, and/or the peptide chain I-C comprises the amino acid sequence as shown in SEQ ID NO: ID NO: 3 or 10.
In a further embodiment of the bispecific antibody, the first antigen-binding domain is an scFv, and the second antigen-binding domain is a Fab.
In a further embodiment of the bispecific antibody, the bispecific antibody comprises a peptide chain II-A, a peptide chain II-B and a peptide chain II-C; wherein the peptide chain II-A comprises the second VL and a light chain constant region; the peptide  chain II-B comprises the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain II -C comprises: the first VL, the first VH and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the peptide chain II-A comprises from N-terminus to C-terminus the second VL and the light chain constant region; and the peptide chain II-B comprises from N-terminus to C-terminus the second VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain II-C comprises from N-terminus to C-terminus (i) the first VL, the first VH and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the first VH, the first VL and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the adjacent domains of the peptide chain II-A are connected optionally with or without a linker; the adjacent domains of the peptide chain II-B are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain II-C are connected optionally with or without a linker.
In a further embodiment of the bispecific antibody, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) . In a further embodiment of the bispecific antibody, the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In a further embodiment of the bispecific antibody, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In a further embodiment of the bispecific antibody, the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
In a further embodiment of the bispecific antibody, the peptide chain II-A comprises the amino acid sequence as shown in SEQ ID NO: 4, the peptide chain II-B comprises the amino acid sequence as shown in SEQ ID NO: 5 or 7, and/or the peptide chain II-C comprises the amino acid sequence as shown in SEQ ID NO: 6 or 8.
In a further embodiment of the bispecific antibody, the first antigen-binding domain is a Fab, and the second antigen-binding domain is a scFab.
In a further embodiment of the bispecific antibody, the bispecific antibody comprises a peptide chain III-A, a peptide chain III-B and a peptide chain III-C; wherein the peptide chain III-A comprises the first VL and a light chain constant region; the peptide chain  III-B comprises: the first VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain III-C comprises: the second VL, a light chain constant region, the second VH, a heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the peptide chain III-A comprises from N-terminus to C-terminus the first VL and the light chain constant region; the peptide chain III-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain III-C comprises from N-terminus to C-terminus (i) the second VL, the light chain constant region, the second VH, the heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the second VH, the heavy chain CH1 region, the second VL, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the adjacent domains of the peptide chain III-A are connected optionally with or without a linker; the adjacent domains of the peptide chain III-B are connected optionally with or without a linker; and/or the adjacent domains of the peptide chain III-C are connected optionally with or without a linker.
In a further embodiment of the bispecific antibody, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) ; or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In a further embodiment of the bispecific antibody, the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the CH1 region of the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 54.
In a further embodiment of the bispecific antibody, the first antigen-binding domain is a scFab, and the second antigen-binding domain is a Fab.
In a further embodiment of the bispecific antibody, the bispecific antibody comprises a peptide chain IV-A, a peptide chain IV-B and a peptide chain IV-C; wherein the peptide chain IV-A comprises the second VL and a light chain constant region; the peptide chain IV-B comprises: the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain IV-C comprises: the first  VL, a light chain constant region, the first VH, a heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the peptide chain IV-A comprises from N-terminus to C-terminus the second VL and the light chain constant region, and the peptide chain IV-B comprises from N-terminus to C-terminus the second VH, the CH1 region of the heavy chain and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain IV-C comprises from N-terminus to C-terminus (i) the first VL, the light chain constant region, the first VH, the heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) ; or (ii) the first VH, the heavy chain CH1 region, the first VL, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the adjacent domains of the peptide chain IV-A are connected optionally with or without a linker, and the adjacent domains of the peptide chain IV-B are connected optionally with or without a linker; and/or the adjacent domains of the peptide chain IV-C are connected optionally with or without a linker.
In a further embodiment of the bispecific antibody, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) . In a further embodiment of the bispecific antibody, the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In a further embodiment of the bispecific antibody, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68;
In a further embodiment of the bispecific antibody, the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
In a further embodiment of the bispecific antibody, the peptide chain IV-A comprises the amino acid sequence as shown in SEQ ID NO: 4, the peptide chain IV-B comprises the amino acid sequence as shown in SEQ ID NO: 7, and/or the peptide chain IV-C comprises the amino acid sequence as shown in SEQ ID NO: 11.
In a further embodiment of the bispecific antibody, the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the second antigen-binding domain comprises domain swaps in the form of CrossMab.
In a further embodiment of the bispecific antibody, the bispecific antibody  comprises a peptide chain V-A, a peptide chain V-B, a peptide chain V-C and a peptide chain V-D; wherein the peptide chain V-A comprises the first VL and a light chain constant region; the peptide chain V-B comprises: the first VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) , and the peptide chain V-C comprises: the second VH, a light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) , the peptide chain V-D comprises: the second VL and a heavy chain CH1 region.
In a further embodiment of the bispecific antibody, the peptide chain V-A comprises from N-terminus to C-terminus the first VL and the light chain constant region; the peptide chain V-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain V-C comprises from N-terminus to C-terminus the second VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) , and/or the peptide chain V-D comprises from N-terminus to C-terminus the second VL and the heavy chain CH1 region.
In a further embodiment of the bispecific antibody, the adjacent domains of the peptide chain V-A are connected optionally with or without a linker, and the adjacent domains of the peptide chain V-B are connected optionally with or without a linker, the adjacent domains of the peptide chain V-C are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain V-D are connected optionally with or without a linker.
In a further embodiment of the bispecific antibody,  the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) . In a further embodiment of the bispecific antibody, the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In a further embodiment of the bispecific antibody, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In a further embodiment of the bispecific antibody, the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54;
In a further embodiment of the bispecific antibody, the peptide chain V-A comprises the amino acid sequence as shown in SEQ ID NO: 1, the peptide chain V-B  comprises the amino acid sequence as shown in SEQ ID NO: 9; the peptide chain V-C comprises the amino acid sequence as shown in SEQ ID NO: 12, and/or the sequence of the peptide chain V-D is shown in SEQ ID NO: 13.
In a further embodiment of the bispecific antibody, the first antigen-binding domain and the second antigen-binding domain are Fab, and the Fab of the first antigen-binding domain comprises domain swaps in the form of CrossMab.
In a further embodiment of the bispecific antibody, the bispecific antibody comprises a peptide chain VI-A, a peptide chain VI-B, a peptide chain VI-C and a peptide chain VI-D; wherein the peptide chain VI-A comprises the second VL and a light chain constant region; the peptide chain VI-B comprises: the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain VI-C comprises: the first VH, a light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) ; the peptide chain VI-D comprises: the first VL and a heavy chain CH1 region.
In a further embodiment of the bispecific antibody, the peptide chain VI-A comprises from N-terminus to C-terminus the second VL and the light chain constant region, and the peptide chain VI-B comprises from N-terminus to C-terminus the second VH, the CH1 region of the heavy chain, and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain VI-C comprises from N-terminus to C-terminus the first VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) , and/or the peptide chain VI-D comprises from N-terminus to C-terminus the first VL and the heavy chain CH1 region.
In a further embodiment of the bispecific antibody, the adjacent domains of the peptide chain VI-A are connected optionally with or without a linker; the adjacent domains of the peptide chain VI-B are connected optionally with or without a linker; the adjacent domains of the peptide chain VI-C are connected optionally with or without a linker; and/or the adjacent domains of the peptide chain VI-D are connected optionally with or without a linker.
In a further embodiment of the bispecific antibody, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) . In a further embodiment of the bispecific antibody, the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In a further embodiment of the bispecific antibody, the peptide linkers each independently comprise the  amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In a further embodiment of the bispecific antibody, the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
In a further embodiment of the bispecific antibody, both the first antigen-binding domain and the second antigen-binding domain are scFvs.
In a further embodiment of the bispecific antibody, the bispecific antibody comprises a peptide chain VII-A and a peptide chain VII-B; wherein the peptide chain VII-A comprises: the first VL, the first VH and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain VII-B comprises: the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the peptide chain VII-A comprises from N-terminus to C-terminus (i) the first VL, the first VH and the first Fc domain monomer (or the second Fc domain monomer) ; or (ii) the first VH, the first VL and the first Fc domain monomer (or the second Fc domain monomer ) ; and/or the peptide chain VII-B comprises from N-terminus to C-terminus (i) the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the second VH, the second VL and the second Fc domain monomer (or the first Fc domain monomer) .
In a further embodiment of the bispecific antibody, the adjacent domains of the peptide chain VII-A are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain VII-B are connected optionally with or without a linker.
In a further embodiment of the bispecific antibody, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
In a further embodiment of the bispecific antibody, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In a further embodiment of the bispecific antibody, the peptide chain VII-A comprises the amino acid sequence as shown in SEQ ID NO: 14, and/or the peptide chain VII-B comprises the amino acid sequence as shown in SEQ ID NO: 8.
In a further embodiment of the bispecific antibody, the bispecific antibody comprises:
(1) peptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1, peptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 2, and peptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 3;
(2) peptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1, peptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 9, and peptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 10;
(3) peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5, and peptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6;
(4) peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7, and peptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8;
(5) peptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, peptide IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and peptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11;
(6) peptide chain V-A comprising the amino acid sequence shown in SEQ ID NO: 1, peptide chain V-B comprising the amino acid sequence shown in SEQ ID NO: 9, peptide chain V-C comprising the amino acid sequence shown in SEQ ID NO: 12, and peptide chain V-D comprising the amino acid sequence as shown in SEQ ID NO: 13; or
(7) peptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and peptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.
In a further embodiment of the bispecific antibody, the bispecific antibody comprises an enhanced tumor suppressive effect relative to a monospecific anti-c-MET antibody and/or a monospecific anti-EGFR antibody; wherein the amino acid sequences of the CDRs of the monospecific anti-c-MET antibody are identical to the amino acid sequences of the CDRs of the first antigen-binding domain, and the amino acid sequences of the CDRs of the monospecific anti-EGFR antibody are identical to the amino acid sequences of the CDRs of the second antigen-binding domain.
In a further embodiment of the bispecific antibody, the tumor suppression effect comprises: inhibition of EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.
The present invention further provides an isolated nucleic acid molecule or set  of nucleic acid molecules, comprising a nucleotide sequence encoding the bispecific antibody disclosed herein. The present invention provides one or more vectors, comprising the isolated nucleic acid molecule or set of nucleic acid molecules encoding the bispecific antibody disclosed herein. In a further embodiment of the one or more vectors, the nucleotide sequences encoding the different peptide chains of the bispecific antibody disclosed herein are located in different vector molecules. In a further embodiment of the one or more vectors, the one or more vectors is a cloning vector or an expression vector.
The present invention further provides a host cell comprising the isolated nucleic acid molecule or set of nucleic acid molecules disclosed herein encoding the bispecific antibody disclosed herein, or the one or more vectors disclosed herein encoding the bispecific antibody disclosed herein. In a further embodiment, the host cell expresses the bispecific antibody disclosed herein.
The present invention further provides a method for preparing the bispecific antibody disclosed herein, comprising culturing the host cell comprising the isolated nucleic acid molecule or set of nucleic acid molecules disclosed herein encoding the bispecific antibody disclosed herein, or the one or more vectors disclosed herein encoding the bispecific antibody disclosed herein under conditions allowing the expression of the bispecific antibody, and recovering the bispecific antibody from the culture of the host cell.
The present invention further provides a conjugate comprising the bispecific antibody disclosed herein and a conjugation moiety linked thereto. In a further embodiment, the conjugation moiety is selected from a therapeutic agent (such as a cytotoxic agent, cytokine, toxin, or radionuclide) .
The present invention further provides a pharmaceutical composition comprising the bispecific antibody disclosed herein, or the isolated nucleic acid molecule or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein, and a pharmaceutically acceptable carrier and/or excipient.
In a further embodiment, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In a further embodiment, the additional pharmaceutically active agent is a drug with antitumor activity. In a further embodiment, the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any  combination thereof. In a further embodiment, the bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as mixed components.
The present invention further provides use of the bispecific antibody disclosed herein, or the isolated nucleic acid molecule or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell disclosed herein, or the conjugate disclosed herein, or the pharmaceutical composition disclosed herein, in the preparation of a medicament, wherein the medicament is used for preventing and/or treating and/or acting as an adjuvant in treating diseases related to c-MET and/or EGFR in a subject, and/or for inhibiting c-MET and/or EGFR activity in vitro or in a subject.
In a further embodiment, the disease related to c-MET and/or EGFR is cancer. In a further embodiment,  the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification. In a further embodiment, cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cavity cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.
In a further embodiment, the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, one or more vectors, host cell, conjugate, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, such as administered simultaneously, separately or sequentially. In a further embodiment, the another pharmaceutically active agent is a drug with an anti-tumor activity. In a further embodiment, the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof. In a further embodiment, the subject is resistant to erlotinib, gefitinib, afatinib, CO-1686, AZD9192 or cetuximab treatment.
The present invention further provides a method for inhibiting the activity of c-MET and/or EGFR in a cell, comprising contacting the cell with the bispecific antibody disclosed herein, or the isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or set of nucleic acid molecules encoding the bispecific antibody disclosed  herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein, or the pharmaceutical composition comprising the bispecific antibody disclosed herein, or the isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein.
In a further embodiment, the cells are cells expressing c-MET and/or EGFR, such as tumor cells.
The present invention further provides a method for preventing and/or treating and/or acting as an adjuvant in treating diseases associated with c-MET and/or EGFR in a subject, wherein the method comprises administering to the subject in need thereof an effective amount of the bispecific antibody disclosed herein, or the isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein, or the pharmaceutical composition comprising the bispecific antibody disclosed herein, or the isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or the one or more vectors encoding the bispecific antibody disclosed herein, or the host cell expressing the bispecific antibody disclosed herein, or the conjugate comprising the bispecific antibody disclosed herein.
In a further embodiment, the disease associated with c-MET and/or EGFR is cancer. In a further embodiment, the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification. In a further embodiment, the cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cavity cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.
In a further embodiment, the method further comprises administering to the  subject a second therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof; optionally, the second therapy may be applied simultaneously, separately or sequentially. In a further embodiment, the subject is resistant to a treatment using erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
DESCRIPTION OF DRAWINGS
Fig. 1A: Schematic diagram of the bispecific antibody structure.
Fig. 1B: Schematic diagram of the bispecific antibody structure.
Fig. 1C: Schematic diagram of the bispecific antibody structure.
Fig. 1D: Schematic diagram of the bispecific antibody structure.
Fig. 2A: Determination of the binding activity of bispecific antibodies to human non-small cell lung cancer cells HCC827.
Fig. 2B: Determination of the binding activity of bispecific antibodies to human non-small cell lung cancer cells HCC827.
Fig. 3A: Determination of the binding activity of bispecific antibodies to human gastric cancer cell line MKN45.
Fig. 3B: Determination of the binding activity of bispecific antibodies to human gastric cancer cell line MKN45.
Fig. 4A: Determination of the binding activity of bispecific antibodies to human lung adenocarcinoma NCI-H1975 cells.
Fig. 4B: Determination of the binding activity of bispecific antibodies to human lung adenocarcinoma NCI-H1975 cells.
DETAILED DESCRIPTION OF THE INVENTION
Antibodies
In one aspect, the application provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR.
In certain embodiments, the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH) , and the first VL and the first VH collectively form a domain capable of specifically binding c-MET. In certain embodiments, the second antigen-binding domain comprises a second VL and a second VH, and the second VL and the second VH together form a domain capable of specifically binding EGFR.
In certain embodiments, said first antigen-binding domain and said second antigen-binding domain are each independently selected from an scFv, a Fab, and an scFab. Unless expressly stated otherwise, the Fab domains may be arranged according to their native orientation or comprise domain substitutions or swaps that promote correct VH and VL pairing (e.g., domain swaps in the Crossmab format) .
In certain embodiments, the domain swap of the CrossMab format is selected from:
a. CrossMabFab: In Fab, the antibody light chain CL-VL and the antibody heavy chain CH1-VH are swapped;
b. CrossMabVH-VL: In Fab, the VL and VH of the antibody are swapped;
c. CrossMabCH1-CL: In Fab, the CH and CL of the antibody are swapped.
CrossMab is a method for preventing mispairing of light and heavy chains, see US patent US9266967B2, which is incorporated herein by reference in its entirety. This method mainly utilizes the principle that the same parts of an antibody repel each other, that is, VH and VH repel each other, CH1 and CH1 repel each other, CL and CL repel each other, and VL and VL repel each other. The purpose of preventing light chain mismatch is achieved by regional interchange of the heavy and light chains of one of the Fab in bispecific antibodies (e.g., light chain CL-VL is exchanged with heavy chain CH1-VH, VL and VH are interchanged, or CH and CL are interchanged) .
In certain embodiments, the bispecific antibody further comprises an Fc domain. In certain embodiments, the Fc domain comprises first and second Fc domain monomers. In certain embodiments, the first and second Fc domain monomers comprise one or more modifications that promote heterodimerization of the Fc domain monomers.
In certain embodiments, the Fc domain comprises a first Fc domain monomer comprising modifications that form a knob structure and a second Fc domain monomer comprising modifications that form a hole structure, wherein the hole structure can be paired with the knob structure to form a heterodimeric Fc domain.
Those skilled in the art may easily understand that the hole structure can be paired with the knob structure to form a “knob in hole structure” , which can be used to reduce heavy chain mismatch in the bispecific antibody. A “knob” structure and a “hole” structure can be introduced by mutation in the corresponding positions of the CH3 domains of two Fc domain monomers to form a specific interaction interface between two Fc domain monomers (see Ridgway et al., Protein Eng., 9: 617-621 (1996) ; WO 2006/028936; incorporated herein by reference in its entirety) . Amino acid substitutions that can be used to form a “knob” structure  include, but are not limited to, S354C and T366W, wherein the  numbering is according to the Eu numbering scheme. Amino acid substitutions that can be used to form a “hole” structure include, but are not limited to, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the Eu numbering scheme. Due to mutual attraction between the “knob” structure and “hole” structure, as well as mutual repulsion between the “knob” structure and the “knob” structure, the “knob in hole structure” can effectively reduce the heavy chain mismatch in the bispecific antibody.
In certain embodiments, the Fc domain monomer is derived from the Fc domain of a human immunoglobulin, and comprises modifications that form a knob structure or a hole structure. In certain embodiments, the human immunoglobulin is IgG1, IgG2, IgG3 or IgG4. In certain embodiments, the Fc domain monomer comprises one or more modifications that form a knob structure, wherein the one or more modifications comprise S354C and/or T366W amino acid substitutions, wherein the numbering is according to the Eu numbering scheme. In certain embodiments, the Fc domain monomer comprises S354 and T366W amino acid substitutions, wherein the numbering is according to the Eu numbering scheme. In certain embodiments, the Fc domain monomer comprises one or more modifications that form a hole structure, wherein the one or more modifications comprise Y349C, T366S, L368A, and/or Y407V amino acid substitutions, wherein the numbering is according to the Eu numbering scheme. In certain embodiments, the Fc domain monomer comprises Y349C, T366S, L368A, and Y407V amino acid substitutions, wherein the numbering is according to the Eu numbering scheme.
In certain embodiments, the Fc domain further has altered effector function (e.g., enhanced ADCC activity) compared to a wild-type Fc region. Said altered effector functions may be introduced by, for example, mutation or chemical modification.
In certain embodiments, the first Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 49 or 51, and the second Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 50 or 52.
In certain embodiments, said first antigen-binding domain and said second antigen-binding domain are each linked to one of the first and second Fc domain monomers.
In certain embodiments, the first antigen-binding domain is linked to the first Fc domain monomer, and the second antigen-binding domain is linked to the second Fc domain monomer. In certain embodiments, the first antigen-binding domain is linked to the second Fc domain monomer, and the second antigen-binding domain is linked to the first Fc domain monomer.
The individual CDRs of an antigen-binding domain disclosed herein can be  determined according to any CDR numbering scheme known in the art.
In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the Kabat numbering scheme. In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the Kabat numbering scheme.
In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the Chothia numbering scheme. In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the Chothia numbering scheme.
In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the MacCallum numbering scheme. In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the MacCallum numbering scheme.
In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the IMGT numbering scheme. In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the IMGT numbering scheme.
In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the AbM numbering scheme. In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the AbM numbering scheme.
In certain embodiments, an antigen-binding domain of an antibody provided herein comprises the LCDR1, LCDR2, and/or LCDR3 of a VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59 as determined by the AHo numbering scheme. In certain  embodiments, an antigen-binding domain of an antibody provided herein comprises the HCDR1, HCDR2, and/or HCDR3 of a VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60 as determined by the AHo numbering scheme.
In certain embodiments, the individual CDRs of an antigen-binding domain of an antibody provided herein are each independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the antigen-binding domain, wherein the structural analysis identifies residues in the variable region (s) predicted to make contact with an epitope region of c-MET or EGFR.
In certain embodiments, the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 17 or 59, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 18 or 60 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the bispecific molecule, wherein the structural analysis identifies residues in the variable region (s) predicted to make contact with an epitope region of c-MET or EGFR.
In certain embodiments, the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 17, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 18 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, Aho, or AbM numbering schemes, or by structural analysis of the bispecific molecule, wherein the structural analysis  identifies residues in the variable region (s) predicted to make contact with an epitope region of c-MET or EGFR.
In certain embodiments, the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 59, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 60 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the bispecific molecule, wherein the structural analysis identifies residues in the variable region (s) predicted to make contact with an epitope region of c-MET or EGFR.
In certain embodiments, the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 17, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 60 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the bispecific molecule, wherein the structural analysis identifies residues in the variable region (s) predicted to make contact with an epitope region of c-MET or EGFR.
In certain embodiments, the instant disclosure provides a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL  amino acid sequence set forth in SEQ ID NO: 59, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 18 and/or wherein the second antigen-binding domain comprises a VL comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the bispecific molecule, wherein the structural analysis identifies residues in the variable region (s) predicted to make contact with an epitope region of c-MET or EGFR.
In certain embodiments of the bispecific antibodies provided herein, the first VL comprises:
LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; wherein the CDRs are defined by the Kabat numbering system;
LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; wherein the CDRs are defined by the Chothia numbering system;
LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 34, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; wherein the CDRs are defined by the Abm numbering system; or
LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; wherein said CDRs are defined by the IMGT numbering system; and/or
the first VH comprises:
HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 39, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 43, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47; wherein the CDRs are defined by the Kabat numbering system;
HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 40, HCDR2  comprising the amino acid sequence set forth in SEQ ID NO: in 44, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47; wherein the CDRs are defined by the Chothia numbering system;
HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 42, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47; wherein the CDRs are defined by the Abm numbering system; or
HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48; where the CDRs are defined by the IMGT numbering system.
In certain embodiments, the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 17 or 59. In certain embodiments, the first VL comprises an amino acid sequence set forth in SEQ ID NO: 17 or 59. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 or 59.
In certain embodiments, the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 18 or 60. In certain embodiments, the first VH comprises an amino acid sequence set forth in SEQ ID NO: 18 or 60. In certain embodiments, the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18 or 60.
In certain embodiments, the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 17 or 59 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100%(e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 18 or 60. In certain embodiments, the first VL comprises an amino acid sequence set forth in SEQ ID NO: 17 or 59 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 18 or 60. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 or 59 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18 or 60.
In certain embodiments, the first VL comprises an amino acid sequence that is at  least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 17 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the first VL comprises an amino acid sequence set forth in SEQ ID NO: 17 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18.
In certain embodiments, the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 59 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the first VL comprises an amino acid sequence set forth in SEQ ID NO: 59 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 59 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 60.
In certain embodiments, the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 17 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the first VL comprises an amino acid sequence set forth in SEQ ID NO: 17 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 60.
In certain embodiments, the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 59 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid  sequence set forth in SEQ ID NO: 18. In certain embodiments, the first VL comprises an amino acid sequence set forth in SEQ ID NO: 59 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 59 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18.
In certain embodiments of the bispecific antibodies provided herein, the second VL comprises:
LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; wherein the CDRs are defined by the Kabat numbering system;
LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; wherein the CDRs are defined by the Chothia numbering system;
LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; wherein the CDRs are defined by the Abm numbering system; or
LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; wherein said CDRs are defined by the IMGT numbering system; and/or
the second VH comprises:
HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; wherein the CDRs are defined by the Kabat numbering system;
HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; wherein the CDRs are defined by the Chothia numbering system;
HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, HCDR2  comprising the amino acid sequence set forth in SEQ ID NO: 31, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; wherein the CDRs are defined by the Abm numbering system; or
HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; where the CDRs are defined by the IMGT numbering system.
In certain embodiments, the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first VL comprises an amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 15.
In certain embodiments, the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the first VH comprises an amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the first VH consists of the amino acid sequence set forth in SEQ ID NO: 16.
In certain embodiments, the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 15 and the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the first VL comprises an amino acid sequence set forth in SEQ ID NO: 15 and the first VH comprises an amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 15 and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 16.
In certain embodiments, the instant disclosure provides a bispecific antibody that cross-competes for binding to c-MET and/or EGFR with any of the antibodies described herein. In certain embodiments, the instant disclosure provides a bispecific antibody that binds to the same or an overlapping epitope of c-MET and/or EGFR as an antibody described herein.
In certain embodiments, the epitope of an antibody can be determined by, e.g.,  NMR spectroscopy, surface plasmon resonanceX-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry) , array-based oligo-peptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping) . For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4) : 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are herein incorporated by reference in their entireties) . Antibody: antigen crystals may be studied using well known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc. ; see, e.g., Meth Enzymol (1985) volumes 114 &115, eds. Wyckoff HW et al. ; U. S. Patent Application No. 2004/0014194) , and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1) : 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10) : 1316-1323, all of which are herein incorporated by reference in their entireties) . Mutagenesis mapping studies may be accomplished using any method known to one of skill in the art. See, e.g., Champe M et al., (1995) and Cunningham BC &Wells JA (1989) for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques. In a specific embodiment, the epitope of an antibody is determined using alanine scanning mutagenesis studies. In addition, bispecific antibodies that recognize and bind to the same or overlapping epitopes of c-MET and/or EGFR can be identified using routine techniques such as an immunoassay, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competition binding assays also can be used to determine whether two antibodies have similar binding specificity for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as c-MET or EGFR. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA) , solid phase direct or indirect enzyme immunoassay (EIA) , sandwich competition assay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253) ; solid phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137: 3614-9) ; solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow E &Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press) ; solid phase direct label RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25 (1) : 7-15) ; solid phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176: 546-52) ; and direct labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32: 77-82) , all of which are  herein incorporated by reference in their entireties. Typically, such an assay involves the use of purified antigen (e.g., c-MET or EGFR) bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually, the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference or antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. A competition binding assay can be configured in a large number of different formats using either labeled antigen or labeled antibody. In a common version of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block the binding of labeled antibodies to the antigen is then measured using radioactive or enzyme labels. For further details see, e.g., Wagener C et al., (1983) J Immunol 130: 2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407 and Antibodies: A Laboratory Manual, Ed Harlow E &Lane D editors supra, pp. 386-389, all of which are herein incorporated by reference in their entireties.
In certain embodiments of the bispecific antibodies provided herein, one, two, or more mutations (e.g., amino acid substitutions) are introduced into an Fc region (e.g., a CH2 domain (residues 231-340 of human IgG1) ) and/or a CH3 domain (residues 341-447 of human IgG1, numbered according to the EU numbering system) and/or a hinge region (residues 216-230, numbered according to the EU numbering system) , to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into an Fc region to, e.g., reduce or ablate effector function of the Fc region. In certain embodiments, the antibodies comprise an Fc region comprising: (i) E233A and L235A (EALA) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (ii) L234A and L235A (LALA) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (iii) L234A L235A D265S (LALADS) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (iv) L234A L235A P329G (LALAPG) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (v) L234A L235A P329A (LALAPA) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (vi) L235E (LE) amino acid substitutions, wherein the numbering is according to the Eu  numbering scheme; (vii) D265A (DS) amino acid substitution, wherein the numbering is according to the Eu numbering scheme; (viii) D265A N297G (DANG) amino acid substitutions, wherein the numbering is according to the Eu numbering scheme; (ix) N297X amino acid substitution, wherein X is any amino acid other than N, wherein the numbering is according to the Eu numbering scheme; (x) N297A/D356E/L358M (NADELM) amino acid substitutions, wherein the numbering is according to Eu numbering scheme; or (x1) D356E L358M (DELM) amino acid substitutions, wherein the numbering is according to Eu numbering scheme.
In certain embodiments, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody described herein having two heavy chain constant regions.
Polypeptides
In another aspect, provided herein are polypeptides comprising one or more LCDR, HCDR, VL, and/or VH amino acid sequences as described above. In certain embodiments, the polypeptide comprises the LCDR1, LCDR2, and/or LCDR3 of the VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59, as determined by any of the methods discussed above. In certain embodiments, the polypeptide comprises the LCDR1, LCDR2, and LCDR3 of the VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17 and 59. In certain embodiments, the polypeptide comprises the LCDR1, LCDR2, and/or LCDR3 amino acid sequences set forth in SEQ ID NOs: 34, 36, and 38; 35, 37, and 38; 19, 21, and 23; or 20, 22, and 23, respectively.
In certain embodiments, the polypeptide comprises the HCDR1, HCDR2, and/or HCDR3 of the VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60, as determined by any of the methods discussed above. In certain embodiments, the polypeptide comprises the HCDR1, HCDR2, and HCDR3 of the VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18 and 60. In certain embodiments, the polypeptide comprises the HCDR1, HCDR2, and/or HCDR3 amino acid sequences set forth in SEQ ID NOs: 39, 43, and 47; 40, 44, and 47; 42, 46, and 47; 41, 45, and 48; 24, 28, and 32; 25, 29, and 32; 27, 31, and 32; or 26, 30, and 33, respectively.
In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the VL amino acid sequence set forth in any one of SEQ ID NOs: 15, 17, and 59. In certain embodiments, the polypeptide comprises the VL amino acid sequence set  forth in any one of SEQ ID NOs: 15, 17, and 59.
In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18, and 60. In certain embodiments, the polypeptide comprises the VH amino acid sequence set forth in any one of SEQ ID NOs: 16, 18, and 60.
Fab-scFv structure
In certain embodiments, the first antigen-binding domain is a Fab and the second antigen-binding domain is an scFv.
In certain embodiments, the bispecific antibody comprises polypeptide chain I-A, polypeptide chain I-B, and polypeptide chain I-C; wherein (a) the elements comprised in polypeptide chain I-A comprise a first VL and a first CL; the elements comprised in polypeptide chain I-B comprise a first VH, a first heavy chain CH1 region, and a first Fc monomer; and the elements comprised in polypeptide chain I-C comprise a second VL, a second VH, a second heavy chain CH1 hinge region, and a second Fc monomer; or (b) the elements comprised in polypeptide chain I-A comprise a first VL and a CL; the elements comprised in polypeptide chain I-B comprise a first VH, a first heavy chain CH1 region, and a second Fc monomer; and the elements comprised in polypeptide chain I-C comprise a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc monomer.
In certain embodiments, (a) the elements comprised in polypeptide chain I-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain I-B from the N-terminal end to the C-terminal end comprises the first VH, the first heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain I-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, the second heavy chain CH1 hinge region, and the second Fc monomer, or (ii) the second VH, the second VL, the second heavy chain CH1 hinge region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain I-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain I-B from the N-terminal end to the C-terminal end comprise the first VH, the first heavy chain CH1, and the second Fc monomer; and/or the elements comprised in polypeptide chain I-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, the second heavy chain CH1 hinge region, and the first Fc monomer, or (ii) the second VH, the second VL, the second heavy  chain CH1 hinge region, and the first Fc monomer.
In certain embodiments, the adjacent elements comprising the polypeptide chain I-A are optionally connected to each other by peptide linkers or not; the adjacent elements comprising the polypeptide chain I-B are optionally connected to each other by peptide linkers or not; and/or the adjacent elements comprising the polypeptide chain I-C are optionally connected by a peptide linker or not.
In certain embodiments, the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) . In certain embodiments, each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues. In certain embodiments, each peptide linker may independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues. In certain embodiments, each peptide linker may independently comprise the peptide GGGG (SEQ ID NO: 61) . In certain embodiments, the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In certain embodiments, the peptide linkers each independently comprises the amino acid sequence shown in SEQ ID NOs: 55-58, 61 and 63-68.
In certain embodiments, the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53. In certain embodiments, the first heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54. In certain embodiments, the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16, the first VL comprises the amino acid sequence shown in SEQ ID NO: 15, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the first VH comprises the amino acid sequence shown  in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
In certain embodiments, the polypeptide chain I-A comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain I-B comprises the amino acid sequence shown in SEQ ID NO: 2 or 9, and/or the polypeptide chain I-C comprises the amino acid sequence shown in SEQ ID NO: 3 or 10.
scFv-Fab structure
In certain embodiments, the first antigen-binding domain is an scFv and the second antigen-binding domain is an Fab.
In certain embodiments, the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein (a) the elements comprised in polypeptide chain II-A comprise a second VL and a second CL; the elements comprised in polypeptide chain II-B comprise a second VH, a second heavy chain CH1 region, and a first Fc monomer; the elements comprised in polypeptide chain II-C comprise a first VL, a first VH, a first heavy chain CH1 hinge region, and a second Fc monomer, or (b) the elements comprised in polypeptide chain II-A comprise a second VL and a second CL; the elements comprised in polypeptide chain II-B comprise a second VH, a second heavy chain CH1 region, and a second Fc monomer; and the elements comprised in polypeptide chain II-C comprise a  first VL, a first VH, a first heavy chain CH1 hinge region, and a first Fc monomer.
In certain embodiments, (a) the elements comprised in the polypeptide chain II-A from the N-terminal end to the C-terminal end comprise the second VL and the second CL; the elements comprised in polypeptide chain II-B from the N-terminal end to the C-terminal end comprise the second VH, the second heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain II-C from the N-terminal end to the C-terminal end comprise: (i) the first VL, the first VH, the first heavy chain CH1 hinge region, and the second Fc monomer, or (ii) the first VH, the first VL, the first heavy chain CH1 hinge region, and the second Fc monomer, or (b) the elements comprised in polypeptide chain II-A from the N-terminal end to the C-terminal end comprise the second VL and the second CL; the elements comprised in polypeptide chain II-B from the N-terminal end to the C-terminal end comprise the second VH, the second heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain II-C from the N-terminal end to the C-terminal end comprise: (i) the first VL, the first VH, the first heavy chain CH1 hinge region, and the first Fc monomer, or (ii) the first VH, the first VL, the first heavy chain CH1 hinge region, and the first Fc monomer.
In certain embodiments, the adjacent elements comprised in the polypeptide chain II-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain II-B are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in the polypeptide chain II-C are optionally connected to each other by a peptide linker or not.
In certain embodiments, the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) . In certain embodiments, each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues. In certain embodiments, the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In certain embodiments, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In certain embodiments, the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53, and/or the first and second heavy chain CH1 region  comprises the amino acid sequence shown in SEQ ID NO: 54, and/or the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16, the first VL comprises the amino acid sequence shown in SEQ ID NO: 15, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
In certain embodiments, the peptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4, the peptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 5 or 7, and/or the peptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 6 or 8.
Fab-scFab structure
In certain embodiments, the first antigen-binding domain is a Fab and the second antigen-binding domain is a scFab (single chain Fab) .
In certain embodiments, the bispecific antibody comprises polypeptide chain III-A, polypeptide chain III-B, and polypeptide chain III-C; wherein (a) the elements comprised in  polypeptide chain III-A comprise the first VL and the first CL; the elements comprised in polypeptide chain III-B comprise the first VH, the first heavy chain CH1 region, and the first Fc monomer; and the elements comprised in polypeptide chain III-C comprise the second VL, the second CL, the second VH, the second heavy chain CH1 region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain III-A comprise the first VL and the first CL; the elements comprised in polypeptide chain III-B comprise the first VH, the first heavy chain CH1 region, and the second Fc monomer; and the elements comprised in polypeptide chain III-C comprise the second VL, the second CL, the second VH, the second heavy chain CH1 region, and the first Fc monomer.
In certain embodiments, (a) the elements comprised in polypeptide chain III-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain III-B from the N-terminal end to the C-terminal end comprise the first VH, the first heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain III-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second CL, the second VH, the second heavy chain CH1 region, and the second Fc monomer; or (ii) the second VH, the second heavy chain CH1 region, the second VL, the second CL, and the second Fc monomer; or (b) the elements comprised in polypeptide chain III-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain III-B from the N-terminal end to the C-terminal end comprise the first VH, the first heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain III-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second CL, the second VH, the second heavy chain CH1 region, and the first Fc monomer; or (ii) the second VH, the second heavy chain CH1 region, the second VL, the second CL, and the first Fc monomer.
In certain embodiments, the adjacent elements comprising the polypeptide chain III-A are optionally connected to each other by a peptide linker or not, and the adjacent elements comprising the polypeptide chain III-B are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprising the polypeptide chain III-C are optionally connected to each other by a peptide linker or not.
In certain embodiments, the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) . In certain embodiments, each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues. In certain embodiments, the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In certain embodiments, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In certain embodiments, the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53, and/or the first and second heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54, and/or the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16, the first VL comprises the amino acid sequence shown in SEQ ID NO: 15, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises  the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
scFab-Fab structure
In certain embodiments, the first antigen-binding domain is a scFab and the second antigen-binding domain is a Fab.
In certain embodiments, the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B; and polypeptide chain IV-C, wherein (a) the elements comprised in  polypeptide chain IV-A comprise the second VL and second CL; the elements comprised in polypeptide chain IV-B comprise the second VH, the second heavy chain CH1 region, and the first Fc monomer; and the elements comprised in polypeptide chain IV-C comprise the first VL, the first CL, the first VH, the first heavy chain CH1 region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain IV-A comprise the second VL and second CL; the elements comprised in polypeptide chain IV-B comprise the second VH, the second heavy chain CH1 region, and the second Fc monomer; and the elements comprised in polypeptide chain IV-C comprise the first VL, the first CL, the first VH, the first heavy chain CH1 region, and the first Fc monomer.
In certain embodiments, (a) the elements comprised in polypeptide chain IV-A from the N-terminal end to the C-terminal end comprise the second VL and the second CL; the elements comprised in polypeptide chain IV-B from the N-terminal end to the C-terminal end comprise the second VH, the second heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain IV-C from the N-terminal end to the C-terminal end comprise the first VL, the first CL, the first VH, the first heavy chain CH1 region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain IV-A from the N-terminal end to the C-terminal end comprise the second VL and the second CL; the elements comprised in polypeptide chain IV-B from the N-terminal end to the C-terminal end comprise the second VL and the second CL, the second VH, the second heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain IV-C from the N-terminal end to the C-terminal end comprise (i) the first VL, the first CL, the  first VH, the first heavy chain CH1 region, and the first Fc monomer.
In certain embodiments, the adjacent elements comprising the polypeptide chain IV-A are optionally connected to each other by a peptide linker or not; the adjacent elements of the polypeptide chain IV-B are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprising the polypeptide chain IV-C are optionally connected to each other by a peptide linker or not.
In certain embodiments, the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) . In certain embodiments, each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues. In certain embodiments, the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In certain embodiments, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In certain embodiments, the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53, and/or the first and second heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54, and/or the first heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16, the first VL comprises the amino acid sequence shown in SEQ ID NO: 15, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VL comprises the amino acid sequence shown in SEQ ID NO: 59 and the first VH comprises the amino acid sequence shown in SEQ ID NO: 60. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID  NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
In certain embodiments, the peptide chain IV-A comprises the amino acid sequence shown in SEQ ID NO: 4, the peptide chain IV-B comprises the amino acid sequence shown in SEQ ID NO: 7, and/or the peptide chain IV-C comprises the amino acid sequence shown in SEQ ID NO: 11.
Fab-Fab (domain swapping including CrossMab form) structures
In certain embodiments, the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the second antigen-binding domain comprises a domain swap in the form of a CrossMabFab.
In certain embodiments, the Fab of the second antigen-binding domain comprises a domain swap of the VH and VL in the form of CrossMabVH-VL.
In certain embodiments, the Fab of the second antigen-binding domain comprises a domain swap of the CH1 and CL in the form of CrossMabCH1-CL.
In certain embodiments, the bispecific antibody in the form of CrossMabCH1-CL comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D; wherein (a) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a first Fc monomer; the elements comprised in polypeptide chain V-C comprise a second VH, a second CL, a second heavy chain CH1 hinge  region, and a second Fc monomer; and the polypeptide chain V-D comprise a second VL and a second heavy chain CH1 region; or (b) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise the first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-C comprise the second VH, the second CL, the second heavy chain CH1 hinge region, and the first Fc monomer; and the elements comprised in polypeptide chain V-D comprise the second VL and second heavy chain CH1 region.
In certain embodiments, (a) the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the first CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain V-C comprise from N-terminus to C-terminus the second VH, the second CL, the second heavy chain CH1 hinge region, and the second Fc monomer, and/or the elements comprised in polypeptide chain V-D comprise from N-terminus to C-terminus the second VL and second heavy chain CH1 region; or (b) the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-C comprise from N-terminus to C-terminus the second VH, the second CL, the second heavy chain CH1 hinge region, and the first Fc monomer, and/or the elements comprised in polypeptide chain V-D comprise from N-terminus to C-terminus the second VL and second heavy chain CH1 region.
In certain embodiments, the adjacent elements comprised in the polypeptide chain V-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-B are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-C are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in the polypeptide chain V-D are optionally connected to each other by a peptide linker or not.
In certain embodiments, the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) . In certain embodiments, each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues. In certain embodiments, the peptide linkers  each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In certain embodiments, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In certain embodiments, the first and second CL comprises the amino acid sequence shown in SEQ ID NO: 53, and/or the first and second heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54, and/or the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16, the first VL comprises the amino acid sequence shown in SEQ ID NO: 15, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the  amino acid sequence shown in SEQ ID NO: 51.
In certain embodiments, the polypeptide chain V-A comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain V-B comprises the amino acid sequence shown in SEQ ID NO: 9, the polypeptide chain V-C comprises the amino acid sequence shown in SEQ ID NO: 12, and/or the polypeptide chain V-D comprises the amino acid sequence shown in SEQ ID NO: 13.
Fab (contains domain swapping in the form of CrossMab) -Fab structures
In certain embodiments, the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the first antigen-binding domain comprises a domain swap in the form of a CrossMab.
In certain embodiments, the Fab of the first antigen-binding domain comprises a domain swap in the form of CrossMabCH1-CL.
In certain embodiments, the bispecific antibody comprises polypeptide chain VI-A, polypeptide chain VI-B, polypeptide chain VI-C, and polypeptide chain VI-D; wherein (a) the elements comprised in polypeptide chain VI-A comprise the second VL and the CL; the elements comprised in polypeptide chain VI-B comprise the second VH, the heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain VI-C comprise the first VH, the CL, first heavy chain CH1 hinge region, and the second Fc monomer; and the elements comprised in polypeptide chain VI-D comprise the first VL and the heavy chain CH1 region; or (b) the elements comprised in polypeptide chain VI-A comprise the second VL and the CL; the elements comprised in polypeptide chain VI-B comprise the second VH, the heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain VI-C comprise the first VH, the CL, the first heavy chain CH1 hinge region, and the first Fc monomer; and the elements comprised in polypeptide chain VI-D comprise first VL and the heavy chain CH1 region.
In certain embodiments, (a) the elements comprised in polypeptide chain VI-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain VI-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain VI-C from the N-terminal end to the C-terminal end comprise the first VH, the CL, the first heavy chain CH1 hinge region, and the second Fc monomer; and/or the elements comprised in polypeptide chain VI-D from the N-terminal end to the C-terminal end comprise the first VL and the heavy chain CH1 region; or (b) the elements comprised in polypeptide chain VI-A from the N-terminal end to the C-terminal end comprises the second VL and the CL; the peptide  chain VI-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain VI-C from the N-terminal end to the C-terminal end comprise the first VH, the CL, the first heavy chain CH1 hinge region, and the first Fc monomer; and/or the elements comprised in polypeptide chain VI-D from the N-terminal end to the C-terminal end comprises the first VL and the heavy chain CH1 region.
In certain embodiments, the adjacent elements comprised in the polypeptide chain VI-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain VI-B are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain VI-C are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in  the polypeptide chain VI-D are optionally connected to each other by a peptide linker or not.
In certain embodiments, the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) . In certain embodiments, each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) . In certain embodiments, the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
In certain embodiments, the peptide linkers each independently comprises the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In certain embodiments, the CL comprises the amino acid sequence shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54, and/or the first heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16, the first VL comprises the amino acid sequence shown in SEQ ID NO: 15, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL  comprises the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
In certain embodiments, the bispecific antibody in the form of CrossMabVH-VL comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D; wherein (a) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a first Fc monomer; the elements comprised in polypeptide chain V-C comprise a second VL, a second heavy chain CH1 hinge region, and a second Fc monomer; and the polypeptide chain V-D comprise a second VH and second CL; or (b) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-C comprise the second VL, a second heavy chain CH1 hinge region, and the first Fc monomer; and the elements comprised in polypeptide chain V-D comprise the second VH and second CL.
In certain embodiments, (a) the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the first CL; the elements comprised  in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain V-C comprise from N-terminus to C-terminus the second VL, the second heavy chain CH1 hinge region, and the second Fc monomer, and/or the elements comprised in polypeptide chain V-D comprise from N-terminus to C-terminus the second VH and second CL; or (b) the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the first CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-C comprise from N-terminus to C-terminus the second VL, the second heavy chain CH1 hinge region, and the first Fc monomer, and/or the elements comprised in polypeptide chain V-D comprise from N-terminus to C-terminus the second VH and second CL.
In certain embodiments, the adjacent elements comprised in the polypeptide chain V-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-B are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-C are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in the polypeptide chain V-D are optionally connected to each other by a peptide linker or not.
In certain embodiments, the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) . In certain embodiments, each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues. In certain embodiments, the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In certain embodiments, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In certain embodiments, the first and second CL comprise the amino acid sequence shown in SEQ ID NO: 53, and/or the first and second heavy chain CH1 region comprise the amino acid sequence shown in SEQ ID NO: 54, and/or the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16, the first VL comprises the amino acid sequence shown in SEQ ID NO: 15, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
In certain embodiments, the bispecific antibody in the form of CrossMabFab-Fab comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D; wherein (a) the elements comprised in polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a first Fc monomer; the elements comprised in polypeptide chain V-C comprise a second VL, a second CL, a second heavy chain CH1 hinge region, and a second Fc monomer; and the polypeptide chain V-D comprise a second VH and second heavy chain CH1 region; or (b) the elements comprised in polypeptide chain V-A  comprise a first VL and a first CL; the elements comprised in polypeptide chain V-B comprise first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-C comprise the second VL, the second CL, the second heavy chain CH1 hinge region, and the first Fc monomer; and the elements comprised in polypeptide chain V-D comprise the second VH and second heavy chain CH1 region.
In certain embodiments, (a) the elements comprised in polypeptide chain V-A from the N-terminal end to the C-terminal end comprise the first VL and the first CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain V-C comprise from N-terminus to C-terminus the second VL, the second CL, the second heavy chain CH1 hinge region, and the second Fc monomer, and/or the elements comprised in polypeptide chain V-D comprise from N-terminus to C-terminus the second VH and second heavy chain CH1 region; or (b) the elements comprised in polypeptide chain V-A comprise from N-terminus to C-terminus the first VL and the first CL; the elements comprised in polypeptide chain V-B comprise from N-terminus to C-terminus the first VH, the first heavy chain CH1 region, and the second Fc monomer; the elements comprised in polypeptide chain V-C comprise from N-terminus to C-terminus the second VL, the second CL, the second heavy chain CH1 hinge region, and the first Fc monomer, and/or the elements comprised in polypeptide chain V-D comprise from N-terminus to C-terminus the second VH and second heavy chain CH1 region.
In certain embodiments, the adjacent elements comprised in the polypeptide chain V-A are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-B are optionally connected to each other by a peptide linker or not; the adjacent elements comprised in the polypeptide chain V-C are optionally connected to each other by a peptide linker or not; and/or the adjacent elements comprised in  the polypeptide chain V-D are optionally connected to each other by a peptide linker or not.
In certain embodiments, the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker) . In certain embodiments, each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues. In certain embodiments, the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of  the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In certain embodiments, the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
In certain embodiments, the first and second CL comprise the amino acid sequence shown in SEQ ID NO: 53, and/or the first and second heavy chain CH1 region comprise the amino acid sequence shown in SEQ ID NO: 54, and/or the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16, the first VL comprises the amino acid sequence shown in SEQ ID NO: 15, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51. scFv-scFv structures
In certain embodiments, the first antigen-binding domain and the second antigen-binding domain are both scFv.
In certain embodiments, the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein (a) the elements comprised in polypeptide chain VII-A comprise the first VL, the first VH, the first heavy chain CH1 hinge region, and the first Fc monomer; and the elements comprised in polypeptide chain VII-B comprise the second VL, the second VH, the second heavy chain CH1 hinge region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain VII-A comprise the first VL, the first VH, the first heavy chain CH1 hinge region and the second Fc monomer; and the elements comprised in polypeptide chain VII-B comprise the second VL, the second VH, the second heavy chain CH1 hinge region, and the first Fc monomer.
In certain embodiments, (a) the elements comprised in polypeptide chain VII-A from the N-terminal end to the C-terminal end comprise (i) the first VL, the first VH, the first heavy chain CH1 hinge region, and the first Fc monomer, or (ii) the first VH, the first VL, the first heavy chain CH1 hinge region, and the first Fc monomer; and/or the elements comprised in polypeptide chain VII-B from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, the second heavy chain CH1 hinge region, and the second Fc monomer, or (ii) the second VH, the second VL, the second heavy chain CH1 hinge region, and the second Fc monomer; or (b) the elements comprised in polypeptide chain VII-A from the N-terminal end to the C-terminal end comprise (i) the first VL, the first VH, the first heavy chain CH1 hinge region, and the second Fc monomer, or (ii) the first VH, the first VL, the first heavy chain CH1 hinge region, and the second Fc monomer; and/or the elements comprised in polypeptide chain VII-B from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, the second heavy chain CH1 hinge region, and the first Fc monomer, or (ii) the second VH, the second VL, the second heavy chain CH1 hinge region, and the first Fc monomer.
In certain embodiments, the adjacent elements comprising the polypeptide chain VII-A are optionally connected to each other by a peptide linker or not, and/or the adjacent elements comprising the polypeptide chain VII-B are optionally connected to each other by a peptide linker or not.
In certain embodiments, the peptide linkers are each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide  linker) . In certain embodiments, each of the peptide linkers independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues. In certain embodiments, the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57) , which may be represented by (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) . In certain embodiments, the peptide linkers each independently comprises the amino acid sequence shown in SEQ ID NO: 55-58, 61, or 63-68.
In certain embodiments, the first and second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 16, the first VL comprises the amino acid sequence shown in SEQ ID NO: 15, the second VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 17.
In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16 and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50 and the second Fc monomer comprises the amino acid  sequence shown in SEQ ID NO: 49. In certain embodiments, the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52 and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51.
In certain embodiments, the polypeptide chain VII-A comprises the amino acid sequence shown in SEQ ID NO: 14, and/or the polypeptide chain VII-B comprises the amino acid sequence shown in SEQ ID NO: 8.
In certain exemplary embodiments, bispecific antibody comprising the ADC comprises:
a polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 3;
a polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 9, and a polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 10;
a polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5, and a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6;
a polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8;
a polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11;
a polypeptide chain V-A comprising the amino acid sequence shown in SEQ ID NO: 1,  a polypeptide chain V-B comprising the amino acid sequence shown in SEQ ID NO: 9, a polypeptide chain V-C comprising the amino acid sequence shown in SEQ ID NO: 12, and a polypeptide chain V-D comprising the amino acid sequence shown in SEQ ID NO: 13; or
a polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and a polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.
In certain embodiments, the bispecific antibody has an enhanced tumor suppressive effect relative to the monospecific anti-c-MET antibody and/or the monospecific anti- EGFR antibody; wherein the CDR amino acid sequences of CDRs of the monospecific anti-c-MET are identical to the amino acid sequences of CDRs sequence of the first antigen-binding domain, and the amino acid sequences of CDRs sequence of the monospecific anti-EGFR antibody are identical to the CDR amino acid sequences of CDRs of the second antigen-binding domain.
In certain embodiments, the tumor suppressive effects include: Inhibition of EGFR and c-MET signaling, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.
Polynucleotides, Vectors, and Methods of Producing Antibodies
In another aspect, the application provides an isolated nucleic acid molecule or set of nucleic acid molecules comprising a nucleotide sequence encoding a bispecific antibody as described above.
According to the codon degeneracy in the field, in some embodiments, the nucleotide sequence can be replaced according to the codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.
In another aspect, the present application provides a vector comprising an isolated nucleic acid molecule or set of nucleic acid molecules as described above.
In certain embodiments, vectors of the invention are, for example, plasmids, cosmids, phages, lentiviruses, and the like. In certain embodiments, the vector is capable of expressing a bispecific antibody of the invention in a subject (e.g., a mammal, e.g., a human) .
For example, when the bispecific antibody of the present invention comprises two peptide chains, the vector comprises a first nucleotide sequence encoding the first peptide chain of the bispecific antibody of the present invention and a second nucleotide sequence encoding the second peptide chain of the bispecific antibody of the present invention, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence exist in different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.
For example, when the bispecific antibody of the present invention comprises three peptide chains, the vector comprises a first nucleotide sequence encoding the first peptide chain of the bispecific antibody of the present invention, a second nucleotide sequence encoding the second peptide chain of the bispecific antibody of the present invention and a third nucleotide sequence encoding the third peptide chain of the bispecific antibody of the present invention, wherein the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence exist in the  same or on different vectors. When the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence, a second vector comprising the second nucleotide sequence, and a third vector comprising the third nucleotide sequence.
For example, when the bispecific antibody of the present invention comprises four peptide chains, the vector comprises a first nucleotide sequence encoding the first peptide chain of the bispecific antibody of the present invention, a second nucleotide sequence encoding the second peptide chain of the bispecific antibody of the present invention, a third nucleotide sequence encoding the third peptide chain of the bispecific antibody of the present invention and a fourth nucleotide sequence encoding the fourth peptide chain of the bispecific antibody of the present invention, wherein the first nucleotide sequence, the second nucleotide sequence, said third nucleotide sequence and said fourth nucleotide sequence are present on the same or different vectors from each other. When the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence and the fourth nucleotide sequence exist in different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence, a second vector comprising the second nucleotide sequence, a third vector comprising the third nucleotide sequence, and a fourth vector comprising the fourth nucleotide sequence.
In certain embodiments, the nucleotide sequences encoding the different peptide chains of the bispecific antibody are located on different vector molecules. In certain embodiments, the vector is a cloning vector or an expression vector.
In another aspect, the application provides a host cell comprising an isolated nucleic acid molecule or set of nucleic acid molecules as described above, or a vector as described above. Host cells can be eukaryotic cells (e.g., mammalian cells, insect cells, yeast cells) or prokaryotic cells (e.g., E. coli) . Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the invention is a mammalian cell, such as a CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44) .
The bispecific antibody of the present invention can be prepared by various methods known in the art, for example, by genetic engineering and recombination techniques. For example, a DNA molecule encoding the peptide chain of the bispecific antibody of the present invention is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, which is then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions, and express the bispecific antibody of the  present invention.
In another aspect, the present application provides a method for preparing the above-mentioned bispecific antibody, which comprises, under conditions that allow the expression of the bispecific antibody, culturing the above-mentioned host cell, and recovering the bispecific antibody from the culture of the host cell.
Therapeutic Applications
A bispecific antibody of the invention may be derivatized, e.g. linked to another molecule (e.g. another polypeptide or protein) . Typically, derivatization of antibodies does not adversely affect their binding to EGFR and c-MET. Accordingly, the bispecific antibodies of the invention are also intended to include such derivatized forms. For example, a bispecific antibody of the invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage or otherwise) to one or more other molecular moieties, such as another antibody, a detection reagent, a pharmaceutical reagent, and/or proteins or polypeptides (e.g., avidin or polyhistidine tags) capable of mediating the binding of the bispecific antibody to another molecule.
Accordingly, in one aspect, the present application provides a conjugate comprising the bispecific antibody as described above and a conjugation moiety linked thereto.
In certain embodiments, the conjugation moiety is selected from a therapeutic agent (e.g., a cytotoxic agent, cytokine, toxin, or radionuclide) .
In certain embodiments, the conjugation moiety is selected from substances capable of improving the biological properties of the bispecific antibody (such as increasing the serum half-life) , such as chemical groups such as polyethylene glycol (PEG) , formazan group or ethyl group, or sugar group.
In another aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody as described above, or the isolated nucleic acid molecule or set of nucleic acid molecules as described above, or the vector as described above, or the host cell as described above, or the conjugate as described above, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments, the pharmaceutical compositions further comprise an additional pharmaceutically active agent.
In certain embodiments, the additional pharmaceutically active agent is a drug having antineoplastic activity.
In certain embodiments, the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
In certain embodiments, the bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as mixed components.
In certain embodiments, the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate in the pharmaceutical composition of the invention is sufficient (e.g., in a subject) to exert a tumor suppressive effect (e.g., a tumor suppressive effect superior to a monospecific anti-c-MET antibody and/or a monospecific anti-EGFR antibody, wherein the amino acid sequences of CDRs of the monospecific anti-c-MET antibody are identical to the amino acid sequences of CDRs of the first antigen-binding domain, and the amino acid sequences of CDRs of the monospecific anti-EGFR antibody are identical to the amino acid sequences of CDRs of the second antigen-binding domain) .
In certain embodiments, the tumor suppressive effects include: Inhibition of EGFR and c-MET signaling, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.
In another aspect, the application provides use of the bispecific antibody as described above, or the isolated nucleic acid molecule or set of nucleic acid molecules as described above, or the vector as described above, or the host cell as described above, or the conjugate as described above, or the pharmaceutical composition as described above in the preparation of medicine, wherein the medicine is used for preventing and/or treating and/or acting as an adjuvant in treating diseases related to c-MET and/or EGFR, and/or for inhibiting the activity of c-MET and/or EGFR in vitro or in a subject.
In certain embodiments, the disease related to c-MET and/or EGFR is cancer. In certain embodiments, the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification.
In certain embodiments, the cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer,  kidney cancer or head and neck cancer.
In certain embodiments, the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, such as administered simultaneously, separately, or sequentially.
In certain embodiments, the additional pharmaceutically active agent is a drug having antineoplastic activity.
In certain embodiments, the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
In certain embodiments, the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
In another aspect, the present application provides a method for inhibiting the activity of c-MET and/or EGFR in a cell, comprising contacting the cell with the bispecific antibody as described above, or the isolated nucleic acid molecule or set of nucleic acid molecules as described above, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned conjugate, or the above-mentioned pharmaceutical composition.
In certain embodiments, the cells are cells expressing c-MET and/or EGFR, such as tumor cells.
In another aspect, the present application provides a method for preventing and/or treating and/or acting as an adjuvant in treating a disease related to c-MET and/or EGFR in a subject, wherein the method comprises administering to the subject in need thereof an effective amount of the above-mentioned bispecific antibody, or the above-mentioned isolated nucleic acid molecule or set of nucleic acid molecules, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned conjugate, or pharmaceutical composition as described above.
In certain embodiments, the disease related to c-MET and/or EGFR is cancer. In certain embodiments, the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification.
In certain embodiments, the cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal  cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.
In certain embodiments, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and any combination thereof.
In certain embodiments, the second therapy may be applied simultaneously, separately or sequentially to the methods described above.
In certain embodiments, the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
The bispecific antibody of the present invention and the pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablet, pill, suspension, emulsion, solution, gel, capsule, powder, granule, elixir, lozenge, suppository, injection (including solution for injection, sterile powder for injection and concentrated solution for injection) , inhalant, spray, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. Pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is injection. Such injection can be a sterile solution for injection. For example, the sterile solution for injection can be prepared by incorporating in an appropriate solvent a necessary dose of the bispecific antibody or the pharmaceutical composition of the present invention, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusting agent, surfactant, adjuvant, ionic strength enhancer, isotonic agent, preservative, diluent, or any combination thereof) , followed by filter sterilization. In addition, a sterile solution for injection can be prepared as sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) for ease of storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as sterile pyrogen-free water.
Furthermore, the bispecific antibodies of the present invention may be presented in pharmaceutical compositions in unit dosage form for ease of administration.
The bispecific antibody and the pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, groin, intravesical, topical (e.g., powder, ointment, or drops) , or nasal route. However,  for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection) . The skilled artisan will understand that the route and/or mode of administration will vary depending on the intended purpose. In some preferred embodiments, the bispecific antibody and the pharmaceutical composition of the present invention are administered by intravenous injection or injection.
The pharmaceutical composition of the present invention may comprise a “therapeutically effective amount” or “prophylactically effective amount” of the bispecific antibody of the present invention. The “prophylactically effective amount” means an amount sufficient to prevent, arrest, or delay the occurrence of a disease. The “therapeutically effective amount” refers to an amount sufficient to cure, or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the bispecific antibody of the invention may vary according to the following factors: The severity of the disease to be treated, the general state of the patient's immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered at the same time, etc.
In the present invention, dosing regimens can be adjusted to obtain the optimum desired response (e.g., a therapeutic or prophylactic response) . For example, a single dose can be administered, multiple doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
In the present invention, the subject may be a mammal, such as a human.
A bispecific antibody described herein can also be used to assay c-MET and/or EGFR protein levels in a biological sample using classical immunohistological methods known to those of skill in the art, including immunoassays, such as the enzyme linked immunosorbent assay (ELISA) , immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I) , carbon (14C) , sulfur (35S) , tritium (3H) , indium (121In) , and technetium (99Tc) ; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Such labels can be used to label a bispecific antibody described herein. Alternatively, a second antibody that recognizes a bispecific antibody described herein can be labeled and used in combination with a bispecific antibody to detect c-MET and/or EGFR protein levels. Therefore, in certain embodiments, the present disclosure relates to the use of a bispecific antibody of the present disclosure for in vitro detection of c-MET and/or EGFR protein in a biological sample. In a further embodiment, the present disclosure relates to the use of a  bispecific antibody of the disclosure, for assaying and/or detecting c-MET and/or EGFR protein levels in a biological sample in vitro, optionally wherein the bispecific antibody is conjugated to a radionuclide or detectable label, and/or carries a label described herein, and/or wherein an immunohistological method is used.
Assaying for the expression level of c-MET and/or EGFR protein is intended to include qualitatively or quantitatively measuring or estimating the level of c-MET and/or EGFR protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing to the disease associated protein level in a second biological sample) . c-MET and/or EGFR polypeptide expression level in the first biological sample can be measured or estimated and compared to a standard c-MET and/or EGFR protein level, the standard being taken, for example, from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once the “standard” c-MET and/or EGFR polypeptide level is known, it can be used repeatedly as a standard for comparison. Therefore, in a further embodiment, the present disclosure relates to an in vitro method for assaying and/or detecting c-MET and/or EGFR protein levels in a biological sample, comprising qualitatively or quantitatively measuring or estimating the level of c-MET and/or EGFR protein in a biological sample, by an immunohistological method.
As used herein, the term “biological sample” refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing c-MET and/or EGFR. Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Biological samples include peripheral blood mononuclear cells (PBMCs) .
A bispecific antibody described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro assessment and evaluation of cancer (e.g., tumorigenesis) or other disorders may be utilized to predict, diagnose, and monitor to evaluate patient samples, including those known to have or suspected of having cancer or other disorders, or with regard to an anticipated or desired therapeutic response. The assessment and evaluation of cancer or other disorders are also useful in determining the suitability of a patient for a clinical trial of a drug or for the administration of a particular chemotherapeutic agent, a radiotherapeutic agent, or an antibody, including combinations thereof, versus a different agent or antibody. This type of prognostic and diagnostic monitoring and assessment is already in practice utilizing  antibodies against the HER2 protein in breast cancer (HercepTestTM, Dako) where the assay is also used to evaluate patients for antibody therapy using In vivo applications include directed cell therapy and immune system modulation and radio imaging of immune responses. Therefore, in certain embodiments, the present disclosure relates to a bispecific antibody and/or pharmaceutical composition of the present disclosure for use as a diagnostic. In certain embodiments, the present disclosure relates to a bispecific antibody and/or pharmaceutical composition of the present disclosure for use in a method for the prediction, diagnosis, and/or monitoring of a subject having or suspected to have cancer or another disorder, and/or with regard to an anticipated or desired therapeutic response. In another embodiment, the present disclosure relates to the use of a bispecific antibody of the disclosure, for predicting, diagnosing, and/or monitoring of a subject having or suspected to have cancer or another disorder, and/or with regard to an anticipated or desired therapeutic response by assaying and/or detecting c-MET and/or EGFR protein levels in a biological sample of the subject in vitro.
In certain embodiments, a bispecific antibody of the present disclosure can be used in immunohistochemistry of biopsy samples. In certain embodiments, the method is an in vitro method. In another embodiment, a bispecific antibody of the present disclosure can be used to detect levels of c-MET and/or EGFR, or levels of cells which contain c-MET and/or EGFR on their membrane surface, the levels of which can then be linked to certain disease symptoms. Bispecific antibodies described herein may carry a detectable or functional label and/or may be conjugated to a radionuclide or detectable label. When fluorescence labels are used, currently available microscopy and fluorescence-activated cell sorter analysis (FACS) or combination of both methods procedures known in the art may be utilized to identify and to quantitate the specific binding members. Bispecific antibodies described herein may carry or may be conjugated to a fluorescence label. Exemplary fluorescence labels include, for example, reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. The Bispecific antibody of the present disclosure may carry or may be conjugated to a radioactive label or radionuclide, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 67Cu, 90Y, 99Tc, 111In, 117Lu, 121I, 124I, 125I, 131I, 198Au, 211At, 213Bi, 225Ac, and 186Re. When radioactive labels are used, currently available counting procedures known in the art may be utilized to identify and quantitate the specific binding of a bispecific antibody to c-MET and/or EGFR. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques as known in the art. This can be achieved by contacting a sample or a control sample with bispecific antibody under conditions  that allow for the formation of a complex between the bispecific antibody and c-MET and/or EGFR. Any complexes formed between the bispecific antibody and c-MET and/or EGFR are detected and compared in the sample and the control. In light of the specific binding of the bispecific antibodies described herein for c-MET and EGFR, the bispecific antibodies can be used to specifically detect c-MET and/or EGFR. The bispecific antibodies described herein can also be used to purify c-MET and/or EGFR via immunoaffinity purification. Also included herein is an assay system which may be prepared in the form of a test kit, kit, or kit-of-parts for the quantitative analysis of the extent of the presence of, for instance, c-MET and/or EGFR ligand complexes. The system, test kit, kit, or kit-of-parts may comprise a labeled component, e.g., a labeled antibody, and one or more additional immunochemical reagents.
Kits
Also provided are kits comprising one or more bispecific antibodies described herein, or pharmaceutical compositions or conjugates thereof. In a specific embodiment, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as one or more bispecific antibodies provided herein. In certain embodiments, the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein. In certain embodiments, the kits may contain a T cell mitogen, such as, e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA) , or a TCR complex stimulating antibody, such as an anti-CD3 antibody and anti-CD28 antibody. Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Also provided are kits that can be used in the above methods. In certain embodiments, a kit comprises a bispecific antibody described herein, preferably purified antibody, in one or more containers. In a specific embodiment, kits described herein contain a substantially isolated c-MET and/or EGFR antigen as a control. In another specific embodiment, the kits described herein further comprise a control antibody which does not react with c-MET or EGFR antigen. In another specific embodiment, kits described herein contain one or more elements for detecting the binding of an antibody to a c-MET and/or EGFR antigen (e.g., the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody can be conjugated to a detectable substrate) . In specific  embodiments, a kit provided herein can include a recombinantly produced or chemically synthesized c-MET and/or EGFR antigen. The antigen provided in the kit can also be attached to a solid support. In a more specific embodiment, the detecting means of the above-described kit includes a solid support to which a c-MET and/or EGFR antigen is attached. Such a kit can also include a non-attached reporter-labeled anti-human antibody or anti-mouse/rat antibody. In this embodiment, binding of the bispecific antibody to the antigen can be detected by binding of the said reporter-labeled antibody. In certain embodiments, the present disclosure relates to the use of a kit of the present disclosure for in vitro assaying and/or detecting c-MET and/or EGFR antigen in a biological sample.
Specific embodiments of the present invention further include:
Embodiment 1. A bispecific antibody or bispecific antigen-binding fragment thereof comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR, optionally wherein the bispecific antibody binds to c-MET and EGFR with increased affinity relative to other known antibodies.
Embodiment 2. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 1, wherein the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH) , the first VL and the first VH together form a domain capable of specifically binding c-MET; the second antigen-binding domain includes a second VL and a second VH, which together form a specific domain that binds EGFR;
wherein the first VL comprises:
i. a complementarity determining region (CDR) -L1 comprising the amino acid sequence shown in SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDRs are defined by the Kabat numbering system;
ii. a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDRs are defined by the Chothia numbering system;
iii. a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36, and a CDR-L3  comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDRs are defined by the Abm numbering system; or
iv. a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 35, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 37, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDRs are defined by the IMGT numbering system;
and /or
the first VH contains:
i. a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 39, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 43, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47, wherein the CDRs are defined by the Kabat numbering system;
ii. a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 40, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 44, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47, wherein the CDRs are defined by the Chothia numbering system;
iii. a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 42, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 46, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47, wherein the CDRs are defined by the Abm numbering system; or
iv. a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 41, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 45, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 48, where the CDRs are defined by the IMGT numbering system.
Embodiment 3. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 2, wherein the second VL comprises:
i. a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDRs are defined by the Kabat numbering system;
ii. a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDRs are defined by the Chothia numbering system;
iii. a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDRs are defined by the Abm numbering system; or
a CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 20, a CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 22, and a CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDRs are defined by the IMGT numbering system; and/or
the second VH contains:
i. a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 24, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 28, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32, wherein the CDRs are defined by the Kabat numbering system;
ii. a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32, wherein the CDRs are defined by the Chothia numbering system;
iii. a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 27, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 31, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32, wherein the CDRs are defined by the Abm numbering system; or
iv. a CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 26, a CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 30, and a CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 33, where the CDRs are defined by the IMGT numbering system.
Embodiment 4. The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 2-3, wherein (i) the first VL comprises the amino acid sequence shown in SEQ ID NO: 17 and the first VH comprises the amino acid sequence shown in SEQ ID NO: 18; or (ii) the first VL comprises the amino acid sequence shown in SEQ ID NO: 59 and the first VH comprises the amino acid sequence shown in SEQ ID NO: 60.
Embodiment 5. The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 2-4, wherein the second VL comprises the amino acid sequence shown in SEQ ID NO: 15, and/or the second VH comprises the amino acid sequence shown in SEQ ID NO: 16.
Embodiment 6. The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 5, wherein the bispecific antibody or its antigen-binding fragment further comprises an Fc dimer comprising first and second Fc monomers, and the first and second Fc monomers each independently comprise a modification of one or more amino acids that promotes heterodimerization of the first and second Fc monomers.
Embodiment 7. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 6, wherein the Fc comprises a first Fc monomer containing amino acid modifications capable of forming a knob structure and a second Fc monomer containing amino acid modifications capable of forming a hole structure, wherein the hole structure is capable of pairing with the knob structure to form a heterodimeric Fc dimer.
Embodiment 8. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 6 or 7, wherein (a) the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 50; or (b) the first Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc monomer comprises the amino acid sequence shown in SEQ ID NO: 52.
Embodiment 9. The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 6-8, wherein the first antigen-binding domain and the first Fc monomer are connected and the second antigen-binding domain is connected to the second Fc monomer; or the first antigen-binding domain and the second Fc monomer are connected and the second antigen-binding domain is connected to the first Fc monomer.
Embodiment 10. The bispecific antibody or bispecific antigen-binding fragment thereof described in any one of embodiments 1-9, wherein the first antigen-binding domain is a Fab and the second antigen-binding domain is an scFv.
Embodiment 11. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 10, wherein the bispecific antibody comprises polypeptide chain I-A, polypeptide chain I-B, and polypeptide chain I-C; wherein
i. the elements comprised in polypeptide chain I-A from the N-terminal end to the C-terminal end comprise the first VL and CL; the elements comprised in polypeptide chain I-B from the N-terminal end to the C-terminal end comprise the first VH, the heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain I-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, and the second Fc monomer; or (ii) the second VH, the second VL, and the second Fc monomer; or
ii. the elements comprised in polypeptide chain I-A from the N-terminal end to the C-terminal end comprise the first VL and CL; the elements comprised in polypeptide chain I-B from the N-terminal end to the C-terminal end comprise the first VH, the heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain I-C from the N-terminal end to the C-terminal end comprise (i) the second VL, the second VH, and the first Fc monomer; or (ii) the second VH, the second VL, and the first Fc monomer.
Embodiment 12. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 10 or 11, wherein (a) the polypeptide chain I-A comprises the amino acid sequence shown in SEQ ID NO: 1; the polypeptide chain I-B comprises the amino acid sequence shown in SEQ ID NO: 2; and/or the polypeptide chain I-C comprises the amino acid sequence shown in SEQ ID NO: 3; or (b) the polypeptide chain I-A comprises the amino acid sequence shown in SEQ ID NO: 1; the polypeptide chain I-B comprises the amino acid sequence shown in SEQ ID NO: 9; and/or the polypeptide chain I-C comprises the amino acid sequence shown in SEQ ID NO: 10.
Embodiment 13. The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 9, wherein the first antigen-binding domain is an scFv, and the second antigen-binding domain is a Fab.
Embodiment 14. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 13, wherein
i. the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein the elements comprised in polypeptide chain II-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain II-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the first Fc monomer; and/or the elements comprised in polypeptide chain II-C from N-terminal end to C-terminal end comprise:
ii. the first VL, the first VH, and the second Fc monomer; or
iii. the first VH, the first VL, and the second Fc monomer; or
iv. the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein the elements comprised in polypeptide chain II-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain II-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain II-C from the N-terminal end to the C-terminal end comprise:
v. the first VL, the first VH, and the first Fc monomer; or
vi. the first VH, the first VL, and the first Fc monomer.
Embodiment 15. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 13 or 14, wherein (a) the polypeptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4; the polypeptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 5; and/or the polypeptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 6; or (b) the polypeptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4; the polypeptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 7; and/or the polypeptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 8.
Embodiment 16. The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-9, wherein the first antigen-binding domain is an scFab, and the second antigen-binding domain is a Fab.
Embodiment 17. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 16, wherein
the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C; wherein the elements comprised in polypeptide chain IV-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain IV-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain IV-C from the N-terminal end to the C-terminal end comprise:
the first VL, the CL, the first VH, the heavy chain CH1 region, and the first Fc monomer; or
the first VH, the heavy chain CH1 region, the first VL, the CL, and the first Fc monomer; or
the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C; wherein the elements comprised in polypeptide chain IV-A from the N-terminal end to the C-terminal end comprise the second VL and the CL; the elements comprised in polypeptide chain IV-B from the N-terminal end to the C-terminal end comprise the second VH, the heavy chain CH1 region, and the second Fc monomer; and/or the elements comprised in polypeptide chain IV-C from the N-terminal end to the C-terminal end comprise:
the first VL, the CL, the first VH, the heavy chain CH1 region, and the the first Fc  monomer; or
the first VH, the heavy chain CH1 region, the first VL, the CL, and the first Fc monomer.
Embodiment 18. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 16 or 17, wherein the polypeptide chain IV-A comprises the amino acid sequence shown in SEQ ID NO: 4; the polypeptide chain IV-B comprises the amino acid sequence shown in SEQ ID NO: 7; and/or the polypeptide chain IV-C comprises the amino acid sequence shown in SEQ ID NO: 11.
Embodiment 19. The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-9, wherein the first antigen-binding domain and the second antigen-binding domain are each a Fab, and wherein the second antigen-binding domain Fab comprises a CrossMab form of domain swapping.
Embodiment 20. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 19, wherein
the bispecific antibody comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D;
wherein the elements comprised in polypeptide chain V-A from the N-terminal end to the C-terminal end comprise the first VL and CL; the elements comprised in polypeptide chain V-B from the N-terminal end to the C-terminal end comprise the first VH, the heavy chain CH1 region, and the first Fc monomer; the elements comprised in polypeptide chain V-C from the N-terminal end to the C-terminal end comprise the second VH, the CL, and the second Fc monomer; and/or the elements comprised in polypeptide chain V-D from the N-terminal end to the C-terminal end comprise the second VL and heavy chain CH1 region; or
(b) the bispecific antibody comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D;
wherein the elements comprised in polypeptide chain V-A from the N-terminal end to the C-terminal end comprise the first VL and the CL; the elements comprised in polypeptide chain V-B from the N-terminal end to the C-terminal end comprise the first VH, the heavy chain CH1 region, and the the second Fc monomer; the elements comprised in polypeptide chain V-C from the N-terminal end to the C-terminal end comprise the second VH, the CL, and the first Fc monomer; and/or the elements comprised in polypeptide chain V-D from the N-terminal end to the C-terminal end comprise the second VL and heavy chain CH1 region.
Embodiment 21. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 19 or 20, wherein the polypeptide chain V-A comprises the amino acid sequence shown in SEQ ID NO: 1; the polypeptide chain V-B comprises the amino acid sequence shown in SEQ ID NO: 9; the polypeptide chain V-C comprises the amino acid sequence shown in SEQ ID NO: 12; and/or the polypeptide chain V-D comprises the amino acid sequence shown  in SEQ ID NO: 13.
Embodiment 22. The bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-9, wherein both the first antigen-binding domain and the second antigen-binding domain are scFv.
Embodiment 23. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 22, wherein
the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B;wherein the elements comprised in polypeptide chain VII-A from the N-terminal end to the C-terminal end comprise:
the first VL, the first VH, and the first Fc monomer, or
the first VH, the first VL, and the first Fc monomer; and/or
the elements comprised in polypeptide chain VII-B rom the N-terminal end to the C-terminal end comprise:
(x) the second VL, the second VH, and said second Fc monomer, or
(y) the second VH, the second VL, and the second Fc monomer; or
(b) the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein the polypeptide chain VII-A from the N-terminal end to the C-terminal end comprise:
the first VL, the first VH, and the second Fc monomer, or
the first VH, the first VL, and the second Fc monomer; and/or
the elements comprised in polypeptide chain VII-B contains from the N-terminal end to the C-terminal end comprise:
(x) the second VL, the second VH, and said first Fc monomer, or
(y) the second VH, the second VL, and the first Fc monomer.
Embodiment 24. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 22 or 23, wherein the polypeptide chain VII-A comprises the amino acid sequence shown in SEQ ID NO: 14, and/or the polypeptide chain VII-B comprises the amino acid sequence shown in SEQ ID NO: 8.
Embodiment 25. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 11, 14, 17, 20 or 23, wherein adjacent elements comprising each of the polypeptide chains are optionally connected through a peptide linker or not.
Embodiment 26. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 25, wherein each peptide linker is independently the same peptide linker or a different peptide linker, wherein each peptide linker selected from the group  consisting of a rigid peptide linker and a flexible peptide linker.
Embodiment 27. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 26, wherein each peptide linker is independently selected from peptide linkers comprising one or more glycine (G) and/or serine (S) residues.
Embodiment 28. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 27, wherein each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of a peptide linker subunit comprising the amino acid sequence GGGGS (SEQ ID NO: 57) .
Embodiment 29. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 28, wherein each peptide linker independently comprises the amino acid sequence shown in SEQ ID NO: 55-58, or 63-68.
Embodiment 30. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 11, 14, 17, 20 or 23, wherein the CL comprises the amino acid sequence shown in SEQ ID NO: 53; and/or the heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54.
Embodiment 31. The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 30, wherein the bispecific antibody or its antigen-binding fragment comprises:
polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1, polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 2 or 9, and/or polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 3 or 10;
polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5 or 7, and/or polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6 or 8;
polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and/or polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11;
polypeptide chain V-A comprising the amino acid sequence shown in SEQ ID NO: 1, polypeptide chain V-B comprising the amino acid sequence shown in SEQ ID NO: 9, polypeptide chain V-C comprising the amino acid sequence shown in SEQ ID NO: 12, and/or polypeptide chain V-D comprising the amino acid sequence shown in SEQ ID NO: 13; or
polypeptide chain VII-A comprises the amino acid sequence shown in SEQ ID NO: 14, and/or polypeptide chain VII-B comprising the amino acid sequence of SEQ ID NO: 8.
Embodiment 32. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 28, wherein the bispecific antibody or antigen-binding fragment thereof comprises:
i. the polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 2, and the polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 3;
ii. the polypeptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 9, and the polypeptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 10;
iii. the polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, the polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5, and the polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6;
iv. the polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, the polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7, and the polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8;
v. the polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, the polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and the polypeptide chain IV-C with the amino acid sequence shown in SEQ ID NO: 11;
vi. the polypeptide chain V-A comprising the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain V-B comprising the amino acid sequence shown in SEQ ID NO: 9, the polypeptide chain V-C comprising the amino acid sequence shown in SEQ ID NO:  12, and the polypeptide chain V-D comprising the amino acid sequence shown in SEQ ID NO: 13; or
vii. the polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and the polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.
Embodiment 33. The bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 30, wherein the bispecific antibody or its antigen-binding fragment has an enhanced tumor inhibitory effect compared to the monospecific anti-c-MET antibody and/or the monospecific anti-EGFR antibody; and wherein
i. the amino acid sequences of the CDRs of the monospecific anti-c-MET antibody are the same as the amino acid sequences of the CDRs of the first antigen-binding domain and the amino acid sequence of the CDRs of the monospecific anti-EGFR antibody are the same as the amino acid sequences of the CDRs of the second antigen-binding domain, or
ii. the amino acid sequences of the CDRs of the monospecific anti-c-MET antibody are the same as the amino acid sequences of the CDRs of the second antigen-binding domain and the amino acid sequence of the CDRs of the monospecific anti-EGFR antibody are the same as the amino acid sequences of the CDRs of the first antigen-binding domain.
Embodiment 34. The bispecific antibody or bispecific antigen-binding fragment thereof of embodiment 33, wherein the tumor inhibitory effect includes inhibition of the EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or complement-dependent cytotoxicity (CDC) activity.
Embodiment 35. An isolated nucleic acid molecule or set of nucleic acid molecules, comprising a nucleotide sequence encoding the bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-34.
Embodiment 36. A vector comprising the isolated nucleic acid molecule or set of nucleic acid molecules of embodiment 35.
Embodiment 37. The vector according to embodiment 36, wherein the nucleotide sequences encoding the different peptide chains of the bispecific antibody are located in different vector molecules.
Embodiment 38. The vector according to embodiment 36 or 37, wherein the vector is a cloning vector or an expression vector.
Embodiment 39. A host cell comprising the isolated nucleic acid molecule or set of nucleic acid molecules of embodiment 35, or the vector of any one of embodiments 36-38.
Embodiment 40. A method for preparing the bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1-34, comprising culturing the host cell of embodiment 39 under conditions allowing the expression of the bispecific antibody or bispecific antigen-binding fragment thereof, and recovering the bispecific antibody from the culture of the host cell.
Embodiment 41. A pharmaceutical composition containing one or more bispecific antibodies or bispecific antigen-binding fragment thereof according to any one of embodiments 1-34 and one or more pharmaceutically acceptable carriers and/or excipients.
Embodiment 42. The pharmaceutical composition of embodiment 41, wherein the pharmaceutical composition further comprises one or more additional pharmaceutically active agents selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
Embodiment 43. Use of the bispecific antibody according to any one of embodiments 1 to 34 or the pharmaceutical composition according to embodiment 41 or 42 in preparation of a medicament, wherein the medicament is used to prevent and/or treat and/or assist in the treatment of diseases related to c-MET and/or EGFR in a subject, and/or for inhibiting c-MET cells or activity of c-MET and/or EGFR in vitro or in the body of a subject; wherein the diseases related to c-MET and/or EGFR are EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations and/or c-MET cancers associated with MET gene amplification.
Embodiment 44. The use of according to embodiment 43, wherein the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, liver cell carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, and head and neck cancer.
Embodiment 45. The use of according to embodiment 43 or 44, wherein said bispecific antibody or bispecific antigen-binding fragment thereof or pharmaceutical composition thereof and another pharmaceutically active agent are administered in combination, either simultaneously, separately or sequentially; wherein the another pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c- MET or VEGF inhibitors, chemotherapy drugs, or any combination thereof.
Embodiment 46. The use of any one of embodiments 43-45, wherein the medicament is used in a subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192 or cetuximab.
Embodiment 47. A method for inhibiting the activity of c-MET and/or EGFR in cells, comprising combining the cells with the bispecific antibody or bispecific antigen-binding fragment thereof of any one of embodiments 1-34 or the composition or the pharmaceutical composition of embodiment 41 or 42; wherein the cells are cells that express c-MET and/or EGFR.
Embodiment 48. A method for preventing and/or treating and/or adjunctive treatment of a disease associated with c-MET and/or EGFR in a subject, the method comprising administering an effective amount to a subject in need thereof the bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 34 or the pharmaceutical composition according to embodiment 41 or 42, wherein the disease associated with c-MET and/or EGFR is a cancer comprising EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and/or c-MET gene amplification.
Embodiment 49. The method of embodiment 48, wherein the cancer is selected from the group consisting of epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, and prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.
Embodiment 50. The method of embodiment 48 or 49, wherein the method further comprises administering a second therapy to the subject, the second therapy being selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof; optionally, the second therapy can be applied simultaneously, separately or sequentially.
Embodiment 51. The method of any one of embodiments 47-50, wherein the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
Embodiment 52. A bispecific antibody or bispecific antigen-binding fragment thereof according to any one of embodiments 1 to 34 or the pharmaceutical composition according to embodiment 41 or 42, for preventing and/or treating and/or adjunctive treatment of a disease associated with c-MET and/or EGFR in a subject, wherein the disease associated with c-MET and/or EGFR is a disease comprising EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and/or c-MET gene amplification cancer.
Embodiment 53. The bispecific antibody or bispecific antigen-binding fragment thereof or the pharmaceutical composition of embodiment 52, wherein the cancer is selected from the group consisting of epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, and prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.
Embodiment 54. The bispecific antibody or bispecific antigen-binding fragment thereof or the pharmaceutical composition of embodiment 52 or 53, wherein the method further comprises administering a second therapy to the subject, the second therapy being selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof; optionally, the second therapy can be applied simultaneously, separately or sequentially.
Embodiment 55. The bispecific antibody or bispecific antigen-binding fragment thereof or the pharmaceutical composition of any one of embodiments 52-54, wherein the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
Acronyms
CDR Complementarity Determining Regions in Immunoglobulin Variable Regions
FR Antibody framework region: Amino acid residues in antibody variable regions other than CDR residues
VH Antibody heavy chain variable region
VL Antibody light chain variable region
IgG immunoglobulin G
IMGT Based on the numbering system of The international ImMunoGeneTics information (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003.
Kabat Alignment and numbering system for immunoglobulins proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) .
Chothia The immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia &Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) .
Abm The immunoglobulin numbering system proposed by Martin et al., see, e.g., Martin AC, et al. Modeling antibody hypervariable loops: a combined algorithm. Proc Natl Acad Sci U S A. 1989 Dec; 86 (23) : 9268-72.
mAb Monoclonal antibodies
EC50 Concentration that produces 50%efficacy or binding
IC50 concentration that produces 50%inhibition
PCR polymerase chain reaction
HRP Horseradish peroxidase
ADCC antibody-dependent cytotoxicity
FACS Flow Cytometry
HCDR1 Complementarity-determining region 1 in the variable region of an immunoglobulin heavy chain
HCDR2 Complementarity-determining region 2 in the variable region of an immunoglobulin heavy chain
HCDR3 Complementarity-determining region 3 in the variable region of an immunoglobulin heavy chain
LCDR1 Complementarity-determining region 1 in the variable region of an immunoglobulin light chain
LCDR2 Complementarity-determining region 2 in the variable region of an immunoglobulin light chain
LCDR3 Complementarity-determining region 3 in the variable region of an immunoglobulin light Chains
Definitions
In the present disclosure, unless otherwise specified, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. In addition, the operation steps in cell culture, biochemistry, nucleic acid chemistry, and immunology laboratories used in this paper are all routine steps widely used in the corresponding fields. Meanwhile, in order to better understand the present disclosure, definitions and explanations of related terms are provided below.
As used herein, the term “antibody” is used in the broadest sense to include various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule may be composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC) . Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also comprising a “D” region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH) . The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3) . Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL) . The light chain constant region consists of one domain, CL. The constant domains are not directly involved in antibody-antigen binding, but exhibit a variety of effector functions, such as mediating the binding of immunoglobulin with host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q) . The VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) , interspersed with more conserved regions called framework regions (FRs) . Each VH and VL is composed of 3 CDRs and 4 FRs arranged from the amino terminal to the carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form the antigen-binding site, respectively. The allocation of amino acids in each region or domain can follow the definition of Kabat, Sequences of Proteins of Immunological Interest  (National Institutes of Health, Bethesda, Md. (1987 and 1991) ) , or Chothia &Lesk (1987) J. Mol. Biol. 196: 901-917 and Chothia et al. (1989) Nature 342: 878-883.
The term “antibody” further includes embodiments in which heavy chain constant domains may comprise a C-terminal lysine or lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide. The term further includes embodiments in which the N-terminal amino acid of the antibody variable domains has undergone cyclization to pyroglutamate. Thus, in a composition comprising antibodies as disclosed herein, various species of the antibodies therein may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and/or comprise an N-terminal glutamine or glutamic acid or cyclization of the N-terminal amino acid to pyroglutamate.
Herein, unless the context clearly dictates otherwise, when the term “antibody” is referred to, it includes not only whole antibodies but also antigen-binding fragments of antibodies.
As used herein, the term “complementarity determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example according to the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268 –9272) , the MacCallum numbering system (MacCallum et al., (1996) J Mol Biol 262: 732-745, see also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains, ” in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001) ) , the AHo numbering system (Honegger and Plückthun, A., J. Mol. Biol. 309: 657-670 (2001) ) , or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003) . For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003) .
In the present invention, the CDRs comprised in the antibody of the present invention or an antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise CDRs identified by the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering system.
The following general rules disclosed in www. bioinf. org. uk : Prof. Andrew C. R. Martin's Group and reproduced below may be used to define the CDRs in an antibody sequence that includes those amino acids that specifically interact with the amino acids comprising the  epitope in the antigen to which the antibody binds. There are rare examples where these generally constant features do not occur; however, the Cys residues are the most conserved feature.
The entire amino acid sequence of the VH is commonly numbered according to Kabat while the three CDRs within the variable region may be defined according to any one of the aforementioned numbering schemes. In particular embodiments, the numbering of the amino acid positions in the VH may be sequential beginning with amino acid position 1 and continuing  sequentially to the end of the sequence or according to Kabat. Unless specified otherwise, the amino acid positions in the VH and VL herein are defined according to sequential numbering.
The numbering of the amino acid positions in the heavy chain constant domain may be sequential beginning with amino acid position 1 and continuing sequentially to the end of the sequence or according to Eu numbering. The IgG1 heavy chain constant domain amino acid sequence has 330 amino acids sequentially numbered 1 to 330. The corresponding sequence numbered according to Eu begins with position number 118 and ends with position number 447. Unless specified otherwise, the amino acid positions in the heavy and light chains herein are defined according to sequential numbering.
As used herein, the term “framework region” or “FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
The term “antibody” is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes) , IgA1, IgA2, IgD, IgE or IgM antibodies.
As used herein, the term “bispecific antibody” refers to an antibody with binding specificities for two different antigens (or epitopes) , which comprises two antibodies with binding specificities for different antigens (or epitopes) . Antigen-binding domains, thereby being able to bind two different binding sites and/or target molecules. Each antigen-binding domain comprised in the bispecific antibody can be independently selected from a full-length antibody (such as an IgG antibody) or an antigen-binding fragment thereof (such as Fv, Fab, scFab or scFv) . In some cases, the individual antigen-binding domains are linked by a peptide linker.
As used herein, the term “Fv fragment” means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. The six CDRs are generally believed to confer antigen-binding specificity to an antibody. However, even a variable region (such as the Fd fragment, which comprises only three CDRs specific for an antigen) is capable of recognizing and binding antigens, although perhaps with a lower affinity than the full binding site.
As used herein, the term “Fc fragment” means a fragment formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain antibody fragments. The Fc fragment of an antibody has several different functions but is not involved in antigen binding.
As used herein, the term “scFv” refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art peptide linkers consist of the repeated GGGGS amino acid sequence (SEQ ID NO: 57) or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 (SEQ ID NO: 58) can be used, but variants thereof can also be used. In some cases, there may also be a disulfide bond between the VH and VL of the scFv.
As used herein, the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains, usually consisting of one peptide chain comprising VL and CL and another peptide chain comprising VH and CH1. However, those skilled in the art understand that the Fab domains may be arranged according to the natural orientation described above, but may also comprise domain substitutions or exchanges that promote correct VH and VL pairing (such as a domain exchange in the form of a CrossMabs) ; the term “scFab” refers to, a single polypeptide chain comprising VL, VH, CL and CH1 domains, wherein the adjacent domains are connected optionally by a linker. In the typical structure of scFab, the single polypeptide chain comprised in scFab starts from the N-terminal to the C-terminal comprises: (1) VL, CL, VH and CH1, where CL and VH are usually linked by a peptide linker (e.g., a flexible peptide linker) , or (2) VH, CH1, VL and CL, where CH1 and VL usually connected by a peptide linker (e.g., a flexible peptide linker) .
As used herein, the terms “monoclonal antibody” , and “mAb” have the same meaning and are used interchangeably to refer to one antibody molecule from a group of highly homogeneous antibody molecules or a fragment of an antibody, that is, a population of antibody molecules that are identical except for natural mutations that may arise spontaneously. mAbs are highly specific for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least 2 or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is characterized as being obtained from a highly homogeneous population of antibodies and is not to be construed as requiring that the antibody be prepared by any particular method.
As used herein, the term “bispecific antibody” or “BsAb” refers to an antibody having two distinct binding domains that enable the biantibody to bind to two different antigens or two different epitopes of the same antigen simultaneously.
As used herein, the term “CrossMab” refers to a method in the construction of bispecific antibodies that enables the correct association of the light chains and their cognate  heavy chains by exchange of heavy-chain and light-chain domains within the antigen-binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. The three possible “CrossMab” formats are: CrossMabFab, which refers to crossover or exchange in position of the complete VH-CH1 and VL-CL domains of one half of the bispecific antibody; CrossMabVH-VL, which refers to the crossover or exchange in position of only the VH and VL domains of one half of the bispecific antibody; and CrossMabCH1-CL, which refers to the crossover or exchange in position of the CH1 and CL domains within the Fab region of one half of the bispecific antibody. CrossMab antibodies have been described or claimed in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010136172, WO2010145792 and WO2013026831. The term "CrossMab" antibody is generally recognized in the art; e.g., see Brinkmannand Kontennann, MAbs 9 (2) : 182-212 (2017) ; Kontermannand Brinkmann, Drug Discovery Today 20 (7) : 838-846 (2015) ; Schaefer et al., PNAS, 108 11187-1191 (2011) ; Kleinet al., MAbs 8 (6) : 1010-1020 (2016) ; and Klein et al., MAbs 4 (6) : 653-663 (2012) .
As used herein, the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) or the half-maximal effect concentration (EC50) of the interaction.
The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the “association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187) . The ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473) . KD, kon and kdis values can be measured by any effective method. In certain embodiments, dissociation constants can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method) . Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used to measure dissociation constants.
As used herein, the term “vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to  those skilled in the art and include, but are not limited to: Plasmid; Phagemid; Cosmid; Artificial chromosome, such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) ; bacteriophage such as lambda phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses) , adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus) , poxviruses, baculoviruses, papillomaviruses, Polyoma vacuolar virus (e.g., SV40) . A vector can comprise a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also comprise an origin of replication.
Expression and cloning vectors comprise nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes origins of replication or autonomously replicating sequences. The term “expression vector” as used herein refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors comprise sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) , and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) .
As used herein, the term “host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or such as fibroblasts, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells (such as CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cells ) , ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells and other animal cells.
As used herein, the term “identity” is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by an adenine, or both a position in each of the polypeptides is occupied by lysine) , then the molecules are identical at that position. “Percent identity” between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of compared positions multiplied by 100. For example, two sequences are  60%identical if 6 out of 10 positions match. For example, the DNA sequences CTGACT and CAGGTT share 50%identity (3 out of a total of 6 positions match) . Typically, comparisons are made when two sequences are aligned for maximum identity. Such alignments can be achieved using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453 which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc. ) . The algorithm of E. Meyers and W. Miller (Comput. The algorithm of Appl Biosci., 4: 11-17 (1988) ) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine the percent identity between two amino acid sequences by using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the algorithm of Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970) ) that has been integrated into the GAP program of the GCG software package (available at www. gcg. com) can be used to determine the percent identity between two amino acid sequences by using the Blossum 62 matrix or PAM250 matrix with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.
The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E. S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991) ) , which is incorporated herein by reference. In the present invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.
As used herein, the term “pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) , and include but are not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline) , alcohols and polyols (e.g., glycerol) , and the like.  Stabilizer has the meaning generally understood by those skilled in the art, and it can stabilize the desired activity of the active ingredient in the medicine, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose) , amino acids (such as glutamic acid, glycine) , proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate) , etc.
As used herein, the term “prevention” refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a tumor) in a subject. As used herein, the term “treatment” refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of this invention, a beneficial or desired clinical outcome includes, but is not limited to, relief of symptoms, reduction of the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delay or slowing of the progression, amelioration or palliation of the disease status, and relief of symptoms (whether partial or total) , whether detectable or not. Additionally, “treatment” can also refer to prolonging survival as compared to expected survival if not receiving treatment.
As used herein, the term “subject” refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., human) has a tumor, or is at risk of having a disease as described above.
As used herein, the term “effective amount” refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing a disease (for example, a tumor) refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (for example, a tumor) ; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent an existing disease or complications. Determining such an effective amount is well within the capability of those skilled in the art. For example, amounts effective for therapeutic use will depend on the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly, etc.
As used herein, the term “effector function (effector function) ” refers to those biological activities that can be attributed to the antibody Fc region (native sequence Fc region or amino acid sequence variant Fc region) , and which are associated with the antibody varies with antibody isotype. Examples of antibody effector functions include, but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC) , complement-dependent cytotoxicity (CDC) , antibody-dependent cellular phagocytosis (ADCP) , cell surface receptors such as B cell receptor, B cell activation, cytokine secretion, half-life/clearance rate of  antibodies and antigen-antibody complexes, etc. Methods of altering the effector function of antibodies are known in the art, for example by introducing mutations in the Fc region.
As used herein, the term “antibody-dependent cell-mediated cytotoxicity (ADCC) ” refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcR) present on cytotoxic cells such as natural killer (NK) cells, neutrophils, or macrophages, allowing these cytotoxic effector cells to specifically bind to the antigen-attached target cells, and then kill the target cells by secreting cytotoxins.
As used herein, the term “combination therapy” comprises combining a bispecific antibody or pharmaceutical composition of the invention with one or more additional active therapeutic agents (e.g. chemotherapeutic agents) of a second therapy or other prophylactic or therapeutic modalities (e.g. radiotherapy) .
In such combination therapies, the various active agents often have different complementary mechanisms of action, and the combination therapy may result in a synergistic effect. Combination therapy includes therapeutic agents that affect the immune response (e.g., enhance or activate the response) and therapeutic agents that affect (e.g., inhibit or kill) tumor/cancer cells. Combination therapy reduces the likelihood of drug-resistant cancer cells developing. Combination therapy may allow dose reduction of one or more of the agents to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder or condition.
As used herein, “combination” includes therapies that may be administered separately, e.g., formulated separately for separate administration (e.g., may be provided in a kit) , as well as therapies that may be administered together in a single formulation (i.e., a “co-formulation” ) . In certain embodiments, the bispecific antibodies of the invention may be administered sequentially. In other embodiments, the bispecific antibodies can be administered simultaneously. The bispecific antibodies of the invention may be used in any combination with at least one other (active) agent.
The terms “cancer” and “tumor” are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant sites in the body through the lymphatic system or bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.
Sequence information
A description of the sequences referred to in this application is provided in the table below.











The invention will now be described with reference to the following examples, which are intended to illustrate the invention, not limit it.
Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Laboratory Guide to Molecular Biology, 3rd Edition, John Wiley &Sons, Inc., 1995.
EXAMPLE 1
Preparation of anti-EGFR and anti-c-MET bispecific antibodies and control antibodies.
a. Construction and expression of recombinant anti-EGFR and anti-c-MET bispecific antibodies
(1) Fab-scFv-Fc (KIH) bispecific antibodies
The Fab-scFv-Fc (KIH) bispecific antibodies comprise the Fab-scFv-Fc (KIH) bispecific antibody structure. The knob-in-hole (KIH) structure is used between the heavy chains to prevent mismatching. The schematic diagram of the structure is shown in Fig. 1A. The amino acid sequences comprising the anti-EGFR and anti-c-MET bispecific Fab-scFv-Fc (KIH) bispecific antibodies are shown in Table 1.
Table 1: Fab-scFv-Fc (KIH) bispecific antibody amino acid sequences
The above amino acid sequences were sent to Genscript Biotechnology Co., Ltd. for codon optimization and DNA synthesis, and the light and heavy chains of the bispecific antibody were cloned into a pKL GS expression vector (containing two expression cassettes; designed by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. and gene synthesis by GenScript) . The promoter of the first expression cassette is the mouse CMV promoter and the polyA signal sequence is the sv40 polyA signal sequence. The promoter of the second expression cassette is the mouse CMV promoter and the polyA signal sequence is the thymidine kinase (TK) polyA signal sequence. The scFv-Fc of each bispecific antibody was separately cloned into  a pKL5 Vector (containing 1 expression cassette; designed by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. and gene synthesis by GenScript; the promoter is the mouse CMV promoter and the  polyA signal sequence is the CMV polyA signal sequence) or pKL8 expression vector (containing 1 expression cassette; the promoter is mouse CMV promoter and the polyA signal sequence is the CMV polyA signal sequence) . For each plasmid pair of the plasmid expressing the light and heavy chains of a bispecific antibody and the plasmid expressing the bispecific antibody scFv-Fc as shown in Table 1, the plasmid pair was co-transfected into CHO-K1 cells (obtained from American Type Culture Collection (ATCC) , Manasas, VA) and the cells were cultured in a cell culture medium for expression of the bispecific antibodies. After allowing expression of the bispecific antibodies for a period of time, the bispecific antibodies were harvested from the culture medium and Protein A (MabSelectTM SuRe, GE) chromatography was used to capture the bispecific antibodies. The bispecific antibodies were then purified using cation chromatography (CPX, Merck KGaA) to provide bispecific antibodies 07B, 10B, 38B, and 49B.
(2) Fab-scFab-Fc (KIH) bispecific antibodies
The Fab-scFab-Fc (KIH) bispecific antibodies comprise the Fab-scFab-Fc (KIH) bispecific antibody structure, and the KIH structure is used between the heavy chains to prevent mismatching. In order to increase stability, a pair of disulfide bonds were introduced into the scFab-Fc chain, that is, the Q at position 106 of scFab-Fc was mutated to C, and the G at position 304 of scFab-Fc was mutated to C.
A schematic diagram of the structure is shown in Fig. 1B. The amino acid sequences comprising the anti-EGFR and anti-c-MET Fab-scFab-Fc (KIH) bispecific antibodies are shown in Table 2.
Table 2: Fab-scFab-Fc (KIH) bispecific antibody amino acid sequence
The above amino acid sequences were sent to Genscript Biotechnology Co., Ltd. for codon optimization and DNA synthesis. The light and heavy chains of the bispecific antibody were cloned into the pKLGS expression vector of Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd., and the scFab-Fc of the bispecific antibody was cloned into the pKL5 expression vector of Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. . The plasmid pair of the plasmid expressing the light and heavy chains of the bispecific antibody and the plasmid expressing the scFab-Fc of  the bispecific antibody were co-transfected into CHO-K1 cells (ATCC) and the cells were cultured in cell culture medium for expression of the bispecific antibody 41B. After allowing expression of the bispecific antibodies for a period of time, the bispecific antibodies were harvested from the culture medium and captured by Protein A (MabSelectTM SuRe LX, GE) affinity chromatography. The bispecific antibodies were then purified using cation chromatography (CPX, Merck KGaA) to provide bispecific antibody 41B.
(3) CrossMab bispecific antibody
In this embodiment, the bispecific antibody comprises the CrossMabCH1-CL bispecific antibody structure, and the KIH structure is used between the heavy chains to prevent mismatching. The structural diagram is shown in Fig. 1C. The amino acid sequences comprising the anti-EGFR and anti-c-MET CrossMabCH1-CL bispecific antibodies are shown in Table 3.
Table 3: CrossMabCH1-CL bispecific antibody amino acid sequences
The above amino acid sequences were sent to Genscript Biotechnology Co., Ltd. for codon optimization and DNA synthesis. The light and heavy chains of bispecific antibody 55B were cloned into the pKLGS expression vector of Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd, and the VL-CH1 and VH-CL-Fc of the bispecific antibody were cloned into another pKLGS expression vector. The two pKLGS plasmids were co-transfected into CHO-K1 cells (ATCC) and the cells were cultured in cell culture medium for expression of the bispecific antibody 55B. After allowing expression of the bispecific antibodies for a period of time, the bispecific antibodies were harvested from the culture medium and Protein A (MabSelectTM SuRe, GE) affinity chromatography was used to capture the bispecific antibodies. The bispecific antibodies were then purified using cation chromatography (CPX, Merck KGaA) to provide bispecific antibody 55B.
(4) scFv-Fc (KIH) bispecific antibodies
The scFv-Fc (KIH) bispecific antibodies comprise the scFv-Fc (KIH) bispecific  antibody structure, and the KIH structure is used between the heavy chains to prevent mismatching. The schematic diagram of the structure is shown in Fig. 1D. The amino acid sequences comprising the anti-EGFR and anti-c-MET scFv-Fc (KIH) bispecific antibodies are shown in Table 4.
Table 4: scFv-Fc (KIH) bispecific antibody sequence
The above amino acid sequences were sent to Genscript Biotechnology Co., Ltd. for codon optimization and DNA synthesis. The scFv-Fc (knob) and scFv-Fc (hole) of the bispecific antibody were cloned into the pKLGS expression vector of Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. The plasmid expressing the bispecific antibody was was transfected into CHO-K1 cells (ATCC) and the cells were cultured in cell culture medium for expression of bispecific antibody 56B. After allowing expression of the bispecific antibodies for a period of time, the bispecific antibodies were harvested from the culture medium and Protein A (MabSelectTM SuRe LX, GE) affinity Chromatography was used to capture the bispecific antibody. The bispecific antibodies were then purified using cation (CPX, Merck KGaA) chromatography to provide bispecific antibody 56B.
1.1 Construction and expression of monospecific recombinant anti-EGFR monoclonal antibody and monospecific anti-c-MET monoclonal antibody
The amino acid sequences of monospecific anti-EGFR monoclonal antibodies Cetuximab, Zalutumumab, Necitumumab, and Nimotuzumab are from the Immunogeneticswebsite, wherein the International Nonproprietary Name (INN) number for Cetuximab is 7906, the INN number for Zalutumumab is 8605, the INN number for Necitumumab is 9083, and the INN number for Nimotuzumab is 8545. Zalutumumab is a parental monoclonal antibody of the bispecific antibody provided by the present invention. The amino acid sequence of the variable region of Zalutumumab is the same as that of the anti-EGFR portion of the bispecific antibody provided by the present invention. Onartuzumab is a parental monoclonal antibody of the bispecific antibody provided by the present invention. The amino acid sequence of the variable region of Onartuzumab is the same as that of the anti-c-MET portion of the bispecific antibody provided by the present invention. MRG003 is an ADC based on an anti-EGFR  monoclonal antibody in clinical phase II and this ADC has shown good efficacy in patients with nasopharyngeal carcinoma and head and neck cancer. BA03 is the antibody of MRG003. The amino acid sequence of BA03 monoclonal antibody is from Chinese patent CN106999606B; the amino acid sequences of monospecific anti-c-MET monoclonal antibodies Onartuzumab and Telisotuzumab are from thewebsite, wherein the INN number for Onartuzumab is 9368, the INN number for Telisotuzumab is 10366, and the sequence of CE355621 monoclonal antibody sequence is from Chinese patent CN107207564A. To obtain the monospecific monoclonal antibodies, expression plasmids were constructed that encoded the heavy and light chains of the monospecific antibodies. The expression plasmids were separately transfected into CHO-K1 cells and the transfected cells were cultivated in culture medium under suitable conditions for expression of the monospecific antibodies into the culture medium. After allowing expression of the monospecific antibodies for a period of time, the monospecific antibodies were harvested from the culture medium and Protein A (MabSelectTM SuRe LX, GE) affinity Chromatography was used to capture the monospecific antibodies. The monospecific antibodies were then purified using cation (CPX, Merck KGaA) chromatography to provide the monospecific antibodies.
1.2 Hydrophilicity properties of bispecific antibodies
The hydrophilicity of the bispecific antibodies was determined using an Agilent 1260 with TSKgel Butyl-NPR column. Column temperature: 30℃, detection wavelength: 280 nm, flow rate: 0.5 mL/min, mobile phase A: 1.5 mol/L (NH4) 2SO4, mobile phase B: 25 mmol/L Na2HPO4, pH=7.0, 25%IPA. A suitable amount of the test sample was diluted with diluent (0.75 mol/L (NH4) 2SO4) to make a solution of 1.0 mg/mL as the test sample solution. The antibody Tagitanlimab (See WO2017148424) was used as a hydrophilic control and the antibody Sacituzumab (See WO2019114666) was used as a hydrophobic control. Each of the control antibodies (produced by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. ) were diluted with diluent to make a solution of 1.0 mg/mL as the system suitability solution. About 40 μg of sample was injected and analyzed in gradient elution: 0-3 minutes, maintaining mobile phase A of 95%and mobile phase B of 5%; 3-40 min, mobile phase B from 5%to 100%; 40-45 minutes, maintaining mobile phase A of 95%and mobile phase B of 5%. The hydrophobicity value of the test sample was calculated according to the control sample with the formula: (retention time of the test sample -retention time of the hydrophilic control) / (retention time of the hydrophobic control -retention time of the hydrophilic control) , and the smaller the retention time and hydrophobicity value, the better the hydrophilicity of the antibody. The results are shown in  Table 5. The bispecific antibodies all displayed good hydrophilicity. Good hydrophilicity is expected to be beneficial for the production of antibodies, quality control, or conjugation with small molecules, etc., and is expected to enhance the in vivo drug efficacy,  improve the pharmacokinetic properties and the medication safety.
Table 5: Hydrophilicity properties of the bispecific antibodies
EXAMPLE 2
Evaluation of protein binding activity of bispecific antibodies.
2.1 Evaluation of the binding activity of bispecific antibodies against the EGFR protein
Human EGFR-His protein was diluted to 1 μg/mL with Carbonate-Bicarbonate Buffer (CBS) coating solution and added to the wells of a 96-well ELISA microtiter plate at 100 ng/well. The plate was incubated overnight at 4℃. The next day, the 96-well ELISA microtiter plate was washed once with PBST (0.05%Tween-20) and 100 μL of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37℃ for 2 hours. The purified bispecific antibody and Nimotuzumab control antibody were serially diluted with 2%BSA, starting at 10 μg/mL, 3-fold gradient, 11 concentration points. The blocking solution was then removed from the wells and the serially diluted antibody solutions added to the wells of the ELISA microtiter plate. The plate was incubated at 37℃ for 2 hours. Afterwards, the plate was washed 3 times with PBST (0.05%Tween-20) at 320 μL per well. The plate was dried and then 100 μL HRP conjugated Goat anti-human IgG was added to each well, and the plate was incubated at 37 ℃ for 1 hour. Afterwards, the plate was washed 5 times with PBST (0.05%Tween-20) at 320 μL per well. After drying the plate, 100 μL 3, 3', 5, 5'-tetramethylbenzidine (TMB) (Huzhou Yingchuang Biotechnology Co., Ltd. ) chromogenic substrate was added to each well and allowed to react at room temperature for 10 minutes. The reaction was stopped by  adding 50 μL 2 M H2SO4. The plate was read at OD450nm absorbance with a microplate reader. The raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting, and the EC50 of the binding of bispecific antibody and control antibody to EGFR-His was calculated.
All of the tested bispecific antibodies were able to normally bind to EGFR-His protein. The specific results are shown in Table 6.
Table 6: Binding activity of bispecific antibodies to EGFR protein
2.2 Evaluation of the binding activity of bispecific antibodies to c-MET protein 
c-Met protein was added to the wells of a 96-well ELISA microtiter  The plate was incubated overnight at 4℃. The next day, the 96-well ELISA microtiter plate was washed once with PBST (0.05%Tween-20) and 100 μL of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37℃ for 2 hours. The purified bispecific antibody and Telisotuzumabcontrol antibody were serially diluted with 2%BSA, starting at 10 μg/mL, 3-fold gradient, 11 concentration points. The blocking solution was removed and the diluted antibody solution was added to the ELISA microtiter plate. The plate was incubated at37℃ for 2 hours. Afterwards, the plate was washed 3 times with PBST (0.05%Tween-20) at 320 μL per well. The plates were dried and then 100 μL HRP conjugated Goat anti-human IgG was added to each well, and the plate was incubated at 37 ℃ for 1 hour. Afterwards, the plate was washed 5 times with PBST (0.05%Tween-20) at 320 μL per well. After drying the plate, 100 μL 3, 3', 5, 5'-tetramethylbenzidine (TMB) (Huzhou Yingchuang Biotechnology Co., Ltd. ) chromogenic substrate was added to each well and allowed to react at room temperature for 10 minutes. The reaction was stopped by adding 50 μL 2 M H2SO4. The plate was read at OD450nm absorbance with a microplate reader. The raw data were imported into  GraphPad Prism 6 software for nonlinear curve fitting, and the EC50 of the binding of bispecific antibody and control antibody to c-MET-His was calculated. All of the tested bispecific antibodies were able to normally bind to c-MET-His protein. The specific results are shown in Table 7.
Table 7: Binding activity of bispecific antibody to c-MET protein
EXAMPLE 3
Evaluation of cell-binding activity of bispecific antibodies.
Flow cytometry (Beckman, model Cytoflex) was used to detect the binding activity of the bispecific antibodies to human non-small cell lung cancer cell HCC827 (ATCC) displaying high expression of EGFR and low expression of c-MET, human gastric cancer cell MKN45 displaying high expression of c-MET and low expression of EGFR (Nanjing Kebai) and human lung adenocarcinoma cell NCI-H1975 (Nanjing Kebai) displaying low expression of EGFR and c-MET. The protocol is as follows:
The adherent cells were digested with Trypsin-EDTA (0.25%, Shanghai Yuanpei) solution, and the cell density adjusted to 4.0×106/mL. The cells were washed twice with 1%BSA and resuspended in 1%BSA solution. Then 50 μL of cell suspension was added to each well of a 96-well sharp-bottomed plate (the number of cells was 2×105 per well) ; The bispecific antibody was diluted with 1%BSA, starting from a final concentration of 10 μg/mL, under 3-fold serial dilution, with a total of 11 concentration points. Then 50 μL of the diluted antibody was added to the wells of the conical bottom plate and the plate was incubated at 4℃ for 60 minutes. The cells were washed twice with 1%BSA and then 50 μL of diluted secondary antibody was added to each  well, mixed well, and the plate was allowed to incubate at 4℃ for 30 minutes. Afterwards, the cells were washed twice with 1%BSA and then resuspended in 200 μL 1%BSA for flow cytometry detection.
Data processing: The Median PE value was exported into GraphPad Prism 6 software for calculating the EC50. The results are shown in Figs. 2A, 2B, 3A, 3B, 4A, and 4B and Table 8 (antibody hlgG1 is a negative control antibody) . The results showed that the bispecific antibodies could effectively bind to EGFR and c-MET regardless of their expression levels. In HCC827 cells with high expression of EGFR and MKN45 cells with high expression of c-MET, the maximum signal values of bispecific antibodies were stronger than those of EGFR and c-MET monoclonal antibodies, that is, tumor cells could bind more bispecific antibodies than monoclonal antibodies, which may produce stronger tumor suppressor effects.
Table 8: Affinity binding of bispecific antibodies to tumor cells
EXAMPLE 4
Detection of internalizing activity of the bispecific antibodies.
4.1 Detection of internalizing activity by bispecific antibody acid washing method
Flow cytometry (Beckman, model Cytoflex) was used to detect the internalizing activity of the bispecific antibodies on human non-small cell lung cancer cell HCC827 displaying high expression of EGFR and low expression of c-MET, human gastric cancer cell MKN45 displaying high expression of c-MET and low expression of EGFR, and human lung adenocarcinoma cell NCI-H1975 displaying low expression of EGFR and c-MET.
The adherent cells were digested with TrypleTM Express (Thermo) solution  and counted. The cell density was adjusted to 4×106 cells/mL with 1%BSA and 50 μL of cell suspension was added to the wells of a 96-well microtiter plate (the number of cells per well was 2×105 cells/well) . The bispecific antibody was diluted with 1%BSA starting from 5 μg/mL, under 4-fold serial dilution, with a total of 8 concentration points. Then 50 μL of diluted bispecific antibody per well was added to the wells of the 96-well microtiter plate and the plate was incubated at 4℃ for 40 minutes. Afterwards, the wells were washed twice with 1%BSA and the secondary antibody (anti-human F (ab') 2 Alexa Fluor 660) diluted 1: 200 with 1%BSA was added at 50 μL per well. The plates were incubated at 4℃ for 20 minutes, then the wells were washed twice with 1%BSA and the cells therein were resuspended in 150 μL of complete medium. The resuspended cells were incubated at 4℃ and 3℃ for 4 hours after resuspension. Afterwards, the cells were centrifuged at 500g for 3 minutes to remove the medium, and 100 μL of pH 2.0 acid solution was added per well and the plate was incubated for 2 minutes. Next, 60 μL of pH 13 base solution was added for neutralization followed by centrifugation to remove the supernatant fraction. Then,  the cells were resuspended in 1%BSA for FACS analysis.
Data processing: Median FITC values were imported into GraphPad Prism 6 software to calculate EC50. The results are shown in Table 9. The bispecific antibodies showed good internalizing activity on tumor cells and may produce a stronger tumor inhibitory effect.
Table 9: Internalizing activity of bispecific antibody
4.2 Detection of internalizing activity of bispecific antibody withreagent
Flow cytometry (Beckman, model Cytoflex) was used to detect the internalizing activity of bispecific antibodies on human non-small cell lung cancer cell HCC827 displaying high expression of EGFR and low expression of c-MET, human gastric cancer cell MKN45 displaying high expression of c-MET and low expression of EGFR, and human lung adenocarcinoma cell NCI-H1975 displaying low expression of EGFR and c-MET.
The adherent cells were digested with Trypsin-EDTA (0.25%) (Shanghai Yuanpei) solution, the cells were counted, and the cell density was adjusted to 1 × 105 cells/mL with complete culture medium. Then 100 μL cell suspension was added to each well of a 96-well plate (the number  of cells is 1 × 104 cells per well) and incubated at 37℃, CO2 for 24 hours. Then the culture medium was removed and 50 μL of fresh complete culture medium was added to each well; complete culture medium was used to gradually dilute the bispecific antibody, with the final concentrations being 0.52, 2.08, 8.33, 33.30, 100.00, 200.00, 333.33, 500 μg/mL (atotal of 8 concentration points) . reagent (Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. ) in complete culture medium was reduced to 12 μg/mL (the final concentration ofis 3 μg/mL) . The serially diluted bispecific antibodies to be tested were combined with the diluted PHrodo reagent to provide a finalreagent ratio of 1: 1 (30 μL: 30 μL) and incubated at room temperature for 30 minutes in the dark. Then, 50 μL of the bispecific antibody to be tested andreagent mixture was added to the wells of a 96-well plate and the 96-well plate was incubated at 37℃, 5%CO2 for 24 hours. Afterwards, the culture medium was removed, the wells were washed once with sterile PBS, and then 100 μL Trypsin-EDTA (0.25%) was added to each well to release the cells from the wells. Next, 100 μL complete culture medium was added to neutralize the trypsin and the cells were dispersed by pipetting and testing by FACS.
Data processing: The value of Median YL1-H was exported and imported into GraphPad Prism 6 software to calculate EC50. The results are shown in Table 10. The bispecific antibodies have a good internalizing activity on tumor cells and may produce stronger tumor suppressor effect.
Table 10: Internalizing activity of bispecific antibody
EXAMPLE 5
Cross-arm binding activity of bispecific antibodies to EGFR and c-MET.
Human EGFR-His protein was diluted to 1 μg/mL with Carbonate-Bicarbonate Buffer (CBS) coating solution and added to the wells of a 96-well ELISA microtiter plate at 100 ng/well. The plate was incubated overnight at 4℃. The next day, the 96-well ELISA microtiter  plate was washed once with PBST (0.05%Tween-20) and 100 μL of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37℃ for 2 hours. The purified bispecific antibodies were serially diluted with 2%BSA, starting at 10 μg/mL, 3-fold gradient, 11 concentration points.  The blocking solution was removed from the wells and the serially diluted antibody solutions were added to the wells of the ELISA microtiter plate. The plate was incubated at 37℃ for 2 hours. Afterwards, the plate was washed 3 times with PBST (0.05%Tween-20) at 320 μL per well. The plate was dried and then 100 μL of 1 μg/mL human c-MET-His-biotin protein (Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd) was added to each well of the ELISA microtiter plate and the plate was incubated at 37℃ for 2 hours. Afterwards, the wells of the plate were washed 3 times with 320 μL per well of PBST (0.05%Tween-20) . After drying the plate, 100 μL peroxidase-conjugated streptavidin secondary antibody was added to each well of the ELISA microtiter plate and the plate was incubated at 37℃for 1 hour. Afterwards, the wells were washed 5 times with 320 μL per well of PBST (0.05%Tween-20) . The wells were pat dried and 100 μL TMB (Huzhou Yingchuang Biotechnology Co., Ltd. ) chromogenic substrate was added to each well. After 10 minutes at room temperature, 50 μL of 2 M H2SO4 was added to the wells to terminate the reaction, and the plate was read at OD450 nm absorbance with a microplate reader. The raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting, and the EC50 of the bispecific antibody cross-arm binding to c-MET and EGFR was calculated. The experimental results are shown in Table 11.
All the designed bispecific antibody molecules can bind c-MET and EGFR simultaneously, showing that binding to one protein does not affect the binding of the second protein, and that the bispecific antibodies can efficiently bind to two proteins simultaneously. Thus, the bispecific antibodies can inhibit the signaling pathways of tumor cell growth mediated by c-MET and EGFR at the same time.
Table 11: Cross-arm binding activity of bispecific antibodies to EGFR and c-MET
EXAMPLE 6
Bispecific antibody blocks the binding of EGF to EGFR.
Human EGF-His protein was diluted to 1 μg/mL with Carbonate-Bicarbonate Buffer (CBS) coating solution and added to the wells of a 96-well ELISA microtiter plate at 200 ng/well. The plate was incubated overnight at 4℃. The next day, the 96-well ELISA microtiter plate was washed once with PBST (0.05%Tween-20) and 100 μL of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37℃ for 2 hours. The purified bispecific antibodies were serially diluted with 2%BSA, starting at 500 μg/mL, 3-fold gradient, 11 concentration points or starting from 100 μg/mL, 3-fold gradient, 11 concentration points. EGFR-hFc-biotin (Kactus Biosystems) was diluted to 600 ng/mL with 2%BSA (the final concentration of EGFR-hFc-biotin was 300 ng/mL) . The blocking solution was removed from the wells and 50 μL aliquots of the serially diluted bispecific antibody solutions and EGFR-hFc-biotin solution were added to the wells of the ELISA microtiter plate. The plate was incubated at 37℃ for 2 hours. Afterwards, the plate was washed 3 times with PBST (0.05%Tween-20) at 320 μL per well. The plate was dried and then 100 μL of peroxidase-conjugated anti-streptavidin antibody was added to each well of the ELISA microtiter plate and the plate was incubated at 37℃ for 1 hour. Afterwards, the wells of the plate were washed 5 times with 320 μL per well of PBST (0.05%Tween-20) . After drying the plate, 100 μL TMB (Huzhou Yingchuang Biotechnology Co., Ltd. ) chromogenic substrate was added to each well. After 20 minutes at room temperature, 50 μL of 2 M H2SO4 was added to the wells to terminate the reaction, and the plate was read at OD450 nm absorbance with a microplate reader.
The raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting, and the IC50 of bispecific antibodies blocking the binding of EGF to EGFR was calculated.
The experimental results are shown in Table 12. The bispecific antibody molecules can block the binding of EGF to EGFR.
Table 12: Bispecific antibody molecules block the binding of EGF to EGFR
EXAMPLE 7
The bispecific antibody blocks the binding of HGF to c-MET.
Human c-MET-hFc protein (Kactus Biosystems) was diluted to 1 μg/mL with Carbonate-Bicarbonate Buffer (CBS) coating solution and added to the wells of a 96-well ELISA microtiter plate at 100 ng/well. The plate was incubated overnight at 4℃. The next day, the 96-well ELISA microtiter plate was washed once with PBST (0.05%Tween-20) and 100 μL of 2%BSA blocking solution was then added to each well of the ELISA microtiter plate and the plate was incubated at 37℃ for 2 hours. The purified bispecific antibodies were serially diluted with 2%BSA, starting at 500 μg/mL, 3-fold gradient, 11 concentration points. HGF-his solution was diluted to 600 ng/mL with 2%BSA (the final concentration of HGF-his was 300 ng/mL) . The blocking solution was removed from the wells and 50 μL aliquots of the serially diluted antibody solutions and EGFR-hFc-biotin solution were added to the wells of the ELISA microtiter plate. The plate was incubated at 37℃ for 2 hours. Afterwards, the plate was washed 3 times with PBST (0.05%Tween-20) at 320 μL per well. The plate was dried and then 100 μL of HRP-conjugated anti-his secondary antibody was added to each well of the ELISA microtiter plate and the plate was incubated at 37℃ for 1 hour. Afterwards, the wells of the plate were washed 5 times with 320 μL per well of PBST (0.05%Tween-20) . After drying the plate, 100 μL TMB (Huzhou Yingchuang Biotechnology Co., Ltd. ) chromogenic substrate was added to each well. After 10 minutes at room temperature, 50 μL of 2 M H2SO4 was added to the wells to terminate the reaction, and the plate was read at OD450 nm absorbance with a microplate reader.
The raw data were imported into GraphPad Prism 6 software for nonlinear curve fitting, and the IC50 of the bispecific antibody blocking the binding of HGF to c-MET was calculated. The experimental results are shown in Table 13. The bispecific antibody molecules can block the binding of HGF to c-MET.
Table 13: Bispecific antibody molecules block the binding of HGF to c-MET
EXAMPLE 8
Proliferation inhibitory activity of bispecific antibody on human tumor cell.
Adherent human lung cancer cells NCI-H292 cells (Nanjing Kebai) and human bronchoalveolar carcinoma cells NCI-H322M cells (Nanjing Kebai) were digested with Trypsin-EDTA (0.25%, Shanghai Yuanpei) solution and counted. The cell density was adjusted to 5×103 and 1×104 cells/mL, respectively. 100 μL of cell suspension was added to a 96-well microtiter plate (final density 500 cells/well for NCI-H292 cells, 1000 cells/well for NCI-H322M cells) and the 96-well microtiter plate was incubated at 37℃ in a CO2 incubator for 24 hours. Afterwards, the bispecific antibody to be tested and the control antibody were diluted with complete medium, starting at a final concentration of 500 μg/mL, 3-fold gradient, with a total of 11 concentration points. 100 μl of the diluted bispecific and control antibody was added to the 96-well microtiter plate and incubated at 37℃, 5%CO2 for 8 days. Afterwards, 20 μL of Cell Counting-Lite reagent (Nanjing Novizan Biotechnology Co., Ltd. ) was added to each well of the 96-well microtiter plate and the plate was shaken at 600 rpm for 5 minutes at room temperature. A microplate reader was used to detect the luminescence signal value.
The raw data were imported into GraphPad Prism 6 software for calculating the IC50. The results are shown in Table 14. All the bispecific antibodies tested had obvious proliferation inhibitory activity on NCI-H292 and NCI-H322M cells, and IC50 was significantly better than Onartuzumab and Telisotuzumab.
Table 14: Inhibitory activity of bispecific antibody molecules on tumor cell proliferation
The maximum proliferation inhibitory rate of bispecific antibodies on tumor cells is shown in Table 15. All the tested bispecific antibodies had obvious proliferation inhibitory activity on NCI-H292 and NCI-H322M cells, and the maximum proliferation inhibitory rates were higher than that of Onartuzumab.
Table 15: The maximum inhibition rate of tumor cell proliferation by bispecific antibody molecules
The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
Other embodiments are within the following claims.

Claims (91)

  1. A bispecific antibody, comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR;
    wherein the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH) , and the first VL and the first VH collectively form a domain capable of specifically binding to c-MET; and the second antigen-binding domain comprises a second VL and a second VH, and the second VL and the second VH together form a domain capable of specifically binding to EGFR.
  2. The bispecific antibody of claim 1, wherein the first antigen-binding domain and the second antigen-binding domain are each independently selected from scFv, Fab, and scFab.
  3. The bispecific antibody according to claim 1 or 2, further comprising an Fc domain, wherein the Fc domain comprises a first Fc domain monomer and a second Fc domain monomer, and the first and second Fc domain monomers comprise one or more modifications that promote heterodimerization of the Fc domain monomers.
  4. The bispecific antibody of claim 3, wherein the Fc domain comprises a first Fc domain monomer comprising modifications that form a knob structure and a second Fc domain monomer comprising modifications that form a hole structure, wherein the hole structure can be paired with the knob structure to form a heterodimeric Fc domain.
  5. The bispecific antibody of claim 4, wherein the first Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 49 or 51, and the second Fc domain monomer comprises the amino acid sequence as shown in SEQ ID NO: 50 or 52.
  6. The bispecific antibody of any one of claims 1-5, wherein the first antigen-binding domain and the second antigen-binding domain are each linked to one of the first and second Fc domain monomers of the Fc domain.
  7. The bispecific antibody of claim 6, wherein the first antigen-binding domain is linked to the first Fc domain monomer, and the second antigen-binding domain is linked to the second Fc domain monomer; or the first antigen-binding domain is linked to the second Fc domain  monomer, and the second antigen-binding domain is linked to the first Fc domain monomer.
  8. The bispecific antibody of any one of claims 1-7, wherein the first VL comprises LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 17 or 59; and/or the first VH comprises HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 18 or 60.
  9. The bispecific antibody of any one of claims 1-8, wherein:
    the first VL comprises:
    (i) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 34, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 36, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; or
    (ii) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 35, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 37, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 38; and/or
    the first VH comprises:
    (i) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 39, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 43, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47;
    (ii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 40, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47;
    (iii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 42, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 46, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 47; or
    (iv) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 41, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 45, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 48.
  10. The bispecific antibody of any one of claims 1-9, wherein:
    the first VL comprises the amino acid sequence as shown in SEQ ID NO: 17 or 59, and/or the first VH comprises the amino acid sequence as shown in SEQ ID NO: 18 or 60; or
    the first VL comprises the amino acid sequence as shown in SEQ ID NO: 17, the first VH comprises the amino acid sequence as shown in SEQ ID NO: 18; or the first VL comprises the  amino acid sequence as shown in SEQ ID NO: 59, the first VH comprises the amino acid sequence as shown in SEQ ID NO: 60.
  11. The bispecific antibody of any one of claims 1-10, wherein the second VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of a VL amino acid sequence set forth in SEQ ID NO: 15; and/or the VH comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of a VH amino acid sequence set forth in SEQ ID NO: 16.
  12. The bispecific antibody of any one of claims 1-11, wherein:
    the second VL comprises:
    (i) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; or
    (ii) LCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 23; and/or
    the second VH comprises:
    (i) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 24, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32;
    (ii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 25, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 29, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32;
    (iii) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 27, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 31, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 32; or
    (iv) HCDR1 comprising the amino acid sequence as shown in SEQ ID NO: 26, HCDR2 comprising the amino acid sequence as shown in SEQ ID NO: 30, and HCDR3 comprising the amino acid sequence as shown in SEQ ID NO: 33.
  13. The bispecific antibody of any one of claims 1-12, wherein the second VL comprises the amino acid sequence as shown in SEQ ID NO: 15, and/or the second VH comprises the amino acid sequence as shown in SEQ ID NO: 16.
  14. The bispecific antibody of any one of claims 1-13, wherein the first antigen-binding domain is a Fab, and the second antigen-binding domain is an scFv.
  15. The bispecific antibody of claim 14, wherein the bispecific antibody comprises a peptide chain I-A, a peptide chain I-B and a peptide chain I-C; wherein the peptide chain I-A comprises the first VL and a light chain constant region; the peptide chain I-B comprises: the VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain I-C comprises: the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) .
  16. The bispecific antibody of claim 15, wherein the peptide chain I-A comprises from N-terminus to C-terminus the first VL and the light chain constant region; the peptide chain I-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain I-C comprises from N-terminus to C-terminus (i) the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the second VH, the second VL and the second Fc domain monomer (or the first Fc domain monomer) .
  17. The bispecific antibody of claim 15 or 16, wherein the adjacent domains of the peptide chain I-A are connected optionally with or without a linker, and the adjacent domains of the peptide chain I-B are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain I-C are connected optionally with or without a linker.
  18. The bispecific antibody of claim 17, wherein the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from the group consisting of peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown as (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) ; or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  19. The bispecific antibody of any one of claims 15-18, wherein the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
  20. The bispecific antibody of any one of claims 15-19, wherein the peptide chain I-A comprises the amino acid sequence as shown in SEQ ID NO: 1, the peptide chain I-B comprises the amino acid sequence as shown in SEQ ID NO: 2 or 9, and/or the peptide chain I-C comprises the amino acid sequence as shown in SEQ ID NO: ID NO: 3 or 10.
  21. The bispecific antibody of any one of claims 1-13, wherein the first antigen-binding domain is an scFv, and the second antigen-binding domain is a Fab.
  22. The bispecific antibody of claim 21, wherein the bispecific antibody comprises a peptide chain II-A, a peptide chain II-B and a peptide chain II-C; wherein the peptide chain II-A comprises the second VL and a light chain constant region; the peptide chain II-B comprises the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain II -C comprises: the first VL, the first VH and the second Fc domain monomer (or the first Fc domain monomer) .
  23. The bispecific antibody of claim 22, wherein the peptide chain II-A comprises from N-terminus to C-terminus the second VL and the light chain constant region; and the peptide chain II-B comprises from N-terminus to C-terminus the second VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain II-C comprises from N-terminus to C-terminus (i) the first VL, the first VH and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the first VH, the first VL and the second Fc domain monomer (or the first Fc domain monomer) .
  24. The bispecific antibody of claim 22 or 23, wherein the adjacent domains of the peptide chain II-A are connected optionally with or without a linker; the adjacent domains of the peptide chain II-B are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain II-C are connected optionally with or without a linker.
  25. The bispecific antibody of claim 24, wherein the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) ; or the  peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  26. The bispecific antibody of any one of claims 22-25, wherein the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
  27. The bispecific antibody of any one of claims 22-26, wherein the peptide chain II-A comprises the amino acid sequence as shown in SEQ ID NO: 4, the peptide chain II-B comprises the amino acid sequence as shown in SEQ ID NO: 5 or 7, and/or the peptide chain II-C comprises the amino acid sequence as shown in SEQ ID NO: 6 or 8.
  28. The bispecific antibody of any one of claims 1-13, wherein the first antigen-binding domain is a Fab, and the second antigen-binding domain is a scFab.
  29. The bispecific antibody of claim 28, wherein the bispecific antibody comprises a peptide chain III-A, a peptide chain III-B and a peptide chain III-C; wherein the peptide chain III-A comprises the first VL and a light chain constant region; the peptide chain III-B comprises: the first VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain III-C comprises: the second VL, a light chain constant region, the second VH, a heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) .
  30. The bispecific antibody of claim 29, wherein the peptide chain III-A comprises from N-terminus to C-terminus the first VL and the light chain constant region; the peptide chain III-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain III-C comprises from N-terminus to C-terminus (i) the second VL, the light chain constant region, the second VH, the heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the second VH, the heavy chain CH1 region, the second VL, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer) .
  31. The bispecific antibody of claim 29 or 30, wherein the adjacent domains of the peptide chain III-A are connected optionally with or without a linker; the adjacent domains of the peptide  chain III-B are connected optionally with or without a linker; and/or the adjacent domains of the peptide chain III-C are connected optionally with or without a linker.
  32. The bispecific antibody of claim 31, wherein the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) ; or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  33. The bispecific antibody of any one of claims 29-32, wherein the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the CH1 region of the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 54.
  34. The bispecific antibody of any one of claims 1-13, wherein the first antigen-binding domain is a scFab, and the second antigen-binding domain is a Fab.
  35. The bispecific antibody of claim 34, wherein the bispecific antibody comprises a peptide chain IV-A, a peptide chain IV-B and a peptide chain IV-C; wherein the peptide chain IV-A comprises the second VL and a light chain constant region; the peptide chain IV-B comprises: the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain IV-C comprises: the first VL, a light chain constant region, the first VH, a heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) .
  36. The bispecific antibody of claim 35, wherein the peptide chain IV-A comprises from N-terminus to C-terminus the second VL and the light chain constant region, and the peptide chain IV-B comprises from N-terminus to C-terminus the second VH, the CH1 region of the heavy chain and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain IV-C comprises from N-terminus to C-terminus (i) the first VL, the light chain constant region, the first VH, the heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer) ; or (ii) the first VH, the heavy chain CH1 region, the first VL, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer) .
  37. The bispecific antibody of claim 35 or 36, wherein the adjacent domains of the peptide chain IV-A are connected optionally with or without a linker, and the adjacent domains of the peptide chain IV-B are connected optionally with or without a linker; and/or the adjacent domains of the peptide chain IV-C are connected optionally with or without a linker.
  38. The bispecific antibody of claim 37, wherein the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) ; or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  39. The bispecific antibody of any one of claims 35-38, wherein the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
  40. The bispecific antibody of any one of claims 35-39, wherein the peptide chain IV-A comprises the amino acid sequence as shown in SEQ ID NO: 4, the peptide chain IV-B comprises the amino acid sequence as shown in SEQ ID NO: 7, and/or the peptide chain IV-C comprises the amino acid sequence as shown in SEQ ID NO: 11.
  41. The bispecific antibody of any one of claims 1-13, wherein the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the second antigen-binding domain comprises domain swaps in a form of CrossMab.
  42. The bispecific antibody of claim 41, wherein the bispecific antibody comprises a peptide chain V-A, a peptide chain V-B, a peptide chain V-C and a peptide chain V-D; wherein the peptide chain V-A comprises the first VL and a light chain constant region; the peptide chain V-B comprises: the first VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) , and the peptide chain V-C comprises: the second VH, a light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) , the peptide chain V-D comprises: the second VL and a heavy chain CH1 region.
  43. The bispecific antibody of claim 42, wherein the peptide chain V-A comprises from N-terminus to C-terminus the first VL and the light chain constant region; the peptide chain V-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain V-C comprises from N-terminus to C-terminus the second VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) , and/or the peptide chain V-D comprises from N-terminus to C-terminus the second VL and the heavy chain CH1 region.
  44. The bispecific antibody of claim 42 or 43, wherein the adjacent domains of the peptide chain V-A are connected optionally with or without a linker, and the adjacent domains of the peptide chain V-B are connected optionally with or without a linker, the adjacent domains of the peptide chain V-C are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain V-D are connected optionally with or without a linker.
  45. The bispecific antibody of claim 44, wherein the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) ; or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  46. The bispecific antibody of any one of claims 42-45, wherein the light chain constant region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
  47. The bispecific antibody of any one of claims 42-46, wherein the peptide chain V-A comprises the amino acid sequence as shown in SEQ ID NO: 1, the peptide chain V-B comprises the amino acid sequence as shown in SEQ ID NO: 9; the peptide chain V-C comprises the amino acid sequence as shown in SEQ ID NO: 12, and/or the sequence of the peptide chain V-D is shown in SEQ ID NO: 13.
  48. The bispecific antibody of any one of claims 1-13, wherein the first antigen-binding  domain and the second antigen-binding domain are Fab, and the Fab of the first antigen-binding domain comprises domain swaps in the form of CrossMab.
  49. The bispecific antibody of claim 48, wherein the bispecific antibody comprises a peptide chain VI-A, a peptide chain VI-B, a peptide chain VI-C and a peptide chain VI-D; wherein the peptide chain VI-A comprises the second VL and a light chain constant region; the peptide chain VI-B comprises: the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) ; the peptide chain VI-C comprises: the first VH, a light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) ; the peptide chain VI-D comprises: the first VL and a heavy chain CH1 region.
  50. The bispecific antibody of claim 49, wherein the peptide chain VI-A comprises from N-terminus to C-terminus the second VL and the light chain constant region, and the peptide chain VI-B comprises from N-terminus to C-terminus the second VH, the CH1 region of the heavy chain, and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain VI-C comprises from N-terminus to C-terminus the first VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) , and/or the peptide chain VI-D from N-terminus to C-terminus comprises: the first VL and the heavy chain CH1 region.
  51. The bispecific antibody of claim 49 or 50, wherein the adjacent domains of the peptide chain VI-A are connected optionally with or without a linker; the adjacent domains of the peptide chain VI-B are connected optionally with or without a linker; the adjacent domains of the peptide chain VI-C are connected optionally with or without a linker; and/or the adjacent domains of the peptide chain VI-D are connected optionally with or without a linker.
  52. The bispecific antibody of claim 51, wherein the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) ; or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  53. The bispecific antibody of any one of claims 49-52, wherein the light chain constant  region comprises the amino acid sequence as shown in SEQ ID NO: 53, and/or the heavy chain CH1 region comprises the amino acid sequence as shown in SEQ ID NO: 54.
  54. The bispecific antibody of any one of claims 1-13, wherein both the first antigen-binding domain and the second antigen-binding domain are scFvs.
  55. The bispecific antibody of claim 54, wherein the bispecific antibody comprises a peptide chain VII-A and peptide chain VII-B; wherein the peptide chain VII-A comprises: the first VL, the first VH and the first Fc domain monomer (or the second Fc domain monomer) , the peptide chain VII-B comprises: the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) .
  56. The bispecific antibody of claim 55, wherein the peptide chain VII-A comprises from N-terminus to C-terminus (i) the first VL, the first VH and the first Fc domain monomer (or the second Fc domain monomer) ; or (ii) the first VH, the first VL and the first Fc domain monomer (or the second Fc domain monomer) ; and/or the peptide chain VII-B comprises from N-terminus to C-terminus (i) the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) , or (ii) the second VH, the second VL and the second Fc domain monomer (or the first Fc domain monomer) .
  57. The bispecific antibody of claim 55 or 56, wherein the adjacent domains of the peptide chain VII-A are connected optionally with or without a linker, and/or the adjacent domains of the peptide chain VII-B are connected optionally with or without a linker.
  58. The bispecific antibody of claim 57, wherein the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers) ; or the peptide linkers are each independently selected from peptide linkers comprising one or more glycines (G) and/or serines (S) , for example, having the structure shown in (GGGGS) n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NOs: 55-58 or 63-68) .
  59. The bispecific antibody of claim 58, wherein the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 63-68.
  60. The bispecific antibody of any one of claims 55-59, wherein the peptide chain VII-A  comprises the amino acid sequence as shown in SEQ ID NO: 14, and/or the peptide chain VII-B comprises the amino acid sequence as shown in SEQ ID NO: 8.
  61. The bispecific antibody of any one of claims 1-60, comprising:
    (1) peptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1, peptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 2, and peptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 3;
    (2) peptide chain I-A comprising the amino acid sequence shown in SEQ ID NO: 1, peptide chain I-B comprising the amino acid sequence shown in SEQ ID NO: 9, and peptide chain I-C comprising the amino acid sequence shown in SEQ ID NO: 10;
    (3) peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5, and peptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6;
    (4) peptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, peptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7, and peptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8;
    (5) peptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, peptide IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and peptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11;
    (6) peptide chain V-A comprising the amino acid sequence shown in SEQ ID NO: 1, peptide chain V-B comprising the amino acid sequence shown in SEQ ID NO: 9, peptide chain V-C comprising the amino acid sequence shown in SEQ ID NO: 12, and peptide chain V-D comprising the amino acid sequence as shown in SEQ ID NO: 13; or
    (7) peptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and peptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.
  62. The bispecific antibody of any one of claims 1-61, which has an enhanced tumor suppressive effect relative to a monospecific anti-c-MET antibody and/or a monospecific anti-EGFR antibody; wherein the amino acid sequences of the CDRs of the monospecific anti-c-MET antibody are identical to the amino acid sequences of the CDRs of the first antigen-binding domain, and the amino acid sequences of the CDRs of the monospecific anti-EGFR antibody are identical to the amino acid sequences of the CDRs of the second antigen-binding domain.
  63. The bispecific antibody of claim 62, wherein the tumor suppression effect comprises:  inhibition of EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.
  64. An isolated nucleic acid molecule or set of nucleic acid molecules, comprising a nucleotide sequence encoding the bispecific antibody of any one of claims 1-63.
  65. One or more vectors, comprising the isolated nucleic acid molecule or set of nucleic acid molecules of claim 64.
  66. The one or more vectors of claim 65, wherein the nucleotide sequences encoding the different peptide chains of the bispecific antibody are located in different vector molecules.
  67. The one or more vectors of claim 65 or 66, wherein the one or more vectors is a cloning vector or an expression vector.
  68. A host cell comprising the isolated nucleic acid molecule or set of nucleic acid molecules of claim 64, or the one or more vectors of any one of claims 65-67.
  69. A method for preparing the bispecific antibody of any one of claims 1-63, comprising culturing the host cell of claim 68 under conditions allowing the expression of the bispecific antibody, and recovering the bispecific antibody from the culture of the host cell.
  70. A conjugate comprising the bispecific antibody of any one of claims 1-63 and a conjugation moiety linked thereto; optionally wherein the conjugation moiety is selected from a therapeutic agent (such as a cytotoxic agent, cytokine, toxin, or radionuclide) .
  71. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-63, the isolated nucleic acid molecule or set of nucleic acid molecules of claim 64, the one or more vectors of any one of claims 65-67, the host cell of claim 68, or the conjugate of claim 70, and a pharmaceutically acceptable carrier and/or excipient.
  72. The pharmaceutical composition of claim 71, wherein the pharmaceutical composition further comprises an additional pharmaceutically active agent.
  73. The pharmaceutical composition of claim 72, wherein the additional pharmaceutically active agent is a drug with antitumor activity.
  74. The pharmaceutical composition of claim 72, wherein the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, and any combination thereof.
  75. The pharmaceutical composition of claim 72, wherein the bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as mixed components.
  76. Use of the bispecific antibody of any one of claims 1-63, the isolated nucleic acid molecule or set of nucleic acid molecules of claim 64, the one or more vectors of any one of claims 65-67, the host cell of claim 68, the conjugate of claim 70, or the pharmaceutical composition of any one of claims 71-75, in the preparation of a medicament, wherein the medicament is used for preventing and/or treating and/or acting as an adjuvant in treating diseases related to c-MET and/or EGFR in a subject, and/or for inhibiting c-MET and/or EGFR activity in vitro or in a subject.
  77. The use of claim 76, wherein the disease related to c-MET and/or EGFR is cancer.
  78. The use of claim 77, wherein the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification.
  79. The use of claim 77 or 78, wherein cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cavity cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.
  80. The use of claim 76, wherein the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, one or more vectors, host cell, conjugate, or pharmaceutical composition is administered in combination with another pharmaceutically active agent, such as administered simultaneously, separately or sequentially.
  81. The use of claim 80, wherein the another pharmaceutically active agent is a drug with an anti-tumor activity.
  82. The use of claim 80, wherein the additional pharmaceutically active agent is selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, and any combination thereof.
  83. The use of any one of claims 76-82, wherein the subject is resistant to erlotinib, gefitinib, afatinib, CO-1686, AZD9192 or cetuximab treatment.
  84. A method for inhibiting the activity of c-MET and/or EGFR in a cell, comprising contacting the cell with the bispecific antibody of any one of claims 1-63, the isolated nucleic acid molecule or set of nucleic acid molecules of claim 64, the one or more vectors of any one of claims 65-67, the host cell of claim 68, the conjugate of claim 70, or the pharmaceutical composition of any one of claims 71-75.
  85. The method of claim 84, wherein the cells are cells expressing c-MET and/or EGFR, such as tumor cells.
  86. A method for preventing and/or treating and/or acting as an adjuvant in treating diseases associated with c-MET and/or EGFR in a subject, wherein the method comprises administering to the subject in need thereof an effective amount of the bispecific antibody of any one of claims 1-63, the isolated nucleic acid molecule or set of nucleic acid molecules of claim 64, the one or more vectors of any one of claims 65-67, the host cell of claim 68, the conjugate of claim 70, or the pharmaceutical composition of any one of claims 71-75.
  87. The method of claim 86, wherein the disease associated with c-MET and/or EGFR is cancer.
  88. The method of claim 87, wherein the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-MET activating mutations and/or c-MET gene amplification.
  89. The method of claim 87 or 88, wherein the cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cavity cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreas cancer, skin cancer, tongue cancer, esophagus cancer, vagina cancer, cervix cancer, spleen cancer, testis cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.
  90. The method of claim 86, wherein the method further comprises administering to the subject a second therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy and any combination thereof; optionally, the second therapy may be applied simultaneously, separately or sequentially.
  91. The method of any one of claims 86-90, wherein the subject is resistant to a treatment using erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.
PCT/CN2024/103914 2023-07-07 2024-07-05 Egfr/c-met bispecific binding protein and use thereof Pending WO2025011471A1 (en)

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