EGFR / C-MET bispecific binding protein and its use

Abstract

In relation to the field of disease treatment, bispecific antibodies against EGFR and c-MET, the nucleic acid molecules encoding them, and methods for preparing them are provided. These anti-EGFR and c-MET bispecific antibodies have high specificity and high affinity for EGFR and c-MET. The use of these bispecific antibodies in the treatment and diagnosis of diseases is also provided.

Description

[Technical Field]

[0001] This disclosure relates to therapeutic bispecific antibodies, more specifically bispecific antibodies against EGFR and c-MET, and to methods of using them. [Background technology]

[0002] EGFR (epidermal growth factor receptor, abbreviated as 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 approximately 170 kDa. Its main structure includes a ligand-binding extracellular domain, a hydrophobic transmembrane domain, and an intracellular domain containing tyrosine kinase. EGFR is highly expressed in various tumors, including colorectal cancer, head and neck cancer, and non-small cell lung cancer, and is also expressed in normal epithelial tissues such as the skin and lungs. EGFR, which is highly expressed in tumor tissue, undergoes homologous or heterodimerization upon binding to ligands such as EGF or TGFα. This dimerization leads to the activation of tyrosine kinases and protein phosphorylation within tumor cells, mediating gene transcription and the activation of various cellular signaling pathways during cell cycle progression. It plays a crucial role in promoting tumor cell survival, proliferation, migration, and tumor angiogenesis.

[0003] 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 approximately 190 kDa, containing two polypeptide chains linked by disulfide bonds via cleavage: an α-chain (50 kDa) and a β-chain (140 kDa). The MET protein can be divided into a semadomain (semaphorin domain, SEMA), a PSI domain (plexin-semaphorin-integrin domain, PSI), four immunoglobulin-like repeat domains (immunoglobulin-plexin-transcription domains, IPT), a transmembrane region, a perimembrane region (JM), a tyrosine kinase domain (TK), and a carboxyl-terminal tail region (CT). The native ligand for the c-MET receptor is hepatocyte growth factor (HGF), an inactive protein that is converted to its active form by proteolytic cleavage. Abnormal regulation of c-MET has been reported in various cancers, including colorectal cancer, non-small cell lung cancer, gastric cancer, and breast cancer. High activation of c-MET and its downstream signaling pathways has been shown to induce hypergrowth, tumor invasion, and angiogenesis, and is associated with reduced survival rates.

[0004] High molecular weight anti-EGFR antibody drugs such as cetuximab, panitumumab, nimotuzumab, and necitumumab are approved for marketing targeting colorectal cancer, non-small cell lung cancer, and other cancers. EGFR tyrosine kinase inhibitors (TKIs) such as osimertinib, alimertinib, and abitinib are the standard of care for patients with advanced cancer who are EGFR mutation-positive. c-MET-TKIs have also achieved good efficacy in non-small cell lung cancer. The signaling pathways of EGFR and c-MET have both been clinically validated.

[0005] Nearly 60% of tumors resistant to EGFR-TKIs exhibit increased c-MET expression, c-MET amplification, or increased HGF levels of the c-MET ligand (Turke et al., Cancer Cell, 17:77-88, 2010), suggesting that c-MET signaling complements EGFR signaling within tumor cells. Both EGFR and c-MET signal via the same survival and anti-apoptotic pathways (ERK and AKT). Bispecific antibodies that simultaneously target EGFR and c-MET are expected to improve overall efficacy and safety by simultaneously inhibiting the EGFR and c-MET signaling pathways, thereby avoiding tumor development and onset caused by activation of the c-MET signaling pathway after EGFR inhibition.

[0006] Anti-c-MET and anti-EGFR antibodies are known in the art. For example, telisotuzumab vedotin (Teliso-v or ABBV399), disclosed in U.S. Patent No. 10603389, is an anti-c-MET antibody ABT-700 conjugated to vc-MMAE (vedotin), and MRG003 (ADC-3 or MYK-3), disclosed in WO2023040941, is an anti-EGFR antibody BA03 conjugated to vedotin.

[0007] Several EGFR / c-Met bispecific antibodies have been disclosed in the art. The 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 ocrelizumab, which is currently in Phase III trials. The bispecific antibody amivantamab, sold under the trademark RYBREVANT®, is disclosed in U.S. Patent No. 9,593,164. Amivantamab comprises an anti-EGFR Fab comprising the VH and VL of zalutumumab (see U.S. Patent Nos. 7,247,301 and 7,595,378), as well as an anti-cMET Fab comprising the VH and VL of antibody 069 disclosed in U.S. Patent No. 9,068,011. In the art, there is a need 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, for example, by simultaneously blocking the binding of EGF to EGFR and HGF to c-Met.

Summary of the Invention

[0008] The present invention provides a high-affinity EGFR / c-Met bispecific antibody that specifically recognizes or binds EGFR and c-Met and simultaneously blocks the downstream signaling pathways of both EGFR and c-Met by simultaneously blocking the binding of EGF to EGFR and HGF to c-Met, thereby increasing the inhibition of cell proliferation.

[0009] The present invention provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to c-Met and a second antigen-binding domain that specifically binds 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 both the first VL and the first VH together form a domain that can specifically bind to c-Met, and the second antigen-binding domain comprises a second VL and a second VH, and both the second VL and the second VH together form a domain that can specifically bind to EGFR. In a further embodiment, the bispecific antibody binds to c-Met and EGFR with high affinity compared to other known antibodies.

[0010] 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.

[0011] In a further embodiment of the bispecific antibody, the bispecific antibody further comprises an Fc domain, 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.

[0012] In a further embodiment of the bispecific antibody, the Fc domain comprises a first Fc domain monomer comprising a modification that forms a knob structure and a second Fc domain monomer comprising a modification that forms a hole structure, and the hole structure forms a pair with the knob structure and can form a heterodimeric Fc domain.

[0013] In a further embodiment of the bispecific antibody, the first Fc domain monomer comprises an amino acid sequence as set forth in SEQ ID NO: 49 or 51, and the second Fc domain monomer comprises an amino acid sequence as set forth in SEQ ID NO: 50 or 52.

[0014] 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.

[0015] 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.

[0016] In a further embodiment of the bispecific antibody, the first VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17 or 59, and / or the first VH comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 18 or 60.

[0017] In a further embodiment of the bispecific antibody, the first VL is, (i) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 34, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38 (CDR is defined by the Kabat numbering system), (ii) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 34, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38 (CDR is defined by the Chothia numbering system), (iii) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 34, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38 (CDR is defined by the Abm numbering system), or (iv) comprising LCDR1 having an amino acid sequence as shown in SEQ ID NO: 35, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 37, and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 38 (where CDR is defined by the IMGT numbering system), and / or

[0018] The 1st VH is, (v) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 39, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47 (CDR is defined by the Kabat numbering system), (vi) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 40, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 44, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47 (CDR is defined by the Chothia numbering system), (vii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 42, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 46, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47 (CDR is defined by the Abm numbering system), or (viii) comprising HCDR1 having an amino acid sequence as shown in SEQ ID NO: 41, HCDR2 having an amino acid sequence as shown in SEQ ID NO: 45, and HCDR3 having an amino acid sequence as shown in SEQ ID NO: 48 (CDR is defined by the IMGT numbering system).

[0019] In a further embodiment of the bispecific antibody, the first VL is, (ix) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 34, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38, or (x) comprising LCDR1 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38,

[0020] and / or

[0021] The 1st VH is, (xi) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 39, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47. (xii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 40, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 44, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47. (xiii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 42, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 46, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47, or (xiv) Includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 41, HCDR2 having the amino acid sequence shown in SEQ ID NO: 45, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 48.

[0022] In further embodiments of the bispecific antibody, the first VL comprises an amino acid sequence such as that shown in SEQ ID NO: 17 or 59, and / or the first VH comprises an amino acid sequence such as that shown in SEQ ID NO: 18 or 60, or the first VL comprises an amino acid sequence such as that shown in SEQ ID NO: 17 and the first VH comprises an amino acid sequence such as that shown in SEQ ID NO: 18, or the first VL comprises an amino acid sequence such as that shown in SEQ ID NO: 59 and the first VH comprises an amino acid sequence such as that shown in SEQ ID NO: 60.

[0023] In a further embodiment of the bispecific antibody, the second VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 15, and / or VH comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16.

[0024] In a further embodiment of the bispecific antibody, the second VL is, (xv) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 23 (CDR is defined by the Kabat numbering system), (xvi) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 23 (CDR is defined by the Chothia numbering system), (xvii) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 23 (CDR is defined by the Abm numbering system), or (xviii) comprising LCDR1 having an amino acid sequence as shown in SEQ ID NO: 20, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 23 (CDR is defined by the IMGT numbering system), and / or

[0025] 2VH is, (xix) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 24, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32 (CDR is defined by the Kabat numbering system), (xx) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32 (CDR is defined by the Chothia numbering system), (xxi) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 27, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32 (CDR is defined by the Abm numbering system), or (xxii) Includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 26, HCDR2 having the amino acid sequence shown in SEQ ID NO: 30, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 33 (CDR is defined by the IMGT numbering system).

[0026] In a further embodiment of the bispecific antibody, the second VL is, (xxiii) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 23, or (xxiv) comprising LCDR1 having an amino acid sequence as shown in SEQ ID NO: 20, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 23, and / or

[0027] 2VH is, (xxv) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 24, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32. (xxvi) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32. (xxvii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 27, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32, or (xxviii) Includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 26, HCDR2 having the amino acid sequence shown in SEQ ID NO: 30, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 33.

[0028] In a further embodiment of the bispecific antibody, the second VL comprises an amino acid sequence as shown in SEQ ID NO: 15, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 16.

[0029] In a further embodiment of the bispecific antibody, the first antigen-binding domain is Fab and the second antigen-binding domain is scFv.

[0030] In a further embodiment of the bispecific antibody, the bispecific antibody comprises a peptide chain IA, a peptide chain IB, and a peptide chain IC, wherein peptide chain IA comprises a first VL and a light chain constant region, peptide chain IB comprises a first VH, a heavy chain CH1 region, and a first Fc domain monomer (or a second Fc domain monomer), and peptide chain IC comprises a second VL, a second VH, and a second Fc domain monomer (or a first Fc domain monomer).

[0031] In further embodiments of the bispecific antibody, peptide chain IA comprises a first VL and a light chain constant region from the N-terminus to the C-terminus, peptide chain IB comprises a first VH, a heavy chain CH1 region and a first Fc domain monomer (or a second Fc domain monomer) from the N-terminus to the C-terminus, and / or peptide chain IC comprises (i) a second VL, a second VH and a second Fc domain monomer (or a first Fc domain monomer) from the N-terminus to the C-terminus, or (ii) a second VH, a second VL and a second Fc domain monomer (or a first Fc domain monomer).

[0032] In further embodiments of the bispecific antibody, the adjacent domains of peptide chain IA are optionally linked with or without a linker, the adjacent domains of peptide chain IB are optionally linked with or without a linker, and / or the adjacent domains of peptide chain IC are optionally linked with or without a linker.

[0033] In further embodiments of the bispecific antibody, the linkers are either the same peptide linker or different peptide linkers, each independently (e.g., a rigid peptide linker or a flexible peptide linker). In further embodiments of the bispecific antibody, the peptide linker comprises one or more glycine (G) and / or serine (S), for example, (GGGGS). n(wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Each is independently selected from the group consisting of peptide linkers having the structure shown as (SEQ ID NOs. 55-58 or 63-68). In further embodiments of the bispecific antibody, each peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0034] In a further embodiment of the bispecific antibody, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0035] In further embodiments of the bispecific antibody, peptide chain IA comprises an amino acid sequence as shown in SEQ ID NO: 1, peptide chain IB comprises an amino acid sequence as shown in SEQ ID NO: 2 or 9, and / or peptide chain IC comprises an amino acid sequence as shown in SEQ ID NO: 3 or 10.

[0036] In a further embodiment of the bispecific antibody, the first antigen-binding domain is scFv and the second antigen-binding domain is Fab.

[0037] In a further embodiment of the bispecific antibody, the bispecific antibody comprises peptide chain II-A, peptide chain II-B, and peptide chain II-C, wherein peptide chain II-A comprises the second VL and the light chain constant region, peptide chain II-B comprises the second VH, the heavy chain CH1 region, and the first Fc domain monomer (or second Fc domain monomer), and peptide chain II-C comprises the first VL, the first VH, and the second Fc domain monomer (or first Fc domain monomer).

[0038] In further embodiments of the bispecific antibody, peptide chain II-A comprises a second VL and a light chain constant region from the N-terminus to the C-terminus, peptide chain II-B comprises a second VH, a heavy chain CH1 region and a first Fc domain monomer (or second Fc domain monomer) from the N-terminus to the C-terminus, and / or peptide chain II-C comprises (i) a first VL, a first VH and a second Fc domain monomer (or first Fc domain monomer) from the N-terminus to the C-terminus, or (ii) a first VH, a first VL and a second Fc domain monomer (or first Fc domain monomer).

[0039] In a further embodiment of the bispecific antibody, the adjacent domain of peptide chain II-A is optionally linked with or without a linker, the adjacent domain of peptide chain II-B is optionally linked with or without a linker, and / or the adjacent domain of peptide chain II-C is optionally linked with or without a linker.

[0040] In further embodiments of the bispecific antibody, the linkers are either the same peptide linker or different peptide linkers, each independently (e.g., a rigid peptide linker or a flexible peptide linker). In further embodiments of the bispecific antibody, the peptide linker comprises one or more glycine (G) and / or serine (S), for example, (GGGGS). n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Each is independently selected from peptide linkers having the structure shown in (SEQ ID NOs. 55-58 or 63-68). In further embodiments of the bispecific antibody, each peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0041] In a further embodiment of the bispecific antibody, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0042] In a further embodiment of the bispecific antibody, peptide chain II-A comprises an amino acid sequence as shown in SEQ ID NO: 4, peptide chain II-B comprises an amino acid sequence as shown in SEQ ID NO: 5 or 7, and / or peptide chain II-C comprises an amino acid sequence as shown in SEQ ID NO: 6 or 8.

[0043] In a further embodiment of the bispecific antibody, the first antigen-binding domain is Fab and the second antigen-binding domain is scFab.

[0044] In a further embodiment of the bispecific antibody, the bispecific antibody comprises peptide chain III-A, peptide chain III-B, and peptide chain III-C, wherein peptide chain III-A comprises the first VL and the light chain constant region, peptide chain III-B comprises the first VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and peptide chain III-C comprises 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).

[0045] In further embodiments of the bispecific antibody, peptide chain III-A comprises a first VL and a light chain constant region from the N-terminus to the C-terminus, peptide chain III-B comprises a first VH, a heavy chain CH1 region and a first Fc domain monomer (or a second Fc domain monomer) from the N-terminus to the C-terminus, and / or peptide chain III-C comprises (i) a second VL, a light chain constant region, a second VH, a heavy chain CH1 region and a second Fc domain monomer (or a first Fc domain monomer) from the N-terminus to the C-terminus, or (ii) a second VH, a heavy chain CH1 region, a second VL, a light chain constant region and a second Fc domain monomer (or a first Fc domain monomer).

[0046] In a further embodiment of the bispecific antibody, the adjacent domain of peptide chain III-A is optionally linked with or without a linker, the adjacent domain of peptide chain III-B is optionally linked with or without a linker, and / or the adjacent domain of peptide chain III-C is optionally linked with or without a linker.

[0047] In further embodiments of the bispecific antibody, the linkers are either the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker), or the peptide linker comprises one or more glycine (G) and / or serine (S), e.g., (GGGGS) n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Each peptide linker is independently selected from those having the structure shown in (SEQ ID NOs. 55-58 or 63-68), or each peptide linker independently contains the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0048] In a further embodiment of the bispecific antibody, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0049] In a further embodiment of the bispecific antibody, the first antigen-binding domain is scFab and the second antigen-binding domain is Fab.

[0050] In a further embodiment of the bispecific antibody, the bispecific antibody comprises peptide chain IV-A, peptide chain IV-B, and peptide chain IV-C, wherein peptide chain IV-A comprises the second VL and the light chain constant region, peptide chain IV-B comprises the second VH, the heavy chain CH1 region, and the first Fc domain monomer (or second Fc domain monomer), and peptide chain IV-C comprises the first VL, the light chain constant region, the first VH, the heavy chain CH1 region, and the second Fc domain monomer (or first Fc domain monomer).

[0051] In further embodiments of the bispecific antibody, peptide chain IV-A comprises a second VL and a light chain constant region from the N-terminus to the C-terminus, peptide chain IV-B comprises a second VH, a heavy chain CH1 region and a first Fc domain monomer (or second Fc domain monomer) from the N-terminus to the C-terminus, and / or peptide chain IV-C comprises (i) a first VL, a light chain constant region, a first VH, a heavy chain CH1 region and a second Fc domain monomer (or first Fc domain monomer) from the N-terminus to the C-terminus, or (ii) a first VH, a heavy chain CH1 region, a first VL, a light chain constant region and a second Fc domain monomer (or first Fc domain monomer).

[0052] In a further embodiment of the bispecific antibody, the adjacent domain of peptide chain IV-A is optionally linked with or without a linker, the adjacent domain of peptide chain IV-B is optionally linked with or without a linker, and / or the adjacent domain of peptide chain IV-C is optionally linked with or without a linker.

[0053] In further embodiments of the bispecific antibody, the linkers are either the same peptide linker or different peptide linkers, each independently (e.g., a rigid peptide linker or a flexible peptide linker). In further embodiments of the bispecific antibody, the peptide linker comprises one or more glycine (G) and / or serine (S), for example, (GGGGS). n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Each is independently selected from peptide linkers having the structure shown in (SEQ ID NOs. 55-58 or 63-68). In further embodiments of the bispecific antibody, each peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0054] In a further embodiment of the bispecific antibody, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0055] In a further embodiment of the bispecific antibody, peptide chain IV-A comprises an amino acid sequence as shown in SEQ ID NO: 4, peptide chain IV-B comprises an amino acid sequence as shown in SEQ ID NO: 7, and / or peptide chain IV-C comprises an amino acid sequence as shown in SEQ ID NO: 11.

[0056] In further embodiments 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 includes a domain swap in the form of a CrossMab.

[0057] In a further embodiment of the bispecific antibody, the bispecific antibody comprises peptide chain VA, peptide chain VB, peptide chain VC, and peptide chain VD, wherein peptide chain VA comprises a first VL and a light chain constant region, peptide chain VB comprises a first VH, a heavy chain CH1 region, and a first Fc domain monomer (or a second Fc domain monomer), peptide chain VC comprises a second VH, a light chain constant region, and a second Fc domain monomer (or a first Fc domain monomer), and peptide chain VD comprises a second VL and a heavy chain CH1 region.

[0058] In further embodiments of the bispecific antibody, peptide chain VA comprises a first VL and a light chain constant region from the N-terminus to the C-terminus, peptide chain VB comprises a first VH, a heavy chain CH1 region and a first Fc domain monomer (or a second Fc domain monomer) from the N-terminus to the C-terminus, peptide chain VC comprises a second VH, a light chain constant region and a second Fc domain monomer (or a first Fc domain monomer) from the N-terminus to the C-terminus, and / or peptide chain VD comprises a second VL and a heavy chain CH1 region from the N-terminus to the C-terminus.

[0059] In further embodiments of the bispecific antibody, the adjacent domains of peptide chain VA are optionally linked with or without a linker, the adjacent domains of peptide chain VB are optionally linked with or without a linker, the adjacent domains of peptide chain VC are optionally linked with or without a linker, and / or the adjacent domains of peptide chain VD are optionally linked with or without a linker.

[0060] In further embodiments of the bispecific antibody, the linkers are either the same peptide linker or different peptide linkers, each independently (e.g., a rigid peptide linker or a flexible peptide linker). In further embodiments of the bispecific antibody, the peptide linker comprises one or more glycine (G) and / or serine (S), for example, (GGGGS). n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Each is independently selected from peptide linkers having the structure shown in (SEQ ID NOs. 55-58 or 63-68). In further embodiments of the bispecific antibody, each peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0061] In a further embodiment of the bispecific antibody, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0062] In a further embodiment of the bispecific antibody, peptide chain VA comprises an amino acid sequence as shown in SEQ ID NO: 1, peptide chain VB comprises an amino acid sequence as shown in SEQ ID NO: 9, peptide chain VC comprises an amino acid sequence as shown in SEQ ID NO: 12, and / or the sequence of peptide chain VD is shown in SEQ ID NO: 13.

[0063] In further embodiments of the bispecific antibody, the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the first antigen-binding domain includes a domain swap in the form of a CrossMab.

[0064] In a further embodiment of the bispecific antibody, the bispecific antibody comprises peptide chain VI-A, peptide chain VI-B, peptide chain VI-C, and peptide chain VI-D, wherein peptide chain VI-A comprises the second VL and the light chain constant region, peptide chain VI-B comprises the second VH, the heavy chain CH1 region, and the first Fc domain monomer (or second Fc domain monomer), peptide chain VI-C comprises the first VH, the light chain constant region, and the second Fc domain monomer (or first Fc domain monomer), and peptide chain VI-D comprises the first VL and the heavy chain CH1 region.

[0065] In further embodiments of the bispecific antibody, peptide chain VI-A comprises a second VL and a light chain constant region from the N-terminus to the C-terminus, peptide chain VI-B comprises a second VH, a heavy chain CH1 region, and a first Fc domain monomer (or second Fc domain monomer) from the N-terminus to the C-terminus, peptide chain VI-C comprises a first VH, a light chain constant region, and a second Fc domain monomer (or first Fc domain monomer) from the N-terminus to the C-terminus, and / or peptide chain VI-D comprises a first VL and a heavy chain CH1 region from the N-terminus to the C-terminus.

[0066] In further embodiments of the bispecific antibody, the adjacent domains of peptide chain VI-A are optionally linked with or without a linker, the adjacent domains of peptide chain VI-B are optionally linked with or without a linker, the adjacent domains of peptide chain VI-C are optionally linked with or without a linker, and / or the adjacent domains of peptide chain VI-D are optionally linked with or without a linker.

[0067] In further embodiments of the bispecific antibody, the linkers are either the same peptide linker or different peptide linkers, each independently (e.g., a rigid peptide linker or a flexible peptide linker). In further embodiments of the bispecific antibody, the peptide linker comprises one or more glycine (G) and / or serine (S), for example, (GGGGS). n(wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Each is independently selected from peptide linkers having the structure shown in (SEQ ID NOs. 55-58 or 63-68). In further embodiments of the bispecific antibody, each peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0068] In a further embodiment of the bispecific antibody, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0069] In further embodiments of the bispecific antibody, both the first antigen-binding domain and the second antigen-binding domain are scFv.

[0070] In a further embodiment of the bispecific antibody, the bispecific antibody comprises peptide chain VII-A and peptide chain VII-B, wherein peptide chain VII-A comprises the first VL, first VH, and first Fc domain monomer (or second Fc domain monomer), and peptide chain VII-B comprises the second VL, second VH, and second Fc domain monomer (or first Fc domain monomer).

[0071] In a further embodiment of the bispecific antibody, peptide chain VII-A comprises (i) the first VL, first VH, and first Fc domain monomer (or second Fc domain monomer) from the N-terminus to the C-terminus, or (ii) the first VH, first VL, and first Fc domain monomer (or second Fc domain monomer), and / or peptide chain VII-B comprises (i) the second VL, second VH, and second Fc domain monomer (or first Fc domain monomer) from the N-terminus to the C-terminus, or (ii) the second VH, second VL, and second Fc domain monomer (or first Fc domain monomer).

[0072] In a further embodiment of the bispecific antibody, the adjacent domain of peptide chain VII-A is optionally linked with or without a linker, and / or the adjacent domain of peptide chain VII-B is optionally linked with or without a linker.

[0073] In further embodiments of the bispecific antibody, the linkers are either the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker), or the peptide linker comprises one or more glycine (G) and / or serine (S), e.g., (GGGGS) n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Each is independently selected from peptide linkers having the structure shown in (SEQ ID NOs. 55-58 or 63-68).

[0074] In further embodiments of the bispecific antibody, the peptide linker independently comprises the amino acid sequences shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0075] In a further embodiment of the bispecific antibody, peptide chain VII-A comprises an amino acid sequence as shown in SEQ ID NO: 14, and / or peptide chain VII-B comprises an amino acid sequence as shown in SEQ ID NO: 8.

[0076] In further embodiments of the bispecific antibody, the bispecific antibody includes: (1) Peptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, peptide chain IB containing the amino acid sequence shown in SEQ ID NO: 2, and peptide chain IC containing the amino acid sequence shown in SEQ ID NO: 3 (2) Peptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, peptide chain IB containing the amino acid sequence shown in SEQ ID NO: 9, and peptide chain IC containing the amino acid sequence shown in SEQ ID NO: 10 (3) Peptide chain II-A containing the amino acid sequence shown in SEQ ID NO: 4, Peptide chain II-B containing the amino acid sequence shown in SEQ ID NO: 5, and Peptide chain II-C containing the amino acid sequence shown in SEQ ID NO: 6 (4) Peptide chain II-A containing the amino acid sequence shown in SEQ ID NO: 4, peptide chain II-B containing the amino acid sequence shown in SEQ ID NO: 7, and peptide chain II-C containing the amino acid sequence shown in SEQ ID NO: 8. (5) Peptide chain IV-A containing the amino acid sequence shown in SEQ ID NO: 4, peptide chain IV-B containing the amino acid sequence shown in SEQ ID NO: 7, and peptide chain IV-C containing the amino acid sequence shown in SEQ ID NO: 11. (6) Peptide chain VA containing the amino acid sequence shown in SEQ ID NO: 1, peptide chain VB containing the amino acid sequence shown in SEQ ID NO: 9, peptide chain VC containing the amino acid sequence shown in SEQ ID NO: 12, and peptide chain VD containing the amino acid sequence shown in SEQ ID NO: 13, or (7) Peptide chain VII-A containing the amino acid sequence shown in SEQ ID NO: 14, and peptide chain VII-B containing the amino acid sequence shown in SEQ ID NO: 8.

[0077] In a further embodiment of the bispecific antibody, the bispecific antibody exhibits an enhanced tumor suppressor effect compared to a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody, wherein the amino acid sequence of the CDR of the monospecific anti-c-MET antibody is identical to the amino acid sequence of the CDR of the first antigen-binding domain, and the amino acid sequence of the CDR of the monospecific anti-EGFR antibody is identical to the amino acid sequence of the CDR of the second antigen-binding domain.

[0078] In further embodiments of the bispecific antibody, the tumor suppressor 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.

[0079] The present invention further provides an isolated nucleic acid molecule or set of nucleic acid molecules comprising a nucleotide sequence encoding the bispecific antibodies disclosed herein. The present invention also provides one or more vectors comprising an isolated nucleic acid molecule or set of nucleic acid molecules encoding the bispecific antibodies disclosed herein. In a further embodiment of the one or more vectors, the nucleotide sequences encoding different peptide chains of the bispecific antibodies disclosed herein are located in different vector molecules. In a further embodiment of the one or more vectors, the one or more vectors are cloning vectors or expression vectors.

[0080] The present invention further provides a host cell comprising an isolated nucleic acid molecule or set of nucleic acid molecules disclosed herein encoding a bispecific antibody disclosed herein, or one or more vectors disclosed herein encoding a bispecific antibody disclosed herein. In a further embodiment, the host cell expresses a bispecific antibody disclosed herein.

[0081] The present invention further provides a method for preparing a bispecific antibody disclosed herein, the method comprising culturing a host cell containing an isolated nucleic acid molecule or set of nucleic acid molecules disclosed herein encoding a bispecific antibody disclosed herein, or one or more vectors disclosed herein encoding a bispecific antibody, under conditions that enable the expression of the bispecific antibody, and recovering the bispecific antibody from the host cell culture.

[0082] The present invention further provides a complex comprising a bispecific antibody and a complexing moiety linked thereto, as disclosed herein. In further embodiments, the complexing moiety is selected from therapeutic agents (such as cytotoxic agents, cytokines, toxins, or radionuclides).

[0083] The present invention further provides a pharmaceutical composition comprising a bispecific antibody disclosed herein, or an isolated nucleic acid molecule or set of nucleic acid molecules encoding a bispecific antibody disclosed herein, or one or more vectors encoding a bispecific antibody disclosed herein, or a host cell expressing a bispecific antibody disclosed herein, or a complex comprising a bispecific antibody disclosed herein, and a pharmaceutically acceptable carrier and / or excipient.

[0084] In further embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In further embodiments, the additional pharmaceutically active agent is a drug having antitumor activity. In further 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, chemotherapeutic agents, or any combination thereof. In further embodiments, the bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as mixed components.

[0085] The present invention further provides the use of a bispecific antibody disclosed herein, or an isolated nucleic acid molecule or set of nucleic acid molecules encoding a bispecific antibody disclosed herein, or one or more vectors encoding a bispecific antibody disclosed herein, or a host cell disclosed herein, or a complex disclosed herein, or a pharmaceutical composition disclosed herein, in the preparation of a pharmaceutical, the pharmaceutical being used for the prevention and / or treatment of c-MET and / or EGFR-related diseases in a subject, and / or as an adjuvant in the treatment thereof, and / or for the inhibition of c-MET and / or EGFR activity in vitro or in a subject.

[0086] In further embodiments, the disease associated with c-MET and / or EGFR is cancer. In further embodiments, cancer is associated with EGFR-activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET-activating mutations and / or c-MET gene amplification. In further embodiments, 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 cavity cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0087] In further embodiments, a bispecific antibody, an isolated nucleic acid molecule or set of nucleic acid molecules, one or more vectors, host cells, a complex, or a pharmaceutical composition is administered in combination with another pharmaceutically active agent, for example, simultaneously, separately, or sequentially. In further embodiments, the other pharmaceutically active agent is a drug having antitumor activity. In further 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, chemotherapeutic agents, or any combination thereof. In further embodiments, the subject is resistant to erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab treatment.

[0088] The present invention further provides a method for inhibiting intracellular c-MET and / or EGFR activity, the method comprising contacting cells with a pharmaceutical composition comprising a bispecific antibody disclosed herein, or an isolated nucleic acid molecule encoding a bispecific antibody disclosed herein or a set of nucleic acid molecules encoding a bispecific antibody disclosed herein, or one or more vectors encoding a bispecific antibody disclosed herein, or a host cell expressing a bispecific antibody disclosed herein, or a complex comprising a bispecific antibody disclosed herein, or a bispecific antibody disclosed herein, or an isolated nucleic acid molecule encoding a bispecific antibody disclosed herein or a set of nucleic acid molecules encoding a bispecific antibody disclosed herein, or one or more vectors encoding a bispecific antibody disclosed herein, or a host cell expressing a bispecific antibody disclosed herein, or a complex comprising a bispecific antibody disclosed herein.

[0089] In further embodiments, the cells are c-MET and / or EGFR-expressing cells, such as tumor cells.

[0090] The present invention further provides a method for preventing and / or treating c-MET and / or EGFR-related diseases in a subject, and / or a method for acting as an adjuvant in the treatment thereof, the method comprising administering to a subject in need thereof an effective amount of the bispecific antibody disclosed herein, or an isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or a set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or one or more vectors encoding the bispecific antibody disclosed herein, or a host cell expressing the bispecific antibody disclosed herein, or a complex containing the bispecific antibody disclosed herein, or the bispecific antibody disclosed herein, or the bispecific antibody disclosed herein, or an isolated nucleic acid molecule encoding the bispecific antibody disclosed herein or a set of nucleic acid molecules encoding the bispecific antibody disclosed herein, or one or more vectors encoding the bispecific antibody disclosed herein, or a host cell expressing the bispecific antibody disclosed herein, or a complex containing the bispecific antibody disclosed herein.

[0091] In further embodiments, the disease associated with c-MET and / or EGFR is cancer. In further embodiments, cancer is associated with EGFR-activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET-activating mutations and / or c-MET gene amplification. In further embodiments, 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 cavity cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0092] In further embodiments, the method further comprises administering 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, the second therapy may optionally be applied simultaneously, separately, or sequentially. In further embodiments, the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab. [Brief explanation of the drawing]

[0093] [Figure 1A] Schematic diagram of the bispecific antibody structure. [Figure 1B] Schematic diagram of the bispecific antibody structure. [Figure 1C] Schematic diagram of the bispecific antibody structure. [Figure 1D] Schematic diagram of the bispecific antibody structure. [Figure 2A] Determination of the binding activity of a bispecific antibody against human non-small cell lung cancer cells HCC827. [Figure 2B] Determination of the binding activity of a bispecific antibody against human non-small cell lung cancer cells HCC827. [Figure 3A] Determination of the binding activity of a bispecific antibody against the human gastric cancer cell line MKN45. [Figure 3B] Determination of the binding activity of a bispecific antibody against the human gastric cancer cell line MKN45. [Figure 4A] Determination of the binding activity of a bispecific antibody against human lung adenocarcinoma NCI-H1975 cells. [Figure 4B] Determination of the binding activity of a bispecific antibody against human lung adenocarcinoma NCI-H1975 cells. [Modes for carrying out the invention]

[0094] antibody In one embodiment, the present application provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR.

[0095] In certain embodiments, the first antigen-binding domain comprises a first variable light chain region (VL) and a first variable heavy chain region (VH), and the first VL and the first VH together form a domain that can specifically bind to 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 that can specifically bind to EGFR.

[0096]

[0095] In certain embodiments, the first antigen-binding domain and the second antigen-binding domain are each independently selected from scFv, Fab, and scFab. Unless otherwise clearly stated, the Fab domains may be arranged according to their natural orientation or may include domain substitutions or swaps (e.g., domain swaps in the Crossmab format) that promote correct VH and VL pairing.

[0097] In certain embodiments, the domain swap in the CrossMab format is selected from the following: a. CrossMab Fab : For Fab, the antibody light chain CL-VL and the antibody heavy chain CH1-VH are exchanged. b. CrossMab VH-VL : For Fab, the VL and VH of the antibody are exchanged. c. CrossMab CH1-CL : For Fab, the CH and CL of the antibody are exchanged.

[0098] CrossMab is a method for preventing mismatching of light and heavy chains, with reference to U.S. Patent US9266967B2, which is incorporated herein by reference in its entirety. This method primarily utilizes the principle that the same portions of an antibody repel each other, i.e., 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 objective of preventing light chain mismatch is achieved by local mutual exchange of one heavy and light chain of Fab within a bispecific antibody (e.g., light chain CL-VL is exchanged with heavy chain CH1-VH, or VL and VH are exchanged, or CH and CL are exchanged).

[0099] 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 monomer.

[0100] In certain embodiments, the Fc domain comprises a first Fc domain monomer containing a modification that forms a knob structure and a second Fc domain monomer containing a modification that forms a hole structure, the hole structure pairing with the knob structure to form a heterodimer Fc domain.

[0101] Those skilled in the art will readily understand that a hole structure can pair with a knob structure to form a "knob-in-hole" structure, which can be used to reduce heavy chain mismatch in bispecific antibodies. The "knob" and "hole" structures can be introduced by mutation at corresponding positions in the CH3 domains of two Fc domain monomers, forming a specific interaction interface between the two Fc domain monomers (see Ridgway et al., Protein Eng., 9:617-621 (1996), WO2006 / 028936, which are incorporated herein by reference in their entirety). Amino acid substitutions that can be used to form a "knob" structure include, but are not limited to, S354C and T366W, and numbering follows 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, and numbering follows the Eu numbering scheme. In addition to the mutual attraction between the "knob" structure and the "hole" structure, the mutual repulsion between the "knob" structures allows the "knob-in-hole structure" to effectively reduce heavy chain mismatch within bispecific antibodies.

[0102] In certain embodiments, the Fc domain monomer is derived from the Fc domain of a human immunoglobulin and includes modifications that form a knob or hole structure. In certain embodiments, the human immunoglobulin is IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the Fc domain monomer includes one or more modifications that form a knob structure, and one or more modifications include the S354C and / or T366W amino acid substitution, and the numbering follows the Eu numbering scheme. In certain embodiments, the Fc domain monomer includes the S354 and T366W amino acid substitution, and the numbering follows the Eu numbering scheme. In certain embodiments, the Fc domain monomer includes one or more modifications that form a hole structure, and one or more modifications include the Y349C, T366S, L368A, and / or Y407V amino acid substitution, and the numbering follows the Eu numbering scheme. In certain embodiments, the Fc domain monomer includes Y349C, T366S, L368A, and Y407V amino acid substitutions, and the numbering follows the Eu numbering scheme.

[0103] In certain embodiments, the Fc domain further exhibits altered effector function compared to the wild-type Fc region (e.g., enhanced ADCC activity). This alteration of effector function may be introduced, for example, by mutation or chemical modification.

[0104] In certain embodiments, the first Fc domain monomer comprises an amino acid sequence such as that shown in SEQ ID NO: 49 or 51, and the second Fc domain monomer comprises an amino acid sequence such as that shown in SEQ ID NO: 50 or 52.

[0105] In a particular embodiment, the first antigen-binding domain and the second antigen-binding domain are each linked to one of the first and second Fc domain monomers.

[0106] 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.

[0107] The individual CDRs of the antigen-binding domains disclosed herein may be determined according to any CDR numbering scheme known in the art.

[0108] In certain embodiments, the antigen-binding domain of the antibody provided herein comprises LCDR1, LCDR2, and / or LCDR3 of the VL amino acid sequence shown in any one of SEQ ID NOs. 15, 17, and 59, as determined by the Kabat numbering scheme. In certain embodiments, the antigen-binding domain of the antibody provided herein comprises HCDR1, HCDR2, and / or HCDR3 of the VH amino acid sequence shown in any one of SEQ ID NOs. 16, 18, and 60, as determined by the Kabat numbering scheme.

[0109] In certain embodiments, the antigen-binding domain of the antibody provided herein comprises LCDR1, LCDR2, and / or LCDR3 of the VL amino acid sequence shown in any one of SEQ ID NOs. 15, 17, and 59, as determined by the Chothia numbering scheme. In certain embodiments, the antigen-binding domain of the antibody provided herein comprises HCDR1, HCDR2, and / or HCDR3 of the VH amino acid sequence shown in any one of SEQ ID NOs. 16, 18, and 60, as determined by the Chothia numbering scheme.

[0110] In certain embodiments, the antigen-binding domain of the antibody provided herein comprises LCDR1, LCDR2, and / or LCDR3 of the VL amino acid sequence shown in any one of SEQ ID NOs. 15, 17, and 59, as determined by the MacCallum numbering scheme. In certain embodiments, the antigen-binding domain of the antibody provided herein comprises HCDR1, HCDR2, and / or HCDR3 of the VH amino acid sequence shown in any one of SEQ ID NOs. 16, 18, and 60, as determined by the MacCallum numbering scheme.

[0111] In certain embodiments, the antigen-binding domain of the antibody provided herein comprises LCDR1, LCDR2, and / or LCDR3 of the VL amino acid sequence shown in any one of SEQ ID NOs. 15, 17, and 59, as determined by the IMGT numbering scheme. In certain embodiments, the antigen-binding domain of the antibody provided herein comprises HCDR1, HCDR2, and / or HCDR3 of the VH amino acid sequence shown in any one of SEQ ID NOs. 16, 18, and 60, as determined by the IMGT numbering scheme.

[0112] In certain embodiments, the antigen-binding domain of the antibody provided herein comprises LCDR1, LCDR2, and / or LCDR3 of the VL amino acid sequence shown in any one of SEQ ID NOs. 15, 17, and 59, as determined by the AbM numbering scheme. In certain embodiments, the antigen-binding domain of the antibody provided herein comprises HCDR1, HCDR2, and / or HCDR3 of the VH amino acid sequence shown in any one of SEQ ID NOs. 16, 18, and 60, as determined by the AbM numbering scheme.

[0113] In certain embodiments, the antigen-binding domain of the antibody provided herein comprises LCDR1, LCDR2, and / or LCDR3 of the VL amino acid sequence shown in any one of SEQ ID NOs. 15, 17, and 59, as determined by the AHo numbering scheme. In certain embodiments, the antigen-binding domain of the antibody provided herein comprises HCDR1, HCDR2, and / or HCDR3 of the VH amino acid sequence shown in any one of SEQ ID NOs. 16, 18, and 60, as determined by the AHo numbering scheme.

[0114] In certain embodiments, the individual CDRs of the antigen-binding domain of the antibodies provided herein are determined independently, either according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the antigen-binding domain, the structural analysis of which residues within the variable region(s) predicted to contact the c-MET or EGFR epitope region are identified.

[0115] In certain embodiments, the Disclosure provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR, wherein the first antigen-binding domain comprises VL, which includes the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17 or 59, and VH, which includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 18 or 60, and / or the second antigen-binding domain comprises VL, which includes the VL amino acid sequence shown in SEQ ID NO: 15 The VL includes the amino acid sequences LCDR1, LCDR2, and LCDR3, and the VH includes the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO: 16. Each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the bispecific molecule, through which residues within the variable region(s) predicted to contact the epitope region of c-MET or EGFR are identified.

[0116] In certain embodiments, the Disclosure provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR, wherein the first antigen-binding domain comprises VL, which includes the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17, and VH, which includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 18, and / or the second antigen-binding domain comprises the VL amino acid sequence shown in SEQ ID NO: 15 The VL includes the LCDR1, LCDR2, and LCDR3 amino acid sequences, and the VH includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16, where each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, Aho, or AbM numbering schemes, or by structural analysis of the bispecific molecule, by which the structural analysis identifies residues in the variable region(s) predicted to contact the epitope region of c-MET or EGFR.

[0117] In certain embodiments, the Disclosure provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR, wherein the first antigen-binding domain comprises VL, which includes the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 59, and VH, which includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 60, and / or the second antigen-binding domain comprises the VL amino acid sequence shown in SEQ ID NO: 15 The VL includes the LCDR1, LCDR2, and LCDR3 amino acid sequences, and the VH includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16, where each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the bispecific molecule, by which the structural analysis identifies residues in the variable region(s) predicted to contact the epitope region of c-MET or EGFR.

[0118] In certain embodiments, the Disclosure provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR, wherein the first antigen-binding domain comprises VL, which includes the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17, and VH, which includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 60, and / or the second antigen-binding domain comprises the VL amino acid sequence shown in SEQ ID NO: 15 The VL includes the LCDR1, LCDR2, and LCDR3 amino acid sequences, and the VH includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16, where each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the bispecific molecule, by which the structural analysis identifies residues in the variable region(s) predicted to contact the epitope region of c-MET or EGFR.

[0119] In certain embodiments, the Disclosure provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR, wherein the first antigen-binding domain comprises VL, which includes the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 59, and VH, which includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 18, and / or the second antigen-binding domain comprises the VL amino acid sequence shown in SEQ ID NO: 15 The VL includes the LCDR1, LCDR2, and LCDR3 amino acid sequences, and the VH includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16, where each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the bispecific molecule, by which the structural analysis identifies residues in the variable region(s) predicted to contact the epitope region of c-MET or EGFR.

[0120] In certain embodiments of the bispecific antibodies provided herein, the first VL is

[0121] LCDR1 containing the amino acid sequence shown in SEQ ID NO: 34, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38 (CDR is defined by the Kabat numbering system),

[0122] LCDR1 containing the amino acid sequence shown in SEQ ID NO: 34, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38 (CDR is defined by the Chothia numbering system),

[0123] LCDR1 containing the amino acid sequence shown in SEQ ID NO: 34, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38 (CDR is defined by the Abm numbering system), or

[0124] LCDR1 containing the amino acid sequence shown in SEQ ID NO: 35, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38 (where LCDR is defined by the IMGT numbering system), and / or

[0125] The 1st VH is,

[0126] HCDR1 containing the amino acid sequence shown in SEQ ID NO: 39, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47 (CDR is defined by the Kabat numbering system),

[0127] HCDR1 containing the amino acid sequence shown in SEQ ID NO: 40, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 44, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47 (CDR is defined by the Chothia numbering system),

[0128] HCDR1 containing the amino acid sequence shown in SEQ ID NO: 42, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 46, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47 (CDR is defined by the Abm numbering system), or

[0129] This includes HCDR1 containing the amino acid sequence shown in SEQ ID NO: 41, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 45, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 48 (CDR is defined by the IMGT numbering system).

[0130] In certain embodiments, the first VL includes 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 shown in SEQ ID NO: 17 or 59. In certain embodiments, the first VL includes the amino acid sequence shown in SEQ ID NO: 17 or 59. In certain embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO: 17 or 59.

[0131] In certain embodiments, the first VH includes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (for example, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 18 or 60. In certain embodiments, the first VH includes the amino acid sequence shown in SEQ ID NO: 18 or 60. In certain embodiments, the first VH consists of the amino acid sequence shown in SEQ ID NO: 18 or 60.

[0132] In certain embodiments, the first VL includes 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 shown in SEQ ID NO: 17 or 59, and the first VH includes 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 shown in SEQ ID NO: 18 or 60. In a particular embodiment, the first VL consists of the amino acid sequence shown in SEQ ID NO: 17 or 59, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 18 or 60.

[0133] In certain embodiments, the first VL includes 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 shown in SEQ ID NO: 17, and the first VH includes 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 shown in SEQ ID NO: 18. In a particular embodiment, the first VL consists of the amino acid sequence shown in SEQ ID NO: 17, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 18.

[0134] In a particular embodiment, the first VL includes 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 shown in SEQ ID NO: 59, and the first VH includes 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 shown in SEQ ID NO: 60. In a particular embodiment, the first VL consists of the amino acid sequence shown in SEQ ID NO: 59, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 60.

[0135] In a particular embodiment, the first VL includes 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 shown in SEQ ID NO: 17, and the first VH includes 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 shown in SEQ ID NO: 60. In a particular embodiment, the first VL consists of the amino acid sequence shown in SEQ ID NO: 17, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 60.

[0136] In a particular embodiment, the first VL includes 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 shown in SEQ ID NO: 59, and the first VH includes 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 shown in SEQ ID NO: 18. In a particular embodiment, the first VL consists of the amino acid sequence shown in SEQ ID NO: 59, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 18.

[0137] In certain embodiments of the bispecific antibodies provided herein, the second VL is

[0138] LCDR1 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 23 (CDR is defined by the Kabat numbering system),

[0139] LCDR1 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 23 (CDR is defined by the Chothia numbering system),

[0140] LCDR1 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 23 (CDR is defined by the Abm numbering system), or

[0141] LCDR1 containing the amino acid sequence shown in SEQ ID NO: 20, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 23 (where LCDR is defined by the IMGT numbering system), and / or

[0142] 2VH is,

[0143] HCDR1 containing the amino acid sequence shown in SEQ ID NO: 24, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32 (CDR is defined by the Kabat numbering system),

[0144] HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32 (CDR is defined by the Chothia numbering system),

[0145] HCDR1 containing the amino acid sequence shown in SEQ ID NO: 27, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32 (CDR is defined by the Abm numbering system), or

[0146] This includes HCDR1 containing the amino acid sequence shown in SEQ ID NO: 26, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 30, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 33 (CDR is defined by the IMGT numbering system).

[0147] In certain embodiments, the first VL includes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (for example, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VL includes the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO: 15.

[0148] In certain embodiments, the first VH includes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (for example, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 16. In certain embodiments, the first VH includes the amino acid sequence shown in SEQ ID NO: 16. In certain embodiments, the first VH consists of the amino acid sequence shown in SEQ ID NO: 16.

[0149] In certain embodiments, the first VL includes 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 shown in SEQ ID NO: 15, and the first VH includes 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 shown in SEQ ID NO: 16. In a particular embodiment, the first VL consists of the amino acid sequence shown in SEQ ID NO: 15, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 16.

[0150] In certain embodiments, the Disclosure provides a bispecific antibody that cross-competes with any of the antibodies described herein for binding to c-MET and / or EGFR. In certain embodiments, the Disclosure provides a bispecific antibody that binds to the same or overlapping c-MET and / or EGFR epitopes as the antibodies described herein.

[0151] In certain embodiments, the antibody epitope may be determined, for example, by NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange in conjunction with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenic mapping (e.g., site-directed mutagenic mapping). With respect to X-ray crystallography, crystallization may be achieved using any method known in the art (e.g., Giege 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 incorporated herein by reference)). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc., e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al., see 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, eds. Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt The methods may be refined using computer software such as 10):1316-1323 (the whole of which is incorporated herein by reference). Mutagenesis mapping tests may be achieved using any method known to those skilled in the art.For example, see Champe M et al., (1995) and Cunningham BC & Wells JA (1989) for descriptions of mutagenesis techniques, including alanine scanning mutagenesis techniques. In certain embodiments, the epitope of an antibody is determined using an alanine scanning mutagenesis test. 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 immunoassays, i.e., competitive binding assays, by demonstrating the ability of one antibody to block the binding of another antibody to a target antigen. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity to an epitope. Competitive binding can be determined by assays in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen such as c-MET or EGFR. Numerous types of competitive binding assays are known, including, for example, solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competitive assays (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 labeling assays, solid-phase direct labeling sandwich assays (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeling RIA using I-125 labeling (see Morel GA et al., (1988) Mol Immunol 25(1):7-15), and solid-phase direct biotin-avidin EIA (see Cheung RC et al. See al., (1990) Virology 176:546-52, and directly labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference.Typically, such assays involve the use of purified antigens (e.g., c-MET or EGFR) bound to a solid surface or cell carrying either the unlabeled test immunoglobulin or the labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of labeling bound to the solid surface or cell in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, if the competing antibody is present in excess, it will inhibit the specific binding of the reference or antibody to the common antigen by at least 50–55%, 55–60%, 60–65%, 65–70%, 70–75%, or more. Competitive binding assays can consist of a number of different forms using either labeled antigens or labeled antibodies. In a common form of this assay, the antigen is immobilized on a 96-well plate. The ability of the unlabeled antibody to block the binding of the labeled antibody to the antigen is then measured using radiolabeling or enzymatic labeling. For further details, see, for example, 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, edited by Harlow E & Lane D (the above editors), pp. 386-389, all of which are incorporated herein by reference.

[0152] In certain embodiments of the bispecific antibodies provided herein, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1, numbered according to the EU numbering system) and / or the 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 binding, Fc receptor binding, and / or antigen-dependent cell-mediated cytotoxicity.

[0153] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region to reduce or eliminate the effector function of the Fc region, for example. In certain embodiments, the antibody comprises an Fc region including: (i) E233A and L235A (EALA) amino acid substitutions (numbering according to the Eu numbering scheme), (ii) L234A and L235A (LALA) amino acid substitutions (numbering according to the Eu numbering scheme), (iii) L234A, L235A, D265S (LALADS) amino acid substitutions (numbering according to the Eu numbering scheme), (iv) L234A, L235A, P329G (LALAPG) amino acid substitutions (numbering according to the Eu numbering scheme), (v) L234A, L235A, P329A (LALAPA) amino acid substitutions (numbering according to the Eu numbering scheme) (vi) L235E(LE) amino acid substitution (numbering follows the Eu numbering scheme), (vii) D265A(DS) amino acid substitution (numbering follows the Eu numbering scheme), (viii) D265A, N297G(DANG) amino acid substitution (numbering follows the Eu numbering scheme), (ix) N297X amino acid substitution (X is any amino acid other than N, and numbering follows the Eu numbering scheme), (x) N297A / D356E / L358M(NADELM) amino acid substitution (numbering follows the Eu numbering scheme), or (x1) D356E, L358M(DELM) amino acid substitution (numbering follows the Eu numbering scheme).

[0154] In certain embodiments, any of the constant region mutations or modifications described herein may be introduced into one or both heavy chain constant regions of an antibody described herein having two heavy chain constant regions.

[0155] Polypeptide In another embodiment, this specification provides polypeptides comprising one or more LCDR, HCDR, VL, and / or VH amino acid sequences as described above. In a particular embodiment, the polypeptide comprises LCDR1, LCDR2, and / or LCDR3 of the VL amino acid sequence shown in any one of SEQ ID NOs: 15, 17, and 59, as determined by any of the methods described above. In a particular embodiment, the polypeptide comprises LCDR1, LCDR2, and LCDR3 of the VL amino acid sequence shown in any one of SEQ ID NOs: 15, 17, and 59. In a particular embodiment, the polypeptide comprises LCDR1, LCDR2, and / or LCDR3 amino acid sequences shown in SEQ ID NOs: 34, 36, and 38, SEQ ID NOs: 35, 37, and 38, SEQ ID NOs: 19, 21, and 23, or SEQ ID NOs: 20, 22, and 23, respectively.

[0156] In certain embodiments, the polypeptide comprises HCDR1, HCDR2, and / or HCDR3 of the VH amino acid sequence shown in any one of SEQ ID NOs: 16, 18, and 60, as determined by any of the methods described above. In certain embodiments, the polypeptide comprises HCDR1, HCDR2, and HCDR3 of the VH amino acid sequence shown in any one of SEQ ID NOs: 16, 18, and 60. In certain embodiments, the polypeptide comprises HCDR1, HCDR2, and / or HCDR3 amino acid sequences shown in SEQ ID NOs: 39, 43, and 47, SEQ ID NOs: 40, 44, and 47, SEQ ID NOs: 42, 46, and 47, SEQ ID NOs: 41, 45, and 48, SEQ ID NOs: 24, 28, and 32, SEQ ID NOs: 25, 29, and 32, SEQ ID NOs: 27, 31, and 32, or SEQ ID NOs: 26, 30, and 33, respectively.

[0157] 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 shown in any one of SEQ ID NOs: 15, 17, and 59.

[0158] 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 shown in any one of SEQ ID NOs: 16, 18, and 60.

[0159] Fab-scFv structure In a particular embodiment, the first antigen-binding domain is Fab, and the second antigen-binding domain is scFv.

[0160] In a particular embodiment, the bispecific antibody comprises polypeptide chain IA, polypeptide chain IB, and polypeptide chain IC, wherein (a) the elements of polypeptide chain IA include a first VL and a first CL, the elements of polypeptide chain IB include a first VH, a first heavy chain CH1 region, and a first Fc monomer, and the elements of polypeptide chain IC include a second VL, a second VH, a second heavy chain CH1 hinge region, and a second Fc monomer, or (b) the elements of polypeptide chain IA include a first VL and a CL, the elements of polypeptide chain IB include a first VH, a first heavy chain CH1 region, and a second Fc monomer, and the elements of polypeptide chain IC include a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc monomer.

[0161] In a particular embodiment, (a) elements included in polypeptide chain IA include a first VL and a first CL from the N-terminus to the C-terminus, elements included in polypeptide chain IB include a first VH, a first heavy chain CH1 region, and a first Fc monomer from the N-terminus to the C-terminus, and / or elements included in polypeptide chain IC include (i) a second VL, a second VH, a second heavy chain CH1 hinge region, and a second Fc monomer from the N-terminus to the C-terminus, or (ii) a second VH, a second VL, a second heavy chain CH1 hinge region, and a second Fc monomer -Includes or (b) an element included in polypeptide chain IA includes a first VL and a first CL from the N-terminus to the C-terminus, an element included in polypeptide chain IB includes a first VH, a first heavy chain CH1, and a second Fc monomer from the N-terminus to the C-terminus, and / or an element included in polypeptide chain IC includes (i) a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc monomer from the N-terminus to the C-terminus, or (ii) a second VH, a second VL, a second heavy chain CH1 hinge region, and a first Fc monomer.

[0162] In a particular embodiment, adjacent elements in polypeptide chain IA may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain IB may or may not be linked to each other by peptide linkers, and / or adjacent elements in polypeptide chain IC may or may not be linked to each other by peptide linkers.

[0163] In certain embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers). In certain embodiments, each peptide linker independently contains 3 to 55 amino acid residues (e.g., 3 to 10, 10 to 25, 10 to 20, 10 to 17, 10 to 15, 10 to 25, 10 to 35, 10 to 45, 10 to 55). In certain embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S) residues. In certain embodiments, each peptide linker independently contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues. In certain embodiments, each peptide linker independently contains peptide GGGG (SEQ ID NO: 61). In a particular embodiment, the peptide linker comprises one, two, three, four, five, six, seven, eight, nine, or ten tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), each independently, which is (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) may be represented by these formulas. In certain embodiments, the peptide linker each independently comprises the amino acid sequences shown in SEQ ID NOs. 55-58, 61 and 63-68.

[0164] In certain embodiments, the first and second CLs include the amino acid sequence shown in SEQ ID NO: 53. In certain embodiments, the first heavy chain CH1 region includes the amino acid sequence shown in SEQ ID NO: 54. In certain embodiments, the second heavy chain CH1 hinge region includes the amino acid sequence shown in SEQ ID NO: 62.

[0165] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, and the first VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, the first VL includes the amino acid sequence shown in SEQ ID NO: 15, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17.

[0166] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, and the first VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, the first VL includes the amino acid sequence shown in SEQ ID NO: 17, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15.

[0167] In a particular embodiment, 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 a particular embodiment, 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.

[0168] In a particular embodiment, 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 a particular embodiment, 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.

[0169] In a particular embodiment, polypeptide chain IA comprises the amino acid sequence shown in SEQ ID NO: 1, polypeptide chain IB comprises the amino acid sequence shown in SEQ ID NO: 2 or 9, and / or polypeptide chain IC comprises the amino acid sequence shown in SEQ ID NO: 3 or 10.

[0170] scFv-Fab structure In a particular embodiment, the first antigen-binding domain is scFv, and the second antigen-binding domain is Fab.

[0171] In a particular embodiment, the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C, wherein (a) the elements of polypeptide chain II-A include the second VL and second CL, the elements of polypeptide chain II-B include the second VH, the second heavy chain CH1 region, and the first Fc monomer, and the elements of polypeptide chain II-C include the first VL, the first VH, the first heavy chain CH1 hinge region, and the second Fc monomer, or (b) the elements of polypeptide chain II-A include the second VL and second CL, the elements of polypeptide chain II-B include the second VH, the second heavy chain CH1 region, and the second Fc monomer, and the elements of polypeptide chain II-C include the first VL, the first VH, the first heavy chain CH1 hinge region, and the first Fc monomer.

[0172] In a particular embodiment, (a) elements of polypeptide chain II-A include a second VL and a second CL from the N-terminus to the C-terminus, elements of polypeptide chain II-B include a second VH, a second heavy chain CH1 region, and a first Fc monomer from the N-terminus to the C-terminus, and / or elements of polypeptide chain II-C include (i) a first VL, a first VH, a first heavy chain CH1 hinge region, and a second Fc monomer from the N-terminus to the C-terminus, or (ii) a first VH, a first VL, a first heavy chain CH1 hinge region, and a second Fc monomer from the N-terminus to the C-terminus. (b) elements of polypeptide chain II-A include a second VL and a second CL from the N-terminus to the C-terminus, elements of polypeptide chain II-B include a second VH, a second heavy chain CH1 region, and a second Fc monomer from the N-terminus to the C-terminus, and / or elements of polypeptide chain II-C include (i) a first VL, a first VH, a first heavy chain CH1 hinge region, and a first Fc monomer from the N-terminus to the C-terminus, or (ii) a first VH, a first VL, a first heavy chain CH1 hinge region, and a first Fc monomer from the N-terminus to the C-terminus.

[0173] In a particular embodiment, adjacent elements in polypeptide chain II-A may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain II-B may or may not be linked to each other by peptide linkers, and / or adjacent elements in polypeptide chain II-C may or may not be linked to each other by peptide linkers.

[0174] In certain embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker). In certain embodiments, each peptide linker independently contains 3 to 55 amino acid residues (e.g., 3 to 10, 10 to 25, 10 to 20, 10 to 17, 10 to 15, 10 to 25, 10 to 35, 10 to 45, 10 to 55). In certain embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S) residues. In certain embodiments, each peptide linker independently contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which is (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) may be represented by these formulas. In certain embodiments, the peptide linker each independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0175] In a particular embodiment, the first and second CLs include the amino acid sequence shown in SEQ ID NO: 53, and / or the first and second heavy chain CH1 regions include the amino acid sequence shown in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region includes the amino acid sequence shown in SEQ ID NO: 62.

[0176] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, and the first VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, the first VL includes the amino acid sequence shown in SEQ ID NO: 15, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17.

[0177] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, and the first VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, the first VL includes the amino acid sequence shown in SEQ ID NO: 17, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15.

[0178] In a particular embodiment, 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 a particular embodiment, 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.

[0179] In a particular embodiment, 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 a particular embodiment, 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.

[0180] In a particular embodiment, peptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4, peptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 5 or 7, and / or peptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 6 or 8.

[0181] Fab-scFab structure In a particular embodiment, the first antigen-binding domain is Fab, and the second antigen-binding domain is scFab (single-stranded Fab).

[0182] In a particular embodiment, the bispecific antibody comprises polypeptide chain III-A, polypeptide chain III-B, and polypeptide chain III-C, wherein (a) the elements of polypeptide chain III-A include the first VL and the first CL, the elements of polypeptide chain III-B include the first VH, the first heavy chain CH1 region, and the first Fc monomer, and the elements of polypeptide chain III-C include the second VL, the second CL, the second VH, the second heavy chain CH1 region, and the second Fc monomer, or (b) the elements of polypeptide chain III-A include the first VL and the first CL, the elements of polypeptide chain III-B include the first VH, the first heavy chain CH1 region, and the second Fc monomer, and the elements of polypeptide chain III-C include the second VL, the second CL, the second VH, the second heavy chain CH1 region, and the first Fc monomer.

[0183] In a particular embodiment, (a) elements of polypeptide chain III-A include a first VL and a first CL from the N-terminus to the C-terminus, elements of polypeptide chain III-B include a first VH, a first heavy chain CH1 region, and a first Fc monomer from the N-terminus to the C-terminus, and / or elements of polypeptide chain III-C include (i) a second VL, a second CL, a second VH, a second heavy chain CH1 region, and a second Fc monomer from the N-terminus to the C-terminus, or (ii) a second VH, a second heavy chain CH1 region, a second VL, a second CL, and a second Fc monomer. (b) elements of polypeptide chain III-A include the first VL and first CL from the N-terminus to the C-terminus, elements of polypeptide chain III-B include the first VH, first heavy chain CH1 region, and second Fc monomer from the N-terminus to the C-terminus, and / or elements of polypeptide chain III-C include (i) the second VL, second CL, second VH, second heavy chain CH1 region, and first Fc monomer from the N-terminus to the C-terminus, or (ii) the second VH, second heavy chain CH1 region, second VL, second CL, and first Fc monomer.

[0184] In a particular embodiment, adjacent elements in polypeptide chain III-A may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain III-B may or may not be linked to each other by peptide linkers, and / or adjacent elements in polypeptide chain III-C may or may not be linked to each other by peptide linkers.

[0185] In certain embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker). In certain embodiments, each peptide linker independently contains 3 to 55 amino acid residues (e.g., 3 to 10, 10 to 25, 10 to 20, 10 to 17, 10 to 15, 10 to 25, 10 to 35, 10 to 45, 10 to 55). In certain embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S) residues. In certain embodiments, each peptide linker independently contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57), which is (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) may be represented by these formulas. In certain embodiments, the peptide linker each independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0186] In a particular embodiment, the first and second CLs include the amino acid sequence shown in SEQ ID NO: 53, and / or the first and second heavy chain CH1 regions include the amino acid sequence shown in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region includes the amino acid sequence shown in SEQ ID NO: 62.

[0187] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, and the first VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, the first VL includes the amino acid sequence shown in SEQ ID NO: 15, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17.

[0188] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, and the first VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, the first VL includes the amino acid sequence shown in SEQ ID NO: 17, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15.

[0189] In a particular embodiment, 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 a particular embodiment, 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.

[0190] In a particular embodiment, 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 a particular embodiment, 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.

[0191] scFab-Fab structure In a particular embodiment, the first antigen-binding domain is scFab, and the second antigen-binding domain is Fab.

[0192] In a particular embodiment, the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C, wherein (a) the elements of polypeptide chain IV-A include the second VL and the second CL, the elements of polypeptide chain IV-B include the second VH, the second heavy chain CH1 region, and the first Fc monomer, and the elements of polypeptide chain IV-C include the first VL, the first CL, the first VH, the first heavy chain CH1 region, and the second Fc monomer, or (b) the elements of polypeptide chain IV-A include the second VL and the second CL, the elements of polypeptide chain IV-B include the second VH, the second heavy chain CH1 region, and the second Fc monomer, and the elements of polypeptide chain IV-C include the first VL, the first CL, the first VH, the first heavy chain CH1 region, and the first Fc monomer.

[0193] In a particular embodiment, (a) the elements of polypeptide chain IV-A include a second VL and a second CL from the N-terminus to the C-terminus, the elements of polypeptide chain IV-B include a second VH, a second heavy chain CH1 region, and a first Fc monomer from the N-terminus to the C-terminus, and / or the elements of polypeptide chain IV-C include a first VL, a first CL, a first VH, a first heavy chain CH1 region, and a second Fc monomer from the N-terminus to the C-terminus, or (b) the elements of polypeptide chain IV-A include a second VL and a second CL from the N-terminus to the C-terminus, the elements of polypeptide chain IV-B include a second VL and a second CL, a second VH, a second heavy chain CH1 region, and a second Fc monomer from the N-terminus to the C-terminus, and / or the elements of polypeptide chain IV-C include (i) a first VL, a first CL, a first VH, a first heavy chain CH1 region, and a first Fc monomer from the N-terminus to the C-terminus.

[0194] In a particular embodiment, adjacent elements in polypeptide chain IV-A may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain IV-B may or may not be linked to each other by peptide linkers, and / or adjacent elements in polypeptide chain IV-C may or may not be linked to each other by peptide linkers.

[0195] In certain embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker). In certain embodiments, each peptide linker independently contains 3 to 55 amino acid residues (e.g., 3 to 10, 10 to 25, 10 to 20, 10 to 17, 10 to 15, 10 to 25, 10 to 35, 10 to 45, 10 to 55). In certain embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S) residues. In certain embodiments, each peptide linker independently contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which is (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) may be represented by these formulas. In certain embodiments, the peptide linker each independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0196] In a particular embodiment, the first and second CLs include the amino acid sequence shown in SEQ ID NO: 53, and / or the first and second heavy chain CH1 regions include the amino acid sequence shown in SEQ ID NO: 54, and / or the first heavy chain CH1 hinge region includes the amino acid sequence shown in SEQ ID NO: 62.

[0197] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, and the first VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, the first VL includes the amino acid sequence shown in SEQ ID NO: 15, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17.

[0198] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, and the first VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the first VL includes the amino acid sequence shown in SEQ ID NO: 59, and the first VH includes the amino acid sequence shown in SEQ ID NO: 60. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, the first VL includes the amino acid sequence shown in SEQ ID NO: 17, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15.

[0199] In a particular embodiment, 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 a particular embodiment, 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.

[0200] In a particular embodiment, 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 a particular embodiment, 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.

[0201] In a particular embodiment, peptide chain IV-A comprises the amino acid sequence shown in SEQ ID NO: 4, peptide chain IV-B comprises the amino acid sequence shown in SEQ ID NO: 7, and / or peptide chain IV-C comprises the amino acid sequence shown in SEQ ID NO: 11.

[0202] Fab-Fab (domain swapping including CrossMab configuration) structure In a particular embodiment, the first antigen-binding domain and the second antigen-binding domain are Fab, and the Fab of the second antigen-binding domain is CrossMab Fab This includes domain swaps in this form.

[0203] In a particular embodiment, the Fab of the second antigen-binding domain is CrossMab VH-VL This includes VH and VL domain swaps.

[0204] In a particular embodiment, the Fab of the second antigen-binding domain is CrossMab CH1-CL This includes a domain swap between CH1 and CL in this form.

[0205] In a particular embodiment, CrossMab CH1-CLA bispecific antibody in the form of (a) a polypeptide chain VA, polypeptide chain VB, polypeptide chain VC, and polypeptide chain VD, wherein (a) the elements contained in polypeptide chain VA include the first VL and the first CL, the elements contained in polypeptide chain VB include the first VH, the first heavy chain CH1 region, and the first Fc monomer, the elements contained in polypeptide chain VC include the second VH, the second CL, the second heavy chain CH1 hinge region, and the second Fc monomer, and polypeptide chain VD includes the second VL and the second heavy chain CH1 region, or (b) the elements contained in polypeptide chain VA include the first VL and the first CL, the elements contained in polypeptide chain VB include the first VH, the first heavy chain CH1 region, and the second Fc monomer, the elements contained in polypeptide chain VC include the second VH, the second CL, the second heavy chain CH1 hinge region, and the first Fc monomer, and polypeptide chain VD includes the second VL and the second heavy chain CH1 region.

[0206] In a particular embodiment, (a) elements included in polypeptide chain VA include a first VL and a first CL from the N-terminus to the C-terminus, elements included in polypeptide chain VB include a first VH, a first heavy chain CH1 region, and a first Fc monomer from the N-terminus to the C-terminus, elements included in polypeptide chain VC include a second VH, a second CL, a second heavy chain CH1 hinge region, and a second Fc monomer from the N-terminus to the C-terminus, and / or elements included in polypeptide chain VD include a second VL and a second heavy chain CH1 region from the N-terminus to the C-terminus (b) Elements included in polypeptide chain VA include the first VL and CL from the N-terminus to the C-terminus; elements included in polypeptide chain VB include the first VH, the first heavy chain CH1 region, and the second Fc monomer from the N-terminus to the C-terminus; elements included in polypeptide chain VC include the second VH, the second CL, the second heavy chain CH1 hinge region, and the first Fc monomer from the N-terminus to the C-terminus; and / or elements included in polypeptide chain VD include the second VL and the second heavy chain CH1 region from the N-terminus to the C-terminus.

[0207] In a particular embodiment, adjacent elements in polypeptide chain VA may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain VB may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain VC may or may not be linked to each other by peptide linkers, and / or adjacent elements in polypeptide chain VD may or may not be linked to each other by peptide linkers.

[0208] In certain embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker). In certain embodiments, each peptide linker independently contains 3 to 55 amino acid residues (e.g., 3 to 10, 10 to 25, 10 to 20, 10 to 17, 10 to 15, 10 to 25, 10 to 35, 10 to 45, 10 to 55). In certain embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S) residues. In certain embodiments, each peptide linker independently contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57), which is (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) may be represented by these formulas. In certain embodiments, the peptide linker each independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0209] In a particular embodiment, the first and second CLs include the amino acid sequence shown in SEQ ID NO: 53, and / or the first and second heavy chain CH1 regions include the amino acid sequence shown in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region includes the amino acid sequence shown in SEQ ID NO: 62.

[0210] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, and the first VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, the first VL includes the amino acid sequence shown in SEQ ID NO: 15, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17.

[0211] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, and the first VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, the first VL includes the amino acid sequence shown in SEQ ID NO: 17, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15.

[0212] In a particular embodiment, 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 a particular embodiment, 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.

[0213] In a particular embodiment, 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 a particular embodiment, 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.

[0214] In a particular embodiment, polypeptide chain VA comprises the amino acid sequence shown in SEQ ID NO: 1, polypeptide chain VB comprises the amino acid sequence shown in SEQ ID NO: 9, polypeptide chain VC comprises the amino acid sequence shown in SEQ ID NO: 12, and / or polypeptide chain VD comprises the amino acid sequence shown in SEQ ID NO: 13.

[0215] Fab (CrossMab form containing domain swapping)-Fab structure

[0216] In a particular embodiment, the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the first antigen-binding domain includes a domain swap in the form of a CrossMab.

[0217] In a particular embodiment, the Fab of the first antigen-binding domain is CrossMab CH1-CL This includes domain swaps in this form.

[0218] In a particular embodiment, 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 of polypeptide chain VI-A include the second VL and CL, the elements of polypeptide chain VI-B include the second VH, heavy chain CH1 region, and first Fc monomer, the elements of polypeptide chain VI-C include the first VH, CL, first heavy chain CH1 hinge region, and second Fc monomer, and the elements of polypeptide chain VI-D include the first VL and heavy chain CH1 region, or (b) the elements of polypeptide chain VI-A include the second VL and CL, the elements of polypeptide chain VI-B include the second VH, heavy chain CH1 region, and second Fc monomer, the elements of polypeptide chain VI-C include the first VH, CL, first heavy chain CH1 hinge region, and first Fc monomer, and the elements of polypeptide chain VI-D include the first VL and heavy chain CH1 region.

[0219] In a particular embodiment, (a) the elements of polypeptide chain VI-A include a second VL and CL from the N-terminus to the C-terminus, the elements of polypeptide chain VI-B include a second VH, a heavy chain CH1 region, and a first Fc monomer from the N-terminus to the C-terminus, the elements of polypeptide chain VI-C include a first VH, CL, a first heavy chain CH1 hinge region, and a second Fc monomer from the N-terminus to the C-terminus, and / or the elements of polypeptide chain VI-D include a first VL and a heavy chain from the N-terminus to the C-terminus (b) Elements containing a CH1 region or included in polypeptide chain VI-A include a second VL and CL from the N-terminus to the C-terminus; peptide chain VI-B includes a second VH, a heavy chain CH1 region, and a second Fc monomer from the N-terminus to the C-terminus; elements included in polypeptide chain VI-C include a first VH, CL, a first heavy chain CH1 hinge region, and a first Fc monomer from the N-terminus to the C-terminus; and / or elements included in polypeptide chain VI-D include a first VL and a heavy chain CH1 region from the N-terminus to the C-terminus.

[0220] In a particular embodiment, adjacent elements in polypeptide chain VI-A may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain VI-B may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain VI-C may or may not be linked to each other by peptide linkers, and / or adjacent elements in polypeptide chain VI-D may or may not be linked to each other by peptide linkers.

[0221] In certain embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker). In certain embodiments, each peptide linker independently contains 3 to 55 amino acid residues (e.g., 3 to 10, 10 to 25, 10 to 20, 10 to 17, 10 to 15, 10 to 25, 10 to 35, 10 to 45, 10 to 55). In certain embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S). In certain embodiments, each peptide linker independently contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57), which is (GGGGS) n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) (may be represented by sequence numbers 55-58 or 63-68).

[0222] In certain embodiments, the peptide linker each independently comprises the amino acid sequences shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0223] In a particular embodiment, 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.

[0224] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, and the first VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, the first VL includes the amino acid sequence shown in SEQ ID NO: 15, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17.

[0225] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, and the first VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, the first VL includes the amino acid sequence shown in SEQ ID NO: 17, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15.

[0226] In a particular embodiment, 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 a particular embodiment, 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.

[0227] In a particular embodiment, 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 a particular embodiment, 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.

[0228] In a particular embodiment, CrossMab VH-VLA bispecific antibody in the form of (a) a polypeptide chain VA, polypeptide chain VB, polypeptide chain VC, and polypeptide chain VD, wherein (a) the elements contained in polypeptide chain VA include the first VL and the first CL, the elements contained in polypeptide chain VB include the first VH, the first heavy chain CH1 region, and the first Fc monomer, the elements contained in polypeptide chain VC include the second VL, the second heavy chain CH1 hinge region, and the second Fc monomer, and polypeptide chain VD includes the second VH and the second CL, or (b) the elements contained in polypeptide chain VA include the first VL and the first CL, the elements contained in polypeptide chain VB include the first VH, the first heavy chain CH1 region, and the second Fc monomer, the elements contained in polypeptide chain VC include the second VL, the second heavy chain CH1 hinge region, and the first Fc monomer, and polypeptide chain VD includes the second VH and the second CL.

[0229] In a particular embodiment, (a) elements included in polypeptide chain VA include a first VL and a first CL from the N-terminus to the C-terminus, elements included in polypeptide chain VB include a first VH, a first heavy chain CH1 region, and a first Fc monomer from the N-terminus to the C-terminus, elements included in polypeptide chain VC include a second VL, a second heavy chain CH1 hinge region, and a second Fc monomer from the N-terminus to the C-terminus, and / or elements included in polypeptide chain VD include a second VH and a second CL from the N-terminus to the C-terminus (b) elements of polypeptide chain VA include a first VL and a first CL from the N-terminus to the C-terminus, elements of polypeptide chain VB include a first VH, a first heavy chain CH1 region, and a second Fc monomer from the N-terminus to the C-terminus, elements of polypeptide chain VC include a second VL, a second heavy chain CH1 hinge region, and a first Fc monomer from the N-terminus to the C-terminus, and / or elements of polypeptide chain VD include a second VH and a second CL from the N-terminus to the C-terminus.

[0230] In a particular embodiment, adjacent elements in polypeptide chain VA may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain VB may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain VC may or may not be linked to each other by peptide linkers, and / or adjacent elements in polypeptide chain VD may or may not be linked to each other by peptide linkers.

[0231] In certain embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker). In certain embodiments, each peptide linker independently contains 3 to 55 amino acid residues (e.g., 3 to 10, 10 to 25, 10 to 20, 10 to 17, 10 to 15, 10 to 25, 10 to 35, 10 to 45, 10 to 55). In certain embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S) residues. In certain embodiments, each peptide linker independently contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57), which is (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) may be represented by these formulas. In certain embodiments, the peptide linker each independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0232] In a particular embodiment, the first and second CLs include the amino acid sequence shown in SEQ ID NO: 53, and / or the first and second heavy chain CH1 regions include the amino acid sequence shown in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region includes the amino acid sequence shown in SEQ ID NO: 62.

[0233] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, and the first VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, the first VL includes the amino acid sequence shown in SEQ ID NO: 15, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17.

[0234] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, and the first VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, the first VL includes the amino acid sequence shown in SEQ ID NO: 17, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15.

[0235] In a particular embodiment, 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 a particular embodiment, 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.

[0236] In a particular embodiment, 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 a particular embodiment, 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.

[0237] In a particular embodiment, CrossMab Fab-FabA bispecific antibody in the form of (a) a polypeptide chain VA, polypeptide chain VB, polypeptide chain VC, and polypeptide chain VD, wherein (a) the elements of polypeptide chain VA include the first VL and the first CL, the elements of polypeptide chain VB include the first VH, the first heavy chain CH1 region, and the first Fc monomer, the elements of polypeptide chain VC include the second VL, the second CL, the second heavy chain CH1 hinge region, and the second Fc monomer, and polypeptide chain VD includes the second VH and the second heavy chain CH1 region, or (b) the elements of polypeptide chain VA include the first VL and the first CL, the elements of polypeptide chain VB include the first VH, the first heavy chain CH1 region, and the second Fc monomer, the elements of polypeptide chain VC include the second VL, the second CL, the second heavy chain CH1 hinge region, and the first Fc monomer, and polypeptide chain VD includes the second VH and the second heavy chain CH1 region.

[0238] In a particular embodiment, (a) elements included in polypeptide chain VA include a first VL and a first CL from the N-terminus to the C-terminus, elements included in polypeptide chain VB include a first VH, a first heavy chain CH1 region, and a first Fc monomer from the N-terminus to the C-terminus, elements included in polypeptide chain VC include a second VL, a second CL, a second heavy chain CH1 hinge region, and a second Fc monomer from the N-terminus to the C-terminus, and / or elements included in polypeptide chain VD include a second VH and a second heavy chain CH1 region from the N-terminus to the C-terminus (b) elements included in polypeptide chain VA include a first VL and a first CL from the N-terminus to the C-terminus, elements included in polypeptide chain VB include a first VH, a first heavy chain CH1 region, and a second Fc monomer from the N-terminus to the C-terminus, elements included in polypeptide chain VC include a second VL, a second CL, a second heavy chain CH1 hinge region, and a first Fc monomer from the N-terminus to the C-terminus, and / or elements included in polypeptide chain VD include a second VH and a second heavy chain CH1 region from the N-terminus to the C-terminus.

[0239] In a particular embodiment, adjacent elements in polypeptide chain VA may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain VB may or may not be linked to each other by peptide linkers, adjacent elements in polypeptide chain VC may or may not be linked to each other by peptide linkers, and / or adjacent elements in polypeptide chain VD may or may not be linked to each other by peptide linkers.

[0240] In certain embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker). In certain embodiments, each peptide linker independently contains 3 to 55 amino acid residues (e.g., 3 to 10, 10 to 25, 10 to 20, 10 to 17, 10 to 15, 10 to 25, 10 to 35, 10 to 45, 10 to 55). In certain embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S) residues. In certain embodiments, each peptide linker independently contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57), which is (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) may be represented by these formulas. In certain embodiments, the peptide linker each independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0241] In a particular embodiment, the first and second CLs include the amino acid sequence shown in SEQ ID NO: 53, and / or the first and second heavy chain CH1 regions include the amino acid sequence shown in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region includes the amino acid sequence shown in SEQ ID NO: 62.

[0242] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, and the first VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 16, the first VL includes the amino acid sequence shown in SEQ ID NO: 15, the second VH includes the amino acid sequence shown in SEQ ID NO: 18, and the second VL includes the amino acid sequence shown in SEQ ID NO: 17.

[0243] In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, and the first VL includes the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the first VH includes the amino acid sequence shown in SEQ ID NO: 18, the first VL includes the amino acid sequence shown in SEQ ID NO: 17, the second VH includes the amino acid sequence shown in SEQ ID NO: 16, and the second VL includes the amino acid sequence shown in SEQ ID NO: 15.

[0244] In a particular embodiment, 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 a particular embodiment, 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.

[0245] In a particular embodiment, 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 a particular embodiment, 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.

[0246] scFv-scFv structure In a particular embodiment, both the first antigen-binding domain and the second antigen-binding domain are scFv.

[0247] In a particular embodiment, the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B, wherein (a) the elements of polypeptide chain VII-A include a first VL, a first VH, a first heavy chain CH1 hinge region, and a first Fc monomer, and the elements of polypeptide chain VII-B include a second VL, a second VH, a second heavy chain CH1 hinge region, and a second Fc monomer, or (b) the elements of polypeptide chain VII-A include a first VL, a first VH, a first heavy chain CH1 hinge region, and a second Fc monomer, and the elements of polypeptide chain VII-B include a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc monomer.

[0248] In a particular embodiment, (a) elements included in polypeptide chain VII-A include (i) a first VL, first VH, first heavy chain CH1 hinge region, and first Fc monomer from the N-terminus to the C-terminus, or (ii) a first VH, first VL, first heavy chain CH1 hinge region, and first Fc monomer, and / or elements included in polypeptide chain VII-B include (i) a second VL, second VH, second heavy chain CH1 hinge region, and second Fc monomer from the N-terminus to the C-terminus, or (ii) a second VH, second VL, second heavy chain CH1 hinge region, and second Fc monomer (b) elements included in polypeptide chain VII-A include (i) the first VL, first VH, first heavy chain CH1 hinge region, and second Fc monomer from the N-terminus to the C-terminus, or (ii) the first VH, first VL, first heavy chain CH1 hinge region, and second Fc monomer, and / or elements included in polypeptide chain VII-B include (i) the second VL, second VH, second heavy chain CH1 hinge region, and first Fc monomer from the N-terminus to the C-terminus, or (ii) the second VH, second VL, second heavy chain CH1 hinge region, and first Fc monomer.

[0249] In certain embodiments, adjacent elements in polypeptide chain VII-A are optionally linked to each other by peptide linkers or not, and / or adjacent elements in polypeptide chain VII-B are optionally linked to each other by peptide linkers or not.

[0250] In certain embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linker or flexible peptide linker). In certain embodiments, each peptide linker independently contains 3 to 55 amino acid residues (e.g., 3 to 10, 10 to 25, 10 to 20, 10 to 17, 10 to 15, 10 to 25, 10 to 35, 10 to 45, 10 to 55). In certain embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S) residues. In certain embodiments, each peptide linker independently contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of the peptide linker unit GGGGS (SEQ ID NO: 57), which is (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) may be represented by these formulas. In certain embodiments, the peptide linker each independently comprises the amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

[0251] In a particular embodiment, the first and second double-chain CH1 hinge regions include the amino acid sequence shown in SEQ ID NO: 62.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] In certain embodiments, polypeptide chain VII-A comprises the amino acid sequence set forth in SEQ ID NO: 14 and / or polypeptide chain VII-B comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0257] In certain exemplary embodiments, the bispecific antibody comprising an ADC comprises:

[0258] polypeptide chain I-A comprising the amino acid sequence set forth in SEQ ID NO: 1, polypeptide chain I-B comprising the amino acid sequence set forth in SEQ ID NO: 2, and polypeptide chain I-C comprising the amino acid sequence set forth in SEQ ID NO: 3,

[0259] polypeptide chain I-A comprising the amino acid sequence set forth in SEQ ID NO: 1, polypeptide chain I-B comprising the amino acid sequence set forth in SEQ ID NO: 9, and polypeptide chain I-C comprising the amino acid sequence set forth in SEQ ID NO: 10,

[0260] polypeptide chain II-A comprising the amino acid sequence set forth in SEQ ID NO: 4, polypeptide chain II-B comprising the amino acid sequence set forth in SEQ ID NO: 5, and polypeptide chain II-C comprising the amino acid sequence set forth in SEQ ID NO: 6,

[0261] polypeptide chain II-A comprising the amino acid sequence set forth in SEQ ID NO: 4, polypeptide chain II-B comprising the amino acid sequence set forth in SEQ ID NO: 7, and polypeptide chain II-C comprising the amino acid sequence set forth in SEQ ID NO: 8,

[0262] polypeptide chain IV-A comprising the amino acid sequence set forth in SEQ ID NO: 4, polypeptide chain IV-B comprising the amino acid sequence set forth in SEQ ID NO: 7, and polypeptide chain IV-C comprising the amino acid sequence set forth in SEQ ID NO: 11,

[0263] polypeptide chain V-A comprising the amino acid sequence set forth in SEQ ID NO: 1, polypeptide chain V-B comprising the amino acid sequence set forth in SEQ ID NO: 9, polypeptide chain V-C comprising the amino acid sequence set forth in SEQ ID NO: 12, and polypeptide chain V-D comprising the amino acid sequence set forth in SEQ ID NO: 13, or

[0264] Polypeptide chain VII-A containing the amino acid sequence shown in SEQ ID NO: 14, and polypeptide chain VII-B containing the amino acid sequence shown in SEQ ID NO: 8.

[0265] In certain embodiments, the bispecific antibody exhibits an enhanced tumor-suppressing effect compared to a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody, wherein the CDR amino acid sequence of the monospecific anti-c-MET CDR is identical to the amino acid sequence of the CDR sequence of the first antigen-binding domain, and the CDR amino acid sequence of the monospecific anti-EGFR antibody CDR is identical to the CDR amino acid sequence of the CDR sequence of the second antigen-binding domain.

[0266] In certain embodiments, the tumor suppressor effect includes inhibition of EGFR and c-MET signaling, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0267] Methods for producing polynucleotides, vectors, and antibodies In another embodiment, 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.

[0268] In accordance with codon degeneracy in the art, in some embodiments, nucleotide sequences may be substituted according to codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0269] In another embodiment, the present application provides a vector comprising an isolated nucleic acid molecule or a set of nucleic acid molecules as described above.

[0270] In certain embodiments, the vector of the present invention may be, for example, a plasmid, cosmid, phage, or lentivirus. In certain embodiments, the vector may express the bispecific antibody of the present invention in a subject (e.g., a mammal, e.g., a human).

[0271] For example, if the bispecific antibody of the present invention contains two peptide chains, the vector contains 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 the first nucleotide sequence and the second nucleotide sequence reside on the same or different vectors. If the first nucleotide sequence and the second nucleotide sequence reside in different vectors, the vector of the present invention contains a first vector containing the first nucleotide sequence and a second vector containing the second nucleotide sequence.

[0272] For example, if the bispecific antibody of the present invention contains three peptide chains, the vector includes 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, second, and third nucleotide sequences are present in the same vector or on different vectors. If the first, second, and third nucleotide sequences are present on different vectors, the vector of the present invention includes a first vector containing the first nucleotide sequence, a second vector containing the second nucleotide sequence, and a third vector containing the third nucleotide sequence.

[0273] For example, if the bispecific antibody of the present invention contains four peptide chains, the vector includes 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, the third nucleotide sequence, and the fourth nucleotide sequence are present on the same or different vectors. If the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence, and the fourth nucleotide sequence are present in different vectors, the vector of the present invention includes a first vector containing the first nucleotide sequence, a second vector containing the second nucleotide sequence, a third vector containing the third nucleotide sequence, and a fourth vector containing the fourth nucleotide sequence.

[0274] In certain embodiments, the nucleotide sequences encoding different peptide chains of a bispecific antibody are located on different vector molecules. In certain embodiments, the vector is a cloning vector or an expression vector.

[0275] In another embodiment, the present 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. The host cell may be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (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 present invention is a mammalian cell such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0276] The bispecific antibodies of the present invention can be prepared by various methods known in the art, for example, by genetic engineering and recombinant techniques. For example, a DNA molecule encoding a peptide chain of the bispecific antibody of the present invention can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cell is cultured under specific conditions to express the bispecific antibody of the present invention.

[0277] In another aspect, the present application provides a method for preparing the bispecific antibody described above, the method comprising culturing the host cell described above under conditions that allow the expression of the bispecific antibody and recovering the bispecific antibody from the culture of the host cell.

[0278] Therapeutic uses The bispecific antibodies of the present invention may be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein). Usually, the derivatization of antibodies does not adversely affect their binding to EGFR and c-MET. Thus, the bispecific antibodies of the present invention are also intended to include such derivatized forms. For example, the bispecific antibodies of the present invention may be functionally linked (by chemical bonding, gene fusion, non-covalent bonding or another method) to one or more other molecular moieties, such as another antibody, a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., avidin or polyhistidine tag) that can mediate the binding of the bispecific antibody to another molecule.

[0279] Thus, in one aspect, the present application provides a complex comprising a bispecific antibody as described above and a complexing moiety linked thereto.

[0280] In certain embodiments, the complexing moiety is selected from therapeutic agents (e.g., cytotoxic agents, cytokines, toxins, or radionuclides).

[0281] In certain embodiments, the complexed portion is selected from substances that can improve the biological properties of the bispecific antibody (such as increasing its serum half-life), such as chemical groups, such as polyethylene glycol (PEG), a formazan group or an ethyl group, or sugars.

[0282] In another embodiment, the present application provides a pharmaceutical composition comprising a bispecific antibody as described above, or an isolated nucleic acid molecule or set of nucleic acid molecules as described above, or a vector as described above, or a host cell as described above, or a complex as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0283] In certain embodiments, the pharmaceutical composition further comprises additional pharmaceutically active agents.

[0284] In certain embodiments, the additional pharmaceutically active agent is a drug having antitumor activity.

[0285] In certain embodiments, additional pharmacoactive agents are 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.

[0286] In certain embodiments, the bispecific antibody and additional pharmaceutically active agents are provided as separate components or as mixed components.

[0287] In certain embodiments, the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, or complex in the pharmaceutical composition of the present invention is sufficient to exert a tumor suppressor effect (for example, a tumor suppressor effect superior to that of a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody, wherein the amino acid sequence of the CDR of the monospecific anti-c-MET antibody is identical to the amino acid sequence of the CDR of the first antigen-binding domain, and the amino acid sequence of the CDR of the monospecific anti-EGFR antibody is identical to the amino acid sequence of the CDR of the second antigen-binding domain) (for example, in a subject).

[0288] In certain embodiments, the tumor suppressor effect includes inhibition of EGFR and c-MET signaling, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0289] In another aspect, the present application provides the use of, in the preparation of a pharmaceutical, a bispecific antibody as described above, or an isolated nucleic acid molecule or set of nucleic acid molecules as described above, or a vector as described above, or a host cell as described above, or a complex as described above, or a pharmaceutical composition as described above, the pharmaceutical being used for the prevention and / or treatment of diseases related to c-MET and / or EGFR, and / or as an adjuvant in the treatment thereof, and / or for the inhibition of c-MET and / or EGFR activity in vitro or in a subject.

[0290] In certain embodiments, the disease associated with c-MET and / or EGFR is cancer. In certain embodiments, cancer is associated with EGFR-activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET-activating mutations and / or c-MET gene amplification.

[0291] In certain embodiments, 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 cavity cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0292] In certain embodiments, a bispecific antibody, an isolated nucleic acid molecule or set of nucleic acid molecules, a vector, a host cell, a complex, or a pharmaceutical composition is administered in combination with another pharmaceutical active agent, for example, simultaneously, separately, or sequentially.

[0293] In certain embodiments, the additional pharmaceutically active agent is a drug having antitumor activity.

[0294] In certain embodiments, additional pharmacoactive agents are 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.

[0295] In certain embodiments, the subjects are resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.

[0296] In another embodiment, the present application provides a method for inhibiting intracellular c-MET and / or EGFR activity, the method comprising contacting cells with a bispecific antibody as described above, or an 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 complex as described above, or the pharmaceutical composition as described above.

[0297] In certain embodiments, the cells are c-MET and / or EGFR-expressing cells, such as tumor cells.

[0298] In another aspect, the application provides a method for preventing and / or treating c-MET and / or EGFR-related diseases in a subject, and / or a method for acting as an adjuvant in the treatment thereof, the method comprising administering to a subject in need thereof an effective amount of the aforementioned bispecific antibody, or the aforementioned isolated nucleic acid molecule or set of nucleic acid molecules, or the aforementioned vector, or the aforementioned host cell, or the aforementioned complex, or a pharmaceutical composition as described above.

[0299] In certain embodiments, the disease associated with c-MET and / or EGFR is cancer. In certain embodiments, cancer is associated with EGFR-activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET-activating mutations and / or c-MET gene amplification.

[0300] In certain embodiments, 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 cavity cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0301] In certain embodiments, the method further comprises administering a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0302] In certain embodiments, the second therapy may be applied simultaneously with, separately from, or sequentially with the method described above.

[0303] In certain embodiments, the subjects are resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.

[0304] The bispecific antibodies and pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended method of administration and therapeutic use. The pharmaceutical compositions of the present invention must be sterile and stable under manufacturing and storage conditions. The preferred dosage form is injection. Such an injection may be a sterile solution for injection. For example, a sterile solution for injection can be prepared by mixing the required dose of the bispecific antibodies or pharmaceutical compositions of the present invention in a suitable solvent, optionally including, but not limited to, other desired components (pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, sterile solutions for injection may be prepared as sterile lyophilized powders (e.g., by vacuum drying or lyophilization) to facilitate storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0305] Furthermore, the bispecific antibody of the present invention may be presented as a pharmaceutical composition in a unit dosage form to facilitate administration.

[0306] The bispecific antibodies and pharmaceutical compositions of the present invention may be administered by any preferred method known in the art, including, but not limited to, oral, buccal, sublingual, ophthalmic, topical, parenteral, rectal, intrathecal, intracytoplasmic endoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or infusion), or nasal routes. However, for many therapeutic applications, the preferred route / method of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some preferred embodiments, the bispecific antibodies and pharmaceutical compositions of the present invention are administered by intravenous or injectable injection.

[0307] The pharmaceutical composition of the present invention may contain a "therapeutic effective dose" or a "preventive effective dose" of the bispecific antibody of the present invention. A "preventive effective dose" means an amount sufficient to prevent, inhibit, or delay the onset of the disease. A "therapeutic effective dose" means an amount sufficient to cure, or at least partially prevent, the disease and its complications in a patient already suffering from the disease. The therapeutic effective dose of the bispecific antibody of the present invention may vary depending on the following factors: the severity of the disease being treated, the overall state of the patient's immune system, the patient's general condition (e.g., age, weight, and sex), the method of drug administration, and any other treatments administered concurrently.

[0308] In the present invention, the drug regimen may be adjusted to obtain the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single dose may be administered, multiple doses may be administered over time, or the dose may be reduced or increased in proportion to the urgency of the treatment situation.

[0309] In this invention, the subject may be a mammal such as a human.

[0310] The bispecific antibodies described herein may also be used to assay c-MET and / or EGFR protein levels in biological samples using classical immunohistochemical methods known to those skilled in the art, including immunoassays such as 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), luminescence labels such as luminol, and fluorescence labels such as fluorescein and rhodamine, as well as biotin. Such labels may be used to label the bispecific antibodies described herein. Alternatively, a second antibody that recognizes the bispecific antibodies described herein may be labeled and used in combination with the bispecific antibodies to detect c-MET and / or EGFR protein levels. Accordingly, in certain embodiments, the Disclosure relates to the use of the bispecific antibodies of the Disclosure for the in vitro detection of c-MET and / or EGFR proteins in a biological sample. In further embodiments, the Disclosure relates to the use of the bispecific antibodies of the Disclosure for in vitro assay and / or detection of c-MET and / or EGFR protein levels in a biological sample, wherein the bispecific antibodies are optionally conjugated to a radionuclide or a detectable label and / or have a label as described herein, and / or immunohistochemistry is used.

[0311] Assays relating to the expression levels of c-MET and / or EGFR proteins are intended to involve qualitatively or quantitatively measuring or estimating the levels of c-MET and / or EGFR proteins in a first biological sample, either directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing them to disease-related protein levels in a second biological sample). The c-MET and / or EGFR polypeptide expression levels in the first biological sample may be measured or estimated and compared to a standard c-MET and / or EGFR protein level, which is determined, for example, by taking a second biological sample from a non-disabled individual or by averaging levels across a population of non-disabled individuals. As will be understood in the art, once a “standard” c-MET and / or EGFR polypeptide level is determined, it may be repeatedly used as a standard for comparison. Accordingly, in further embodiments, the disclosure relates to an in vitro method for assaying and / or detecting c-MET and / or EGFR protein levels in a biological sample, the method comprising qualitatively or quantitatively measuring or estimating the levels of c-MET and / or EGFR protein in a biological sample by immunohistochemistry.

[0312] As used herein, the term “biological sample” refers to any biological sample obtained from an subject, cell line, tissue, or other cell source that may express c-MET and / or EGFR. Methods for obtaining tissue biopsies and bodily fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Examples of biological samples include peripheral blood mononuclear cells (PBMCs).

[0313] The bispecific antibodies described herein may be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications, which are well known and standard to those skilled in the art and are based on this description. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro determination and evaluation of cancer (e.g., tumorigenesis) or other disorders may be used to evaluate patient samples, including patients who have been found to have or are suspected to have cancer or other disorders, or to predict, diagnose, and monitor expected or desired therapeutic responses. Determination and evaluation of cancer or other disorders is also useful in determining a patient's suitability for clinical trials of drugs or for the administration of specific chemotherapy agents, radiotherapy agents, or antibodies (including combinations thereof) to different drugs or antibodies. This type of prognostic and diagnostic monitoring and determination has already been performed using antibodies against the HER2 protein of breast cancer (HercepTest®, Dako), and this assay is also used to evaluate patients for antibody therapy using Herceptin®. In vivo applications include specific cell therapies and radioimaging of immune system modulation and immune responses. Accordingly, in certain embodiments, the Disclosure relates to the bispecific antibodies and / or pharmaceutical compositions of the Disclosure for use as diagnostic agents. In certain embodiments, the Disclosure relates to the bispecific antibodies and / or pharmaceutical compositions of the Disclosure for use in predictive, diagnostic, and / or monitoring methods for subjects having or suspected to have cancer or another disorder, and / or expected or desired therapeutic response. In other embodiments, the Disclosure relates to the use of the bispecific antibodies of the Disclosure for predictive, diagnostic, and / or monitoring for subjects having or suspected to have cancer or another disorder, and / or expected or desired therapeutic response, by assaying and / or detecting c-MET and / or EGFR protein levels in a biological sample of a subject in vitro.

[0314] In certain embodiments, the bispecific antibodies of this disclosure may be used for immunohistochemistry of biopsy specimens. In certain embodiments, the method is an in vitro method. In other embodiments, the bispecific antibodies of this disclosure may be used to detect levels of c-MET and / or EGFR, or levels of cells containing c-MET and / or EGFR on the cell membrane surface, the levels of which may be associated with certain disease symptoms. The bispecific antibodies described herein may have detectable or functional labels and / or be conjugated to radionuclides or detectable labels. Where fluorescent labels are used, currently available microscopy and fluorescence-activated cell sorting (FACS) or a combination of both methods and procedures known in the art may be used to identify and quantify the specific binding member. The bispecific antibodies described herein may have fluorescent labels or be conjugated to them. Exemplary fluorescent labels include, for example, responsive and conjugated probes, such as aminocoumarin, fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes. The bispecific antibodies of this disclosure may have radiolabeling or radionuclides, 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, or may be compounded with them. If radiolabeling is used, currently available counting procedures known in the art may be used to identify and quantify the specific binding of the bispecific antibodies to c-MET and / or EGFR. If the labeling is an enzyme, detection may be achieved by any of the currently available colorimetric, spectrophotometric, fluorescence spectrophotometric, amperometric, or gas quantification techniques known in the art. This can be achieved by contacting a sample or control sample with a 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 control. Given the specific binding of the bispecific antibodies described herein to c-MET and EGFR, the bispecific antibodies may be used to specifically detect c-MET and / or EGFR. The bispecific antibodies described herein may also be used to purify c-MET and / or EGFR via immunoaffinity purification. This specification also includes assay systems that may be prepared in the form of test kits, kits, or component kits for quantitative analysis of the amount of c-MET and / or EGFR ligand complexes, for example. These systems, test kits, kits, or component kits may include labeled components, e.g., labeled antibodies, and one or more additional immunochemical reagents.

[0315] kit Kits comprising one or more bispecific antibodies, or their pharmaceutical compositions or complexes, as described herein are also provided. In certain embodiments, the herein provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of the pharmaceutical compositions described herein, such as one or more bispecific antibodies provided herein. In certain embodiments, the kit contains the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein. In certain embodiments, the kit may contain T-cell mitogens, such as phytohemagglutinin (PHA) and / or phorbol myristate acetate (PMA), or TCR complex-stimulating antibodies, such as anti-CD3 antibodies and anti-CD28 antibodies. Optionally, such containers may be accompanied by a notice in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceutical or biological products, the notice reflecting the agency's approval for manufacture, use or sale for human administration.

[0316] Kits that can be used in the above-described method are also provided. In certain embodiments, the kit comprises a bispecific antibody, preferably a purified antibody, as described herein in one or more containers. In certain embodiments, the kit described herein contains substantially isolated c-MET and / or EGFR antigen as a control. In another particular embodiment, the kit described herein further comprises a control antibody that does not react with c-MET or EGFR antigen. In yet another particular embodiment, the kit described herein contains one or more elements for detecting the binding of an antibody to c-MET and / or EGFR antigen (for example, the antibody may be conjugated to a detectable substrate, e.g., a fluorescent compound, an enzyme substrate, a radioactive compound or a luminescent compound, or a second antibody that recognizes a first antibody may be conjugated to a detectable substrate). In certain embodiments, the kit provided herein may include a c-MET and / or EGFR antigen that is recombinantly produced or chemically synthesized. The antigen provided in the kit may also be conjugated to a solid support. In more particular embodiments, the detection means of the kit described above comprises a solid support to which the c-MET and / or EGFR antigen is conjugated. Such kits may also include unbound reporter-labeled anti-human antibodies or anti-mouse / rat antibodies. In this embodiment, the binding of a bispecific antibody to an antigen may be detected by the binding of the reporter-labeled antibody. In certain embodiments, the disclosure relates to the use of the kits of the disclosure for in vitro assays and / or detection of c-MET and / or EGFR antigens in biological samples.

[0317] Specific embodiments of the present invention further include: Embodiment 1. A bispecific antibody or a bispecific antigen-binding fragment comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR, wherein the bispecific antibody or the bispecific antigen-binding fragment optionally binds to c-MET and EGFR with higher affinity compared to other known antibodies.

[0318] Embodiment 2. The first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), both of which form a domain capable of specifically binding to c-MET, and the second antigen-binding domain comprises a second VL and a second VH, both of which form a specific domain capable of binding to EGFR.

[0319] The first VL is, i. Complementarity Determination Region (CDR)-L1 containing the amino acid sequence shown in SEQ ID NO: 34, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38 (where the CDR is defined by the Kabat numbering system), ii. CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38 (where the CDR is defined by the Chothia numbering system), iii. CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38 (where the CDR is defined by the Abm numbering system), or iv. A CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 35, a CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 37, and a CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38 (where the CDR is defined by the IMGT numbering system),

[0320] and / or

[0321] The aforementioned first VH is, i. CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 47 (where the CDR is defined by the Kabat numbering system), ii. CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 40, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 47 (where the CDR is defined by the Chothia numbering system), iii. CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 42, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 46, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 47 (where the CDR is defined by the Abm numbering system), or iv. A bispecific antibody or bispecific antigen-binding fragment according to Embodiment 1, comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 48 (where the CDR is defined by the IMGT numbering system).

[0322] Embodiment 3. The second VL is i. CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 23 (where the CDR is defined by the Kabat numbering system), ii. CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 23 (where the CDR is defined by the Chothia numbering system), iii. CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 23 (where the CDR is defined by the Abm numbering system), or

[0323] A CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 20, a CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 22, and a CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 23 (where the CDR is defined by the IMGT numbering system), and / or

[0324] The aforementioned 2VH is, i. CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 24, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 28, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 32 (where the CDR is defined by the Kabat numbering system), ii. CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 25, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 29, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 32 (where the CDR is defined by the Chothia numbering system), iii. CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 27, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 31, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 32 (where the CDR is defined by the Abm numbering system), or iv. A bispecific antibody or bispecific antigen-binding fragment according to Embodiment 2, comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 26, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 30, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 33 (where the CDR is defined by the IMGT numbering system).

[0325] Embodiment 4. A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 2 to 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.

[0326] Embodiment 5. A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 2 to 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.

[0327] Embodiment 6. The bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 1 to 5, wherein the bispecific antibody or its antigen-binding fragment further comprises an Fc dimer containing first and second Fc monomers, and the first and second Fc monomers each independently include one or more amino acid modifications that promote heterodimerization of the first and second Fc monomers.

[0328] Embodiment 7. The bispecific antibody or bispecific antigen-binding fragment according to Embodiment 6, wherein the Fc comprises a first Fc monomer containing an amino acid modification capable of forming a knob structure and a second Fc monomer containing an amino acid modification capable of forming a hole structure, and the hole structure can pair with the knob structure to form a heterodimeric Fc dimer.

[0329] Embodiment 8. (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, the bispecific antibody or bispecific antigen-binding fragment according to Embodiment 6 or 7.

[0330] Embodiment 9. A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 6 to 8, wherein the first antigen-binding domain and the first Fc monomer are linked and the second antigen-binding domain is linked to the second Fc monomer, or the first antigen-binding domain and the second Fc monomer are linked and the second antigen-binding domain is linked to the first Fc monomer.

[0331] Embodiment 10. A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 1 to 9, wherein the first antigen-binding domain is Fab and the second antigen-binding domain is scFv.

[0332] Embodiment 11. The bispecific antibody comprises polypeptide chain IA, polypeptide chain IB, and polypeptide chain IC. i. The elements contained in polypeptide chain IA include the first VL and CL from the N-terminus to the C-terminus, the elements contained in polypeptide chain IB include the first VH, the heavy chain CH1 region, and the first Fc monomer from the N-terminus to the C-terminus, and / or the elements contained in polypeptide chain IC include (i) the second VL, the second VH, and the second Fc monomer from the N-terminus to the C-terminus, or (ii) the second VH, the second VL, and the second Fc monomer, or ii. A bispecific antibody or bispecific antigen-binding fragment according to Embodiment 10, wherein the elements contained in polypeptide chain IA include the first VL and CL from the N-terminus to the C-terminus, the elements contained in polypeptide chain IB include the first VH, the heavy chain CH1 region, and the second Fc monomer from the N-terminus to the C-terminus, and / or the elements contained in polypeptide chain IC include (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 from the N-terminus to the C-terminus.

[0333] Embodiment 12. (a) The polypeptide chain IA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB comprises the amino acid sequence shown in SEQ ID NO: 2, and / or the polypeptide chain IC comprises the amino acid sequence shown in SEQ ID NO: 3, or (b) The polypeptide chain IA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB comprises the amino acid sequence shown in SEQ ID NO: 9, and / or the polypeptide chain IC comprises the amino acid sequence shown in SEQ ID NO: 10, according to Embodiment 10 or 11.

[0334] Embodiment 13. A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 1 to 9, wherein the first antigen-binding domain is scFv and the second antigen-binding domain is Fab.

[0335] Embodiment 14.i. The bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C, wherein the elements of polypeptide chain II-A include the 2VL and CL from the N-terminus to the C-terminus, the elements of polypeptide chain II-B include the 2VH, heavy chain CH1 region, and 1Fc monomer from the N-terminus to the C-terminus, and / or the elements of polypeptide chain II-C include from the N-terminus to the C-terminus ii. The first VL, the first VH, and the second Fc monomer, or iii. comprising 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 of polypeptide chain II-A include the 2VL and CL from the N-terminus to the C-terminus, the elements of polypeptide chain II-B include the 2VH, heavy chain CH1 region, and 2Fc monomer from the N-terminus to the C-terminus, and / or the elements of polypeptide chain II-C include from the N-terminus to the C-terminus v. The first VL, the first VH, and the first Fc monomer, or vi. The bispecific antibody or bispecific antigen-binding fragment according to Embodiment 13, comprising the first VH, the first VL, and the first Fc monomer.

[0336] Embodiment 15. (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, as described in Embodiment 13 or 14.

[0337] Embodiment 16. A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 1 to 9, wherein the first antigen-binding domain is scFab and the second antigen-binding domain is Fab.

[0338] Embodiment 17. The bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C, wherein the elements of polypeptide chain IV-A include the 2VL and CL from the N-terminus to the C-terminus, the elements of polypeptide chain IV-B include the 2VH, heavy chain CH1 region, and 2Fc monomer from the N-terminus to the C-terminus, and / or the elements of polypeptide chain IV-C include from the N-terminus to the C-terminus

[0339] The first VL, CL, the first VH, the heavy chain CH1 region, and the first Fc monomer, or

[0340] The first VH, heavy chain CH1 region, the first VL, CL, and the first Fc monomer are included, or The bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C, wherein the elements of polypeptide chain IV-A include the 2VL and CL from the N-terminus to the C-terminus, the elements of polypeptide chain IV-B include the 2VH, heavy chain CH1 region, and 2Fc monomer from the N-terminus to the C-terminus, and / or the elements of polypeptide chain IV-C include from the N-terminus to the C-terminus

[0341] The first VL, CL, the first VH, the heavy chain CH1 region, and the first Fc monomer, or

[0342] A bispecific antibody or bispecific antigen-binding fragment according to Embodiment 16, comprising the first VH, heavy chain CH1 region, the first VL, CL, and the first Fc monomer.

[0343] Embodiment 18. A bispecific antibody or bispecific antigen-binding fragment according to 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.

[0344] Embodiment 19. A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 1 to 9, wherein the first antigen-binding domain and the second antigen-binding domain are each Fab, and the second antigen-binding domain Fab includes a CrossMab form of domain swapping.

[0345] Embodiment 20. The bispecific antibody comprises polypeptide chain VA, polypeptide chain VB, polypeptide chain VC, and polypeptide chain VD.

[0346] The elements in polypeptide chain VA include the first VL and CL from the N-terminus to the C-terminus, the elements in polypeptide chain VB include the first VH, heavy chain CH1 region, and first Fc monomer from the N-terminus to the C-terminus, the elements in polypeptide chain VC include the second VH, CL, and second Fc monomer from the N-terminus to the C-terminus, and / or the elements in polypeptide chain VD include the second VL and heavy chain CH1 region from the N-terminus to the C-terminus, or

[0347] (b) The bispecific antibody comprises polypeptide chain VA, polypeptide chain VB, polypeptide chain VC, and polypeptide chain VD,

[0348] A bispecific antibody or bispecific antigen-binding fragment according to Embodiment 19, wherein the elements contained in polypeptide chain VA include the first VL and CL from the N-terminus to the C-terminus, the elements contained in polypeptide chain VB include the first VH, heavy chain CH1 region, and second Fc monomer from the N-terminus to the C-terminus, the elements contained in polypeptide chain VC include the second VH, CL, and first Fc monomer from the N-terminus to the C-terminus, and / or the elements contained in polypeptide chain VD include the second VL and heavy chain CH1 region from the N-terminus to the C-terminus.

[0349] Embodiment 21. A bispecific antibody or bispecific antigen-binding fragment according to Embodiment 19 or 20, wherein the polypeptide chain VA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain VB comprises the amino acid sequence shown in SEQ ID NO: 9, the polypeptide chain VC comprises the amino acid sequence shown in SEQ ID NO: 12, and / or the polypeptide chain VD comprises the amino acid sequence shown in SEQ ID NO: 13.

[0350] Embodiment 22. A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 1 to 9, wherein both the first antigen-binding domain and the second antigen-binding domain are scFv.

[0351] Embodiment 23. The bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B, and the elements contained in polypeptide chain VII-A are arranged from the N-terminus to the C-terminus.

[0352] The first VL, the first VH, and the first Fc monomer, or

[0353] comprising the first VH, the first VL, and the first Fc monomer, and / or

[0354] The elements contained in polypeptide chain VII-B are arranged from the N-terminus to the C-terminus. (x) The second VL, the second VH, and the second Fc monomer, or (y) comprising the second VH, the second VL, and the second Fc monomer, or

[0355] (b) The bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B, wherein polypeptide chain VII-A extends from the N-terminus to the C-terminus. The first VL, the first VH, and the second Fc monomer, or comprising the first VH, the first VL, and the second Fc monomer, and / or

[0356] The elements contained in polypeptide chain VII-B are arranged from the N-terminus to the C-terminus. (x) the second VL, the second VH, and the first Fc monomer, or (y) The bispecific antibody or bispecific antigen-binding fragment according to Embodiment 22, comprising the second VH, the second VL, and the first Fc monomer.

[0357] Embodiment 24. A bispecific antibody or bispecific antigen-binding fragment according to 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.

[0358] Embodiment 25. A bispecific antibody or bispecific antigen-binding fragment according to Embodiments 11, 14, 17, 20, or 23, wherein adjacent elements, each comprising the polypeptide chain, are optionally linked via a peptide linker or not.

[0359] Embodiment 26. The bispecific antibody or bispecific antigen-binding fragment according to Embodiment 25, wherein each peptide linker is independently the same peptide linker or a different peptide linker, and each peptide linker is selected from the group consisting of rigid peptide linkers and flexible peptide linkers.

[0360] Embodiment 27. The bispecific antibody or bispecific antigen-binding fragment according to Embodiment 26, wherein each peptide linker is independently selected from peptide linkers comprising one or more glycine (G) and / or serine (S) residues.

[0361] Embodiment 28. The bispecific antibody or bispecific antigen-binding fragment according to Embodiment 27, wherein each peptide linker independently comprises one, two, three, four, five, six, seven, eight, nine, or ten tandem copies of a peptide linker subunit containing the amino acid sequence GGGGS (SEQ ID NO: 57).

[0362] Embodiment 29. The bispecific antibody or bispecific antigen-binding fragment according to Embodiment 28, wherein each peptide linker independently comprises the amino acid sequences shown in SEQ ID NOs. 55-58 or 63-68.

[0363] Embodiment 30. A bispecific antibody or bispecific antigen-binding fragment according to Embodiments 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.

[0364] Embodiment 31. The bispecific antibody or its antigen-binding fragment is Polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 2 or 9, and / or polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 3 or 10, Polypeptide chain II-A containing the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain II-B containing the amino acid sequence shown in SEQ ID NO: 5 or 7, and / or polypeptide chain II-C containing the amino acid sequence shown in SEQ ID NO: 6 or 8. Polypeptide chain IV-A containing the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain IV-B containing the amino acid sequence shown in SEQ ID NO: 7, and / or polypeptide chain IV-C containing the amino acid sequence shown in SEQ ID NO: 11, Polypeptide chain VA containing the amino acid sequence shown in SEQ ID NO: 1, polypeptide chain VB containing the amino acid sequence shown in SEQ ID NO: 9, polypeptide chain VC containing the amino acid sequence shown in SEQ ID NO: 12, and / or polypeptide chain VD containing the amino acid sequence shown in SEQ ID NO: 13, or A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 1 to 30, comprising polypeptide chain VII-A containing the amino acid sequence shown in SEQ ID NO: 14, and / or polypeptide chain VII-B containing the amino acid sequence of SEQ ID NO: 8.

[0365] Embodiment 32. The bispecific antibody or its antigen-binding fragment is i. The polypeptide chain IA comprising the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB comprising the amino acid sequence shown in SEQ ID NO: 2, and the polypeptide chain IC comprising the amino acid sequence shown in SEQ ID NO: 3 ii. The polypeptide chain IA comprising the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB comprising the amino acid sequence shown in SEQ ID NO: 9, and the polypeptide chain IC comprising the amino acid sequence shown in SEQ ID NO: 10 iii. Polypeptide chain II-A containing the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain II-B containing the amino acid sequence shown in SEQ ID NO: 5, and polypeptide chain II-C containing the amino acid sequence shown in SEQ ID NO: 6 iv. Polypeptide chain II-A containing the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain II-B containing the amino acid sequence shown in SEQ ID NO: 7, and polypeptide chain II-C containing the amino acid sequence shown in SEQ ID NO: 8 v. Polypeptide chain IV-A containing the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain IV-B containing the amino acid sequence shown in SEQ ID NO: 7, and polypeptide chain IV-C containing the amino acid sequence shown in SEQ ID NO: 11, vi. The polypeptide chain VA containing the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain VB containing the amino acid sequence shown in SEQ ID NO: 9, the polypeptide chain VC containing the amino acid sequence shown in SEQ ID NO: 12, and the polypeptide chain VD containing the amino acid sequence shown in SEQ ID NO: 13, or vii. A bispecific antibody or bispecific antigen-binding fragment according to Embodiment 28, comprising polypeptide chain VII-A containing the amino acid sequence shown in SEQ ID NO: 14, and polypeptide chain VII-B containing the amino acid sequence shown in SEQ ID NO: 8.

[0366] Embodiment 33. The bispecific antibody or its antigen-binding fragment has an enhanced tumor inhibitory effect compared to a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody. i. The amino acid sequence of the CDR of the monospecific anti-c-MET antibody is the same as the amino acid sequence of the CDR of the first antigen-binding domain, and the amino acid sequence of the CDR of the monospecific anti-EGFR antibody is the same as the amino acid sequence of the CDR of the second antigen-binding domain, or ii. A bispecific antibody or bispecific antigen-binding fragment according to any one of Embodiments 1 to 30, wherein the amino acid sequence of the CDR of the monospecific anti-c-MET antibody is the same as the amino acid sequence of the CDR of the second antigen-binding domain, and the amino acid sequence of the CDR of the monospecific anti-EGFR antibody is the same as the amino acid sequence of the CDR of the first antigen-binding domain.

[0367] Embodiment 34. The bispecific antibody or bispecific antigen-binding fragment according to Embodiment 33, wherein the tumor inhibitory effect comprises inhibition of the EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0368] Embodiment 35. An isolated nucleic acid molecule or set of nucleic acid molecules comprising a nucleotide sequence encoding a bispecific antibody or a bispecific antigen-binding fragment described in any one of Embodiments 1 to 34.

[0369] Embodiment 36. A vector comprising an isolated nucleic acid molecule or a set of nucleic acid molecules as described in Embodiment 35.

[0370] Embodiment 37. The vector according to Embodiment 36, wherein the nucleotide sequences encoding different peptide chains of the bispecific antibody are located within different vector molecules.

[0371] Embodiment 38. The vector according to Embodiment 36 or 37, wherein the vector is a cloning vector or an expression vector.

[0372] Embodiment 39. A host cell comprising an isolated nucleic acid molecule or set of nucleic acid molecules as described in Embodiment 35, or a vector as described in any one of Embodiments 36 to 38.

[0373] Embodiment 40. A method for preparing a bispecific antibody or a bispecific antigen-binding fragment according to any one of Embodiments 1 to 34, comprising culturing the host cells according to Embodiment 39 under conditions that enable the expression of the bispecific antibody or a bispecific antigen-binding fragment, and recovering the bispecific antibody from the culture of the host cells.

[0374] Embodiment 41. A pharmaceutical composition comprising one or more bispecific antibodies or bispecific antigen-binding fragments described in any one of Embodiments 1 to 34, and one or more pharmaceutically acceptable carriers and / or excipients.

[0375] Embodiment 42. The pharmaceutical composition according to 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, chemotherapeutic agents, or any combination thereof.

[0376] Embodiment 43. Use of a bispecific antibody according to any one of Embodiments 1 to 34 or a pharmaceutical composition according to Embodiment 41 or 42 in the preparation of a pharmaceutical, wherein the pharmaceutical is used in a subject to prevent and / or treat a disease related to c-MET and / or EGFR, and / or to support said treatment, and / or to inhibit c-MET cells or c-MET and / or EGFR activity in vitro or in the body of the subject, wherein the disease related to c-MET and / or EGFR is c-MET cancer associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations and / or MET gene amplification.

[0377] Embodiment 44. The use described in 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 cavity cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, and head and neck cancer.

[0378] Embodiment 45. The use according to Embodiment 43 or 44, wherein the bispecific antibody or its bispecific antigen-binding fragment or its pharmaceutical composition and another pharmaceutically active agent are administered in combination, either simultaneously, separately, or sequentially, and the other 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, chemotherapeutic agents, or any combination thereof.

[0379] Embodiment 46. The use according to any one of Embodiments 43 to 45, wherein the pharmaceutical is used in a subject resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.

[0380] Embodiment 47. A method for inhibiting intracellular c-MET and / or EGFR activity, comprising mixing the cells with a bispecific antibody or bispecific antigen-binding fragment thereof described in any one of Embodiments 1 to 34, or a composition or pharmaceutical composition described in Embodiment 41 or 42, wherein the cells are cells expressing c-MET and / or EGFR.

[0381] Embodiment 48. A method for preventing and / or treating a disease associated with c-MET and / or EGFR in a subject, and / or an adjunct method thereof, comprising administering to a subject in need of the same an effective amount of a bispecific antibody or bispecific antigen-binding fragment thereof described in any one of Embodiments 1 to 34 or a pharmaceutical composition described in Embodiment 41 or 42, wherein the disease associated with c-MET and / or EGFR is a cancer comprising an EGFR-activating mutation, EGFR gene amplification, elevated circulating HGF levels, a c-MET-activating mutation, and / or c-MET gene amplification.

[0382] Embodiment 49. The method according to 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 cavity cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0383] Embodiment 50. The method according to Embodiment 48 or 49, further comprising administering a second therapy to the subject, wherein the second therapy is 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, and the second therapy may optionally be applied simultaneously, separately, or sequentially.

[0384] Embodiment 51. The method according to any one of Embodiments 47 to 50, wherein the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.

[0385] Embodiment 52. A bispecific antibody or bispecific antigen-binding fragment thereof according to any one of Embodiments 1 to 34 or a pharmaceutical composition according to Embodiment 41 or 42 for the prevention and / or treatment of c-MET and / or EGFR-related diseases in a subject, and / or adjunct treatment thereof, wherein the c-MET and / or EGFR-related disease is a disease comprising an EGFR-activating mutation, EGFR gene amplification, elevated circulating HGF levels, a c-MET-activating mutation, and / or c-MET gene-amplified cancer.

[0386] Embodiment 53. The bispecific antibody or bispecific antigen-binding fragment thereof or pharmaceutical composition according to 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 cavity cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0387] Embodiment 54. The method further comprises administering a second therapy to the subject, wherein the second therapy is 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, and the second therapy may optionally be applied simultaneously, separately, or sequentially, the bispecific antibody or its bispecific antigen-binding fragment or pharmaceutical composition according to Embodiment 52 or 53.

[0388] Embodiment 55. A bispecific antibody or bispecific antigen-binding fragment thereof, or pharmaceutical composition according to any one of Embodiments 52 to 54, wherein the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab. [Table 1-1] [Table 1-2]

[0389] definition In this disclosure, unless otherwise specified, the scientific and technical terms used have meanings that are generally understood by those skilled in the art. In addition, all laboratory procedure steps in cell culture, biochemistry, nucleic acid chemistry, and immunology used in this document are commonplace procedures widely used in the corresponding fields. Furthermore, for further understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0390] As used herein, the term “antibody” is used in its broadest sense to include a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule consists 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, defining the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, variable and constant regions are linked by “J” regions of about 12 or more amino acids, and the heavy chain also contains “D” regions of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists 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 domain does not directly participate in antibody-antigen binding, but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells), and to the first component of the classical complement system (C1q). The VH and VL regions are also subdivided into highly variable regions called complementarity-determining regions (CDRs), and more conserved regions called framework regions (FRs) may exist between them. Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites.The assignment of amino acids in each region or domain may follow the definitions 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.

[0391] The term “antibody” further includes embodiments in which the heavy chain constant domain contains C-terminal lysine or lacks either C-terminal lysine or C-terminal glycine-lysine dipeptide. The term further includes embodiments in which the N-terminal amino acid of the antibody variable domain undergoes cyclization to pyroglutamic acid. Thus, in compositions comprising antibodies as disclosed herein, the various species of antibodies herein may independently contain C-terminal lysine, lack C-terminal lysine, lack C-terminal glycine-lysine, and / or involve N-terminal glutamine or glutamic acid or cyclization of the N-terminal amino acid to pyroglutamic acid.

[0392] In this specification, unless otherwise explicitly indicated, the term "antibody" refers not only to the entire antibody but also to the antigen-binding fragment of the antibody.

[0393] As used herein, the terms “complementarity-determining region” or “CDR” refer to amino acid residues in the variable region of an antibody that are involved in antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, 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 Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Duebel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001)), the AHo numbering system (Honegger and The system follows either Plueckthun, A., J. Mol. Biol. 309:657-670 (2001) or the IMGT numbering system (Lefranc et al., Dev. Comparet. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, correspondences between different numbering systems are well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparet. Immunol. 27:55-77, 2003).

[0394] In the present invention, the CDR contained in the antibody or its antigen-binding fragment may be determined according to various numbering systems known in the art. In certain embodiments, the antibody or its antigen-binding fragment includes a CDR identified by the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering system.

[0395] The following general rules, disclosed to Prof. Andrew CRMartin's Group at www.bioinf.org.uk and reproduced below, may be used to define CDRs in antibody sequences that include amino acids containing the epitope of the antigen to which the antibody binds and those amino acids that specifically interact with those amino acids. Although there are rare cases in which these general constant features do not arise, Cys residues are the most conserved feature. [Table 2]

[0396] V H The entire amino acid sequence is usually numbered according to Kabat, while the three CDRs within the variable region may be defined according to any one of the numbering schemes described above. In certain embodiments, V H The numbering of amino acid positions in this specification may begin consecutively from amino acid position 1 and continue consecutively to the end of the sequence, or continue according to Kabat. Unless otherwise specified, V in this specification H and V L The amino acid positions in are defined according to sequential numbering.

[0397] The numbering of amino acid positions in the heavy chain constant domain may begin consecutively from amino acid position 1 and continue consecutively to the end of the sequence, or continue according to Eu numbering. The amino acid sequence of the IgG1 heavy chain constant domain has 330 amino acids, numbered consecutively from 1 to 330. The corresponding sequence, numbered according to Eu, begins at position number 118 and ends at position number 447. Unless otherwise specified, amino acid positions in the heavy and light chains herein are defined according to consecutive numbering.

[0398] As used herein, the terms “framework region” or “FR” residues refer to those amino acid residues within the antibody variable region other than the CDR residues defined above.

[0399] The term "antibody" is not limited to any specific method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0400] As used herein, the term “bispecific antibody” refers to an antibody that has binding specificity to two different antigens (or epitopes), and this includes two antibodies that have binding specificity to different antigens (or epitopes). Thereafter, the antigen-binding domain can bind to two different binding sites and / or target molecules. Each antigen-binding domain in a bispecific antibody may be independently selected from a full-length antibody (such as an IgG antibody) or its antigen-binding fragment (such as Fv, Fab, scFab, or scFv). In some cases, the individual antigen-binding domains are linked by a peptide linker.

[0401] As used herein, the term “Fv fragment” refers to an antibody fragment consisting of the VL and VH domains of a single arm of the 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 thought to confer antigen-binding specificity to the antibody. However, even variable regions (such as the Fd fragment, which contains only three CDRs specific to a particular antigen) can recognize and bind to an antigen, although their affinity may be lower than that of the complete binding site.

[0402] As used herein, the term "Fc fragment" refers to the fragments formed by disulfide bonds in the second and third constant regions of the primary heavy chain of an antibody, and in the second and third constant regions of the secondary heavy chain antibody fragment. The Fc fragments of an antibody have several different functions but are not involved in antigen binding.

[0403] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains, where VL and VH are linked by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Preferred prior art peptide linkers consist of a repeating GGGGS amino acid sequence (SEQ ID NO: 57) or a variant thereof. For example, a linker having amino acid sequence (GGGGS)4 (SEQ ID NO: 58) may be used, but variants thereof may also be used. In some cases, a disulfide bond may also be present between VH and VL of the scFv.

[0404] As used herein, the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL, and CH1 domains, typically comprising one peptide chain containing VL and CL, and another peptide chain containing VH and CH1. However, those skilled in the art will understand that while the Fab domains may be arranged according to the natural orientation described above, domain substitutions or exchanges that facilitate accurate VH and VL pair formation (such as domain exchanges in Crossmab form) may also be included, and the term “scFab” refers to a single polypeptide chain containing VL, VH, CL, and CH1 domains, with adjacent domains optionally linked by linkers. In a typical scFab structure, the single polypeptide chain contained in the scFab comprises (1) VL, CL, VH, and CH1 (CL and VH are typically linked by a peptide linker, e.g., a flexible peptide linker) or (2) VH, CH1, VL, and CL (CH1 and VL are typically linked by a peptide linker, e.g., a flexible peptide linker) starting from the N-terminus and extending to the C-terminus.

[0405] As used herein, the terms “monoclonal antibody” and “mAb” are synonymous and are used interchangeably to refer to a single antibody molecule from a highly homogeneous group of antibody molecules or fragments, i.e., from an identical population of antibody molecules excluding spontaneously occurring variations. mAbs are highly specific to a single epitope on an antigen. Polyclonal antibodies are related to monoclonal antibodies and typically contain at least two different antibodies, which usually recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” merely indicates that the antibody is derived from a highly homogeneous population of antibodies and should not be interpreted as requiring that the antibody be prepared by any particular method.

[0406] As used herein, the terms “bispecific antibody” or “BsAb” refer to an antibody having two distinct binding domains that enable it to simultaneously bind to two different antigens or two different epitopes of the same antigen.

[0407] As used herein, the term “CrossMab” refers to a method within the structure of a bispecific antibody that enables precise association of the light chain with its homogeneous heavy chain by exchanging the heavy and light chain domains within half of the antigen-binding fragment (Fab) of the bispecific antibody. This “crossover” maintains antigen-binding affinity but makes the two arms so different that mispairing of the light chains can no longer occur. Three possible “CrossMab” forms are as follows: CrossMab Fab (Refers to crossover or exchange at the location of the complete VH-CH1 and VL-CL domains of half of the bispecific antibody), CrossMab VH-VL (Refers to crossover or exchange at the position of only half of the VH and VL domains of a bispecific antibody), and CrossMab CH1-CL (This refers to cross-mutation or exchange of CH1 and CL domains within the Fab region of half of a bispecific antibody.) CrossMab antibodies are described or claimed in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010136172, WO2010145792 and WO2013026831. The term "CrossMab" antibody is generally recognized in the art; see, for example, Brinkmann and Kontennann, MAbs 9(2):182-212 (2017), Kontermann and Brinkmann, Drug Discovery Today 20(7):838-846 (2015), Schaefer et al., PNAS, 108 11187-1191 (2011), Kleine et al., MAbs 8(6):1010-1020 (2016), and Klein et al., MAbs 4(6):653-663 (2012).

[0408] 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 is expressed by the equilibrium dissociation constant (KD) or half-effect concentration (EC) of the interaction. 50 ) can be represented by

[0409] The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of formation and dissociation of the antigen-binding site / antigen complex. The "binding rate constant" (k a or k on ) and "dissociation rate constant" (k dis or k off Both of these can be calculated from the concentration and the actual binding and dissociation rates (see Malmqvist M, Nature, 1993, 361:186-187). Ratio k dis / k on k is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). on and k dis The value can be measured by any effective method. In certain embodiments, the dissociation constant 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 the dissociation constant.

[0410] As used herein, the term “vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it can achieve the expression of a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection, thereby enabling the expression of its genetic material components within the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, e.g., lambda phages or M13 phages, and animal viruses. 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, and polyoma vacuolar viruses (e.g., SV40). A vector may contain, but is not limited to, a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene, as well as various other elements that control gene expression. In addition, the vector may also contain an origin of replication.

[0411] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, these sequences enable the vector to replicate independently of host chromosomal DNA, and that they contain an origin of replication or an autonomous replication sequence. The term “expression vector,” as used herein, refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence functionally linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be provided by the host cell or by an in vitro expression system. Examples of expression vectors include all those known in the art, e.g., cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0412] As used herein, the term “host cell” refers to cells that may be used to introduce a vector, including, but are not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast or Aspergillus, insect cells such as S2 Drosophila or Sf9, or 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, as well as other animal cells.

[0413] As used herein, the term “identity” is used to refer to the sequence matching between two polypeptides or two nucleic acids. Molecules are identical at a given position if the positions in both sequences being compared are occupied by the same base or amino acid monomer subunit (for example, if the positions in each of two DNA molecules are occupied by adenine, or if the positions in each polypeptide are occupied by lysine). The “identity percentage” between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared, 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 6 positions match). Typically, a comparison is made when the two sequences are aligned to the greatest extent possible. Such alignments can be achieved, for example, using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be easily performed by a computer program 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)), incorporated into the ALIGN program (version 2.0), can also be used to determine the percentage of identity between two amino acid sequences by using the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4. Alternatively, the algorithm by Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)), incorporated into the GAP program of the GCG software package (available at www.gcg.com), can be used to determine the percentage of identity between two amino acid sequences by using a Blossum62 matrix or PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0414] The descriptions of the 20 conventional amino acids mentioned herein follow conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)) (which is incorporated herein by reference). In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Also in this invention, amino acids are generally represented by one- and three-letter abbreviations known in the art. For example, alanine may be represented by A or Ala.

[0415] As used herein, the term “pharmaceutically acceptable carriers and / or excipients” means carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredients, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure-maintaining agents, absorption-delaying agents, and preservatives. For example, phosphate buffers are an example of a pH adjuster. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Sodium chloride is an example of an ionic strength enhancer. Various antimicrobial and antifungal agents are included, but are not limited to, parabens, chlorobutanol, phenol, and sorbic acid. Drugs that maintain osmotic pressure include, but are not limited to, sugars and NaCl. Drugs that delay absorption include, but are not limited to, monostearic acid and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol). Stabilizers have a meaning that is generally understood by those skilled in the art, and can stabilize the desired activity of the active ingredient in a pharmaceutical product. These include, but are not limited to, monosodium glutamate, gelatin, SPGA, sugars (sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose, etc.), amino acids (glutamic acid, glycine, etc.), proteins (dried whey, albumin, or casein, etc.) or their degradation products (lactalbumin hydrolysate, etc.).

[0416] As used herein, the term “prevention” refers to a method performed to prevent or delay the onset of a disease or disorder or symptom (e.g., 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 the present invention, beneficial or desired clinical outcomes include, but are not limited to, symptom reduction, disease severity reduction, stabilization of the disease state (i.e., no further deterioration), delayed or slowed progression, improvement or mitigation of the disease state, and symptom reduction (whether partial or complete), whether detectable or undetectable. Furthermore, “treatment” may also refer to an extension of survival compared to the expected survival time if no treatment is performed.

[0417] As used herein, the term “subject” refers to mammals such as primates, including humans. In certain embodiments, the subject (e.g., humans) is at risk of having tumors or any of the diseases described above.

[0418] As used herein, the term “effective dose” means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective dose for preventing a disease (e.g., a tumor) means an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a tumor), and an effective dose for treating a disease means an amount sufficient to cure, or at least partially prevent, an existing disease or complication. Determining such an effective dose is well done within the capabilities of those skilled in the art. For example, an effective dose for a therapeutic use will depend on the severity of the disease being treated, the overall state of the patient’s own immune system, the patient’s overall condition (e.g., age, weight, and sex), the method of drug administration, and any other treatments administered concurrently.

[0419] As used herein, the term “effector function” refers to the biological activity that may be attributable to the antibody Fc region (either the native sequence Fc region or the amino acid sequence variant Fc region), and these effects, associated with the antibody, vary depending on the 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 phagocytosis (ADCP), cell surface receptors such as B cell receptors, B cell activation, cytokine secretion, and the half-life / clearance rate of antibodies and antigen-antibody complexes. Methods for altering the effector function of an antibody are known in the art, for example, by introducing mutations into the Fc region.

[0420] As used herein, the term “antibody-dependent cell-mediated cytotoxicity (ADCC)” refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on cytotoxic cells, such as natural killer (NK) cells, neutrophils, or macrophages, enabling these cytotoxic effector cells to specifically bind to antigen-binding target cells and subsequently kill them by secreting cytotoxic substances.

[0421] As used herein, the term “combination therapy” includes combining the bispecific antibody or pharmaceutical composition of the present invention with one or more additional active therapeutic agents (e.g., chemotherapeutic agents) of a second therapy or other mode of prophylaxis or treatment (e.g., radiotherapy).

[0422] In such combination therapies, various activators often have different complementary mechanisms of action, and the combination therapy may produce synergistic effects. Combination therapies include therapeutic agents that affect the immune response (e.g., enhance or activate the response) and therapeutic agents that affect tumor / cancer cells (e.g., inhibit or kill them). Combination therapies reduce the likelihood of drug-resistant cancer cells developing. Combination therapies may allow for a reduction or elimination of adverse effects associated with one or more drugs by reducing the dose of one or more drugs. Such combination therapies may have synergistic therapeutic or preventive effects for an underlying disease, disorder, or condition.

[0423] As used herein, “combination” includes therapies that may be administered together in a single formulation (i.e., a “co-formulation”), in addition to therapies that may be administered separately, for example, separately formulated for individual administration (for example, provided in a kit). In certain embodiments, the bispecific antibodies of the present invention may be administered sequentially. In other embodiments, the bispecific antibodies may be administered simultaneously. The bispecific antibodies of the present invention may be used in any combination with at least one other (active) agent.

[0424] The terms "cancer" and "tumor" are used interchangeably to refer to a broad classification of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize distally within the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as quiescent tumors or micrometastases. Cancer also includes hematological malignancies.

[0425] Sequence information A description of the sequences referred to in this application is provided in the following table. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13]

[0426] The present invention will be described in relation to the following embodiments, which are intended to illustrate, not limit, the present invention.

[0427] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in this invention are based on J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., A Laboratory Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. [Examples]

[0428] Example 1 Preparation of anti-EGFR and anti-c-MET bispecific antibodies, as well as a control antibody.

[0429] a. Construction and expression of recombinant anti-EGFR and anti-c-MET bispecific antibodies (1)Fab-scFv-Fc (KIH) bispecific antibody The Fab-scFv-Fc(KIH) bispecific antibody contains the Fab-scFv-Fc(KIH) bispecific antibody structure. A knob-in-hole (KIH) structure is used between heavy chains to prevent mismatches. A schematic diagram of the structure is shown in Figure 1A. Table 1 shows the amino acid sequences containing the anti-EGFR and anti-c-MET bispecific Fab-scFv-Fc(KIH) bispecific antibodies. [Table 4]

[0430] The above amino acid sequence was 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 synthesized 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 cloned separately into either a pKL5 vector (containing one expression cassette, designed by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd., and synthesized by GenScript; the promoter is the mouse CMV promoter, and the polyA signal sequence is the CMV polyA signal sequence) or a pKL8 expression vector (containing one expression cassette, the promoter is the mouse CMV promoter, and the polyA signal sequence is the CMV polyA signal sequence). For each plasmid pair expressing the light and heavy chains of the bispecific antibodies, as well as 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 the American Type Culture Collection (ATCC), Manasas, VA) for the expression of the bispecific antibodies, and the cells were cultured in cell culture medium. After enabling the expression of bispecific antibodies for a certain period, the bispecific antibodies were recovered from the culture medium and captured using protein A (MabSelect® SuRe, GE) chromatography. The bispecific antibodies were then purified using cation chromatography (Eshmuno® CPX, Merck KGaA) to obtain bispecific antibodies 07B, 10B, 38B, and 49B.

[0431] (2)Fab-scFab-Fc (KIH) bispecific antibody The Fab-scFab-Fc(KIH) bispecific antibody contains the Fab-scFab-Fc(KIH) bispecific antibody structure, with the KIH structure used between heavy chains to prevent mismatches. To increase stability, a pair of disulfide bonds was introduced into the scFab-Fc chain. Specifically, 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.

[0432] A schematic diagram of the structure is shown in Figure 1B. The amino acid sequences containing the anti-EGFR and anti-c-MET Fab-scFab-Fc(KIH) bispecific antibodies are shown in Table 2. [Table 5]

[0433] The above amino acid sequence was 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. Plasmid pairs 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) for the expression of bispecific antibody 41B, and the cells were cultured in cell culture medium. After enabling the expression of the bispecific antibody for a certain period, the bispecific antibody was recovered from the culture medium and captured by protein A (MabSelect® SuRe LX, GE) affinity chromatography. Next, the bispecific antibody was purified using cation chromatography (Eshmuno® CPX, Merck KGaA) to obtain bispecific antibody 41B.

[0434] (3)CrossMab bispecific antibody In this embodiment, the bispecific antibody is CrossMab CH1-CL It contains a bispecific antibody structure, with a KIH structure used between heavy chains to prevent mismatch. The structural diagram is shown in Figure 1C. Anti-EGFR and anti-c-MET CrossMab CH1-CL Table 3 shows the amino acid sequences containing bispecific antibodies. [Table 6]

[0435] The above amino acid sequence was sent to Genscript Biotechnology Co., Ltd. for codon optimization and DNA synthesis. The light and heavy chains of bispecific antibody 55B were cloned into a pKLGS expression vector from 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) for the expression of bispecific antibody 55B, and the cells were cultured in cell culture medium. After enabling the expression of the bispecific antibody for a certain period, the bispecific antibody was recovered from the culture medium and captured using protein A (MabSelect® SuRe, GE) affinity chromatography. The bispecific antibody was then purified using cation chromatography (Eshmuno® CPX, Merck KGaA) to obtain bispecific antibody 55B.

[0436] (4)scFv-Fc(KIH) bispecific antibody The scFv-Fc(KIH) bispecific antibody contains the scFv-Fc(KIH) bispecific antibody structure, with the KIH structure used between the heavy chains to prevent mismatches. A schematic diagram of the structure is shown in Figure 1D. The amino acid sequences containing the anti-EGFR and anti-c-MET scFv-Fc(KIH) bispecific antibodies are shown in Table 4. [Table 7]

[0437] The above amino acid sequence was 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 transfected into CHO-K1 cells (ATCC) for the expression of bispecific antibody 56B, and the cells were cultured in cell culture medium. After enabling bispecific antibody expression for a certain period, the bispecific antibody was recovered from the culture medium and captured using protein A (MabSelect® SuRe LX, GE) affinity chromatography. The bispecific antibody was then purified using cation chromatography (Eshmuno® CPX, Merck KGaA) to obtain bispecific antibody 56B.

[0438] 1.1 Construction and expression of monospecific recombinant anti-EGFR monoclonal antibodies and monospecific anti-c-MET monoclonal antibodies The amino acid sequences of the monospecific anti-EGFR monoclonal antibodies cetuximab, saltumumab, necitumumab, and nimotuzumab are from the Immunogenetics (IMGT®) website. The International Nonproprietary Name (INN) number for cetuximab is 7906, for saltumumab it is 8605, for necitumumab it is 9083, and for nimotuzumab it is 8545. Saltumumab is the parent monoclonal antibody of the bispecific antibody provided by this invention. The amino acid sequence of the variable region of saltumumab is the same as the amino acid sequence of the anti-EGFR portion of the bispecific antibody provided by this invention. Onartuzumab is the parent monoclonal antibody of the bispecific antibody provided by this invention. The amino acid sequence of the variable region of onartuzumab is the same as the amino acid sequence of the anti-c-MET portion of the bispecific antibody provided by this invention. MRG003 is a Phase II clinical-stage anti-EGFR monoclonal antibody-based ADC that has shown good efficacy in patients with nasopharyngeal and head and neck cancers. BA03 is an antibody to MRG003. The amino acid sequence of the BA03 monoclonal antibody is from Chinese patent CN106999606B, and the amino acid sequences of the monospecific anti-c-MET monoclonal antibodies onartuzumab and terisotuzumab are from the IMGT® website, with INN number 9368 for onartuzumab and INN number 10366 for terisotuzumab. The sequence of the CE355621 monoclonal antibody is from Chinese patent CN107207564A. To obtain monospecific monoclonal antibodies, expression plasmids encoding the heavy and light chains of the monospecific antibodies were constructed. Expression plasmids were separately transfected into CHO-K1 cells, and the transfected cells were cultured in culture medium under conditions suitable for the expression of monospecific antibodies. After allowing monospecific antibody expression for a certain period, the monospecific antibodies were recovered from the culture medium and captured using protein A (MabSelect® SuRe LX, GE) affinity chromatography.Next, the monospecific antibodies were purified using cation chromatography (Eshmuno® CPX, Merck KGaA) to obtain monospecific antibodies.

[0439] 1.2 Hydrophilic properties of bispecific antibodies The hydrophilicity of the bispecific antibodies was determined using an Agilent 1260 with a TSKgel Butyl-NPR column. Column temperature: 30°C, 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 test sample was diluted with a diluent (0.75 mol / L (NH4)2SO4) to prepare a 1.0 mg / mL solution 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 control antibody (manufactured by Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd.) was diluted with a diluent to prepare a 1.0 mg / mL system-compatible solution. Approximately 40 μg of the sample was injected and analyzed by gradient elution: mobile phase A was maintained at 95% and mobile phase B at 5% for 0-3 minutes, mobile phase B at 5%-100% for 3-40 minutes, and mobile phase A at 95% and mobile phase B at 5% for 40-45 minutes. The hydrophobicity of the test sample was calculated according to the control sample using the formula: (retention time of test sample - retention time of hydrophilic control) / (retention time of hydrophobic control - retention time of hydrophilic control). Smaller retention time and hydrophobicity value indicate better hydrophilicity of the antibody. The results are shown in Table 5. All bispecific antibodies showed good hydrophilicity. We anticipate that good hydrophilicity will be beneficial in antibody production, quality control, and conjugation with small molecules, thereby enhancing in vivo efficacy and improving pharmacokinetic properties and drug safety. [Table 8]

[0440] Example 2 Evaluation of the protein-binding activity of bispecific antibodies. 2.1 Evaluation of the binding activity of bispecific antibodies against EGFR protein Human EGFR-His protein was diluted to 1 μg / mL with a carbonate-bicarbonate buffer (CBS) coated solution and added to each well of a 96-well ELISA microtiter plate at a rate of 100 ng / well. The plate was incubated overnight at 4°C. The following day, the 96-well ELISA microtiter plate was washed once with PBST (0.05% Tween-20), and then 100 μL of 2% BSA blocking solution was added to each well of the ELISA microtiter plate. The plate was incubated at 37°C for 2 hours. The purified bispecific antibody and nimotuzumab control antibody were serially diluted with 2% BSA, starting at 10 μg / mL, in a 3-fold gradient across 11 concentrations. The blocking solution was then 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. The plate was then washed three times with PBST (0.05% Tween-20) at 320 μL per well. The plate was dried, and then 100 μL of HRP-encompassed goat anti-human IgG was added to each well. The plate was incubated at 37°C for 1 hour. The plate was then washed five times with PBST (0.05% Tween-20) at 320 μL per well. After drying the plate, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) (Huzhou Yingchuang Biotechnology Co., Ltd.) chromogenic substrate was added to each well and the reaction was allowed to proceed at room temperature for 10 minutes. The reaction was stopped by adding 50 μL of 2 M H2SO4. The plate was read by absorbance at OD450 nm using a microplate reader. The raw data was imported into GraphPad Prism 6 software for nonlinear curve fitting, and the EC of the binding of bispecific antibodies and control antibodies to EGFR-His was analyzed. 50 The result was calculated.

[0441] All of the bispecific antibodies tested successfully bound to the EGFR-His protein. Specific results are shown in Table 6. [Table 9]

[0442] 2.2 Evaluation of the binding activity of bispecific antibodies against c-MET protein c-Met protein was added to the wells of a 96-well ELISA microtiter. The plate was incubated overnight at 4°C. The following day, the 96-well ELISA microtiter plate was washed once with PBST (0.05% Tween-20), and then 100 μL of 2% BSA blocking solution was added to each well of the ELISA microtiter plate. The plate was incubated at 37°C for 2 hours. The purified bispecific antibody and terisotuzumab control antibody were serially diluted in 11 concentrations using 2% BSA, starting at 10 μg / mL in a 3-fold gradient. The blocking solution was removed, and the diluted antibody solutions were added to the ELISA microtiter plate. The plate was incubated at 37°C for 2 hours. Subsequently, the plate was washed three times with PBST (0.05% Tween-20) at 320 μL per well. The plate was dried, then 100 μL of HRP-complexed goat anti-human IgG was added to each well, and the plate was incubated at 37°C for 1 hour. The plate was then washed five times with PBST (0.05% Tween-20) at 320 μL per well. After drying the plate, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) (Huzhou Yingchuang Biotechnology Co., Ltd.) chromogenic substrate was added to each well, and the reaction was allowed to proceed at room temperature for 10 minutes. The reaction was stopped by adding 50 μL of 2 M H2SO4. The plate was read by microplate reader at OD450 nm absorbance. The raw data was imported into GraphPad Prism 6 software for nonlinear curve fitting, and the EC of the binding of bispecific antibodies and control antibodies to c-MET-His was analyzed. 50The following was calculated. All bispecific antibodies tested successfully bound to the c-MET-His protein. Specific results are shown in Table 7. [Table 10]

[0443] Example 3 Evaluation of the cell-binding activity of bispecific antibodies. Using flow cytometry (Beckman, Cytoflex model), we detected the binding activity of bispecific antibodies to human non-small cell lung cancer cells HCC827 (ATCC) exhibiting high EGFR expression and low c-MET expression, human gastric cancer cells MKN45 (Nanjing Kebai) exhibiting high c-MET expression and low EGFR expression, and human lung adenocarcinoma cells NCI-H1975 (Nanjing Kebai) exhibiting low EGFR and c-MET expression. The protocol is as follows:

[0444] Adherent cells were digested with trypsin-EDTA (0.25%, Shanghai Yuanpei) solution, and the cell density was reduced to 4.0 × 10⁶. 6 The solution was adjusted to 1 / mL. The cells were washed twice with 1% BSA and resuspended in 1% BSA solution. Then, 50 μL of the cell suspension was added to each well of a 96-well sharp-bottom plate (2 × 10 cells per well). 5 The bispecific antibody was diluted in 1% BSA, starting from a final concentration of 10 μg / mL and serially diluting 3-fold for a total of 11 concentrations. 50 μL of the diluted antibody was then added to the wells of a conical-bottom plate, and the plate was incubated at 4°C for 60 minutes. The cells were washed twice with 1% BSA, then 50 μL of the diluted secondary antibody was added to each well and mixed thoroughly, and the plate was incubated at 4°C for 30 minutes. The cells were then washed twice with 1% BSA and resuspended in 200 μL of 1% BSA for flow cytometry detection.

[0445] Data processing: PE median, EC 50The results were exported to GraphPad Prism 6 software for calculation. The results are shown in Figures 2A, 2B, 3A, 3B, 4A, and 4B and Table 8 (antibody hlgG1 is a negative control antibody). The results showed that bispecific antibodies can effectively bind to EGFR and c-MET regardless of their expression levels. In HCC827 cells, which show high EGFR expression, and MKN45 cells, which show high c-MET expression, the maximum signal values ​​of bispecific antibodies were stronger than those of EGFR and c-MET monoclonal antibodies. In other words, tumor cells may bind more to bispecific antibodies than to monoclonal antibodies, which may result in a stronger tumor suppressor effect. [Table 11]

[0446] Example 4 Detection of the internalization activity of bispecific antibodies. 4.1 Detection of internalization activity by bispecific antibody acid washing method Using flow cytometry (Beckman, Cytoflex model), we detected the internalization activity of bispecific antibodies against human non-small cell lung cancer cells HCC827, which exhibit high EGFR expression and low c-MET expression; human gastric cancer cells MKN45, which exhibit high c-MET expression and low EGFR expression; and human lung adenocarcinoma cells NCI-H1975, which exhibit low EGFR and c-MET expression.

[0447] Adherent cells were digested with Tryple® Express (Thermo) solution and counted. Cell density was measured at 4 × 10⁶ in 1% BSA. 6 The cell suspension was adjusted to the required number of cells / mL, and 50 μL of the suspension was added to the wells of a 96-well microtiter plate (2 × 10⁶ cells per well). 5(Cells / well). The bispecific antibody was diluted with 1% BSA, starting at 5 μg / mL and serially diluting fourfold for a total of eight concentrations. Then, 50 μL of the diluted bispecific antibody per well was added to the wells of a 96-well microtiter plate, and the plate was incubated at 4°C for 40 minutes. Subsequently, the wells were washed twice with 1% BSA, and 50 μL of the secondary antibody (anti-human F(ab')2 Alexa Fluor660), diluted 1:200 with 1% BSA, was added per well. The plate was incubated at 4°C for 20 minutes, then the wells were washed twice with 1% BSA, and the cells were resuspended in 150 μL of complete medium. The resuspended cells were incubated at 4°C and 3°C for 4 hours after resuspending. Subsequently, the cells were centrifuged at 500 g for 3 minutes to remove the medium, 100 μL of acidic solution (pH 2.0) was added per well, and the plate was incubated for 2 minutes. Next, 60 μL of basic solution (pH 13) was added for neutralization, followed by centrifugation to remove the supernatant fraction. The cells were then resuspended in 1% BSA for FACS analysis.

[0448] Data processing: FITC median, EC 50 The data was imported into GraphPad Prism 6 software for calculation. The results are shown in Table 9. Bispecific antibodies show good internalization activity on tumor cells and may result in a more potent tumor inhibitory effect. [Table 12]

[0449] 4.2 Detection of bispecific antibody internalization activity using PHrodo® reagent Using flow cytometry (Beckman, Cytoflex model), we detected the internalization activity of bispecific antibodies against human non-small cell lung cancer cells HCC827, which exhibit high EGFR expression and low c-MET expression; human gastric cancer cells MKN45, which exhibit high c-MET expression and low EGFR expression; and human lung adenocarcinoma cells NCI-H1975, which exhibit low EGFR and c-MET expression.

[0450] Adherent cells were digested with trypsin-EDTA (0.25%) (Shanghai Yuanpei) solution, the cells were counted, and the cell density was set to 1 × 10⁻⁶ in complete culture medium. 5 The cell concentration was adjusted to cells / mL. Then, 100 μL of the cell suspension was added to each well of a 96-well plate (cell count: 1 × 10⁶ per well). 4 Cells were incubated at 37°C in CO2 for 24 hours. The culture medium was then removed, and 50 μL of fresh complete culture medium was added to each well. The bispecific antibodies were then gradually diluted using the complete culture medium to final concentrations of 0.52, 2.08, 8.33, 33.30, 100.00, 200.00, 333.33, and 500 μg / mL (a total of 8 concentration levels). The PHrodo® reagent (Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd.) in the complete culture medium was reduced to 12 μg / mL (the final concentration of PHrodo® is 3 μg / mL). The serially diluted bispecific antibodies to be tested were combined with diluted PHrodo reagent to obtain a final 1:1 (30 μL:30 μL) PHrodo® reagent ratio, and incubated in the dark at room temperature for 30 minutes. Next, 50 μL of the bispecific antibody to be tested and the PHrodo® reagent mixture were added to the wells of a 96-well plate, and the 96-well plate was incubated at 37°C and 5% CO2 for 24 hours. After that, the culture medium was removed and the wells were washed once with sterile PBS, and then 100 μL of trypsin-EDTA (0.25%) was added to each well to release the cells from the wells. Next, 100 μL of complete culture medium was added to neutralize the trypsin, and the cells were dispersed by pipetting and tested by FACS.

[0451] Data processing: Export YL1-H median, EC 50 The data was imported into GraphPad Prism 6 software for calculation. The results are shown in Table 10. Bispecific antibodies may exhibit good internalization activity on tumor cells and potentially result in a more potent tumor suppressor effect. [Table 13]

[0452] Example 5 Cross-arm binding activity of bispecific antibodies against EGFR and c-MET. Human EGFR-His protein was diluted to 1 μg / mL with a carbonate-bicarbonate buffer (CBS) coated solution and added to each well of a 96-well ELISA microtiter plate at a rate of 100 ng / well. The plate was incubated overnight at 4°C. The following day, the 96-well ELISA microtiter plate was washed once with PBST (0.05% Tween-20), and then 100 μL of 2% BSA blocking solution was added to each well of the ELISA microtiter plate. The plate was incubated at 37°C for 2 hours. The purified bispecific antibody was serially diluted in 2% BSA, starting at 10 μg / mL, in a 3-fold gradient across 11 concentrations. 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. Subsequently, the plate was washed three times with PBST (0.05% Tween-20) at a rate of 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. The plate was incubated at 37°C for 2 hours. Subsequently, the wells of the plate were washed three times with 320 μL of PBST (0.05% Tween-20) per well. After drying the plate, 100 μL of peroxidase-conjugated streptavidin secondary antibody was added to each well of the ELISA microtiter plate. The plate was incubated at 37°C for 1 hour. Subsequently, the wells were washed five times with 320 μL of PBST (0.05% Tween-20) per well. The wells were gently pressed to dry, and 100 μL of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) chromogenic substrate was added to each well. After 10 minutes at room temperature, the reaction was stopped by adding 50 μL of 2 M H2SO4 to the wells, and the plate was read using a microplate reader at OD450 nm absorbance.The raw data was imported into GraphPad Prism 6 software for nonlinear curve fitting, and the EC of bispecific antibody cross-arm binding to c-MET and EGFR was measured. 50 The result was calculated. The experimental results are shown in Table 11.

[0453] All of the designed bispecific antibody molecules can bind simultaneously to c-MET and EGFR, and binding to one protein does not affect the binding of the second protein. Furthermore, it has been shown that bispecific antibodies can efficiently bind to both proteins simultaneously. Therefore, bispecific antibodies can inhibit the tumor cell proliferation signaling pathway mediated simultaneously by c-MET and EGFR. [Table 14]

[0454] Example 6 Bispecific antibodies block the binding of EGF and EGFR. Human EGF-His protein was diluted to 1 μg / mL with a carbonate-bicarbonate buffer (CBS) coated solution and added to each well of a 96-well ELISA microtiter plate at a rate of 200 ng / well. The plate was incubated overnight at 4°C. The following day, the 96-well ELISA microtiter plate was washed once with PBST (0.05% Tween-20), and then 100 μL of 2% BSA blocking solution was added to each well of the ELISA microtiter plate. The plate was incubated at 37°C for 2 hours. The purified bispecific antibody was serially diluted using 2% BSA, starting at 500 μg / mL and progressing through 11 concentration steps in a 3-fold gradient, or starting at 100 μg / mL and progressing through 11 concentration steps in a 3-fold gradient. 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 serially diluted bispecific antibody solution and EGFR-hFc-biotin solution were added to the wells of an ELISA microtiter plate. The plate was incubated at 37°C for 2 hours. The plate was then washed three times with PBST (0.05% Tween-20) at a rate of 320 μL per well. The plate was dried, and then 100 μL of peroxidase-complexed anti-streptavidin antibody was added to each well of the ELISA microtiter plate. The plate was incubated at 37°C for 1 hour. The wells of the plate were then washed five times with PBST (0.05% Tween-20) at a rate of 320 μL per well. After drying the plate, 100 μL of 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 stop the reaction, and the plate was read using a microplate reader at OD450 nm absorbance.

[0455] The raw data was imported into GraphPad Prism 6 software for nonlinear curve fitting, and the IC of a bispecific antibody that blocks the binding of EGF and EGFR was performed.50 The result was calculated.

[0456] The experimental results are shown in Table 12. The bispecific antibody molecule can block the binding of EGF and EGFR. [Table 15]

[0457] Example 7 Bispecific antibodies block the binding of HGF to c-MET. Human c-MET-hFc protein (Kactus Biosystems) was diluted to 1 μg / mL with a carbonate-bicarbonate buffer (CBS) coated solution and added to each well of a 96-well ELISA microtiter plate at a dose of 100 ng / well. The plate was incubated overnight at 4°C. The following day, the 96-well ELISA microtiter plate was washed once with PBST (0.05% Tween-20), and then 100 μL of 2% BSA blocking solution was added to each well of the ELISA microtiter plate. The plate was incubated at 37°C for 2 hours. The purified bispecific antibody was serially diluted with 2% BSA, starting at 500 μg / mL, in a 3-fold gradient across 11 concentrations. The HGF-his solution was diluted with 2% BSA to 600 ng / mL (the final concentration of HGF-his was 300 ng / mL). The blocking solution was removed from the wells, and 50 μL aliquots of serially diluted antibody solution 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 then washed three times with PBST (0.05% Tween-20) at 320 μL per well. The plate was dried, and then 100 μL of HRP-complexed anti-his secondary antibody was added to each well of the ELISA microtiter plate. The plate was incubated at 37°C for 1 hour. The wells of the plate were then washed five times with PBST (0.05% Tween-20) at 320 μL per well. After drying the plate, 100 μL of TMB (Huzhou Yingchuang Biotechnology Co., Ltd.) chromogenic substrate was added to each well. After 10 minutes at room temperature, the reaction was stopped by adding 50 μL of 2 M H2SO4 to the wells, and the plate was read using a microplate reader at OD450 nm absorbance.

[0458] The raw data was imported into GraphPad Prism 6 software for nonlinear curve fitting, and the IC of a bispecific antibody that blocks the binding of HGF to c-MET was performed. 50The results were calculated. The experimental results are shown in Table 13. The bispecific antibody molecule can block the binding of HGF to c-MET. [Table 16]

[0459] Example 8 Proliferative inhibitory activity of bispecific antibodies against human tumor cells. Adherent human lung cancer cells NCI-H292 (Nanjing Kebai) and human bronchoalveolar cancer cells NCI-H322M (Nanjing Kebai) were digested with trypsin-EDTA (0.25%, Shanghai Yuanpei) solution and counted. Cell density was calculated to be 5 × 10⁻⁶ for each cell type. 3 and 1 × 10 4 The cells were adjusted to the required concentration per mL. 100 μL of the 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 in a 37°C CO2 incubator for 24 hours. Subsequently, the bispecific antibody and control antibody to be tested were diluted in complete medium in a 3-fold gradient, starting from a final concentration of 500 μg / mL, for a total of 11 concentration steps. 100 μL of the diluted bispecific and control antibodies were added to the 96-well microtiter plate and incubated at 37°C in 5% CO2 for 8 days. Then, 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.

[0460] Raw data, 50 The results were imported into GraphPad Prism 6 software for calculation. The results are shown in Table 14. All bispecific antibodies tested showed clear growth inhibitory activity against NCI-H292 and NCI-H322M cells, and IC50. 50It was significantly better than onartuzumab and terisotuzumab. [Table 17]

[0461] Table 15 shows the maximum proliferation inhibition rates of bispecific antibodies against tumor cells. All bispecific antibodies tested exhibited clear proliferation inhibitory activity against NCI-H292 and NCI-H322M cells, and their maximum proliferation inhibition rates were higher than those of onartuzumab. [Table 18]

[0462] The present invention should not be limited in scope by the specific embodiments described herein. In fact, various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the accompanying claims.

[0463] All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein by reference in whole for all purposes to the same extent as if each individual reference (e.g., publication, patent, or patent application) were specifically and individually indicated as being incorporated by reference in whole for all purposes. Other embodiments are within the scope of the following claims:

Claims

1. A bispecific antibody comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR, The bispecific antibody wherein the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), wherein both the first VL and the first VH form a domain capable of specifically binding to c-MET, and the second antigen-binding domain comprises a second VL and a second VH, wherein both the second VL and the second VH form a domain capable of specifically binding to EGFR.

2. The bispecific antibody according to claim 1, wherein the first antigen-binding domain and the second antigen-binding domain are independently selected from scFv, Fab, and scFab, respectively.

3. A 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 monomer.

4. The bispecific antibody according to claim 3, wherein the Fc domain comprises a first Fc domain monomer including a modification that forms a knob structure and a second Fc domain monomer including a modification that forms a hole structure, and the hole structure can pair with the knob structure to form a heterodimer Fc domain.

5. The bispecific antibody according to claim 4, wherein the first Fc domain monomer comprises an amino acid sequence as shown in SEQ ID NO: 49 or 51, and the second Fc domain monomer comprises an amino acid sequence as shown in SEQ ID NO: 50 or 52.

6. The bispecific antibody according to any one of claims 1 to 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 according to 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 according to any one of claims 1 to 7, wherein the first VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17 or 59, and / or the first VH comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 18 or 60.

9. The first VL is, (i) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 34, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 38, or (ii) comprising LCDR1 having an amino acid sequence as shown in SEQ ID NO: 35, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 37, and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 38, and / or The aforementioned first VH is, (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 39, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 43, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47 (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 40, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 44, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47 (iii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 42, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 46, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 47, or (iv) A bispecific antibody according to any one of claims 1 to 8, comprising HCDR1 having an amino acid sequence as shown in SEQ ID NO: 41, HCDR2 having an amino acid sequence as shown in SEQ ID NO: 45, and HCDR3 having an amino acid sequence as shown in SEQ ID NO:

48.

10. The first VL contains an amino acid sequence as shown in SEQ ID NO: 17 or 59, and / or the first VH contains an amino acid sequence as shown in SEQ ID NO: 18 or 60, or A bispecific antibody according to any one of claims 1 to 9, wherein the first VL comprises an amino acid sequence as shown in SEQ ID NO: 17, and the first VH comprises an amino acid sequence as shown in SEQ ID NO: 18, or the first VL comprises an amino acid sequence as shown in SEQ ID NO: 59, and the first VH comprises an amino acid sequence as shown in SEQ ID NO:

60.

11. The bispecific antibody according to any one of claims 1 to 10, wherein the second VL comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 15, and / or the VH comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO:

16.

12. The second VL is, (i) LCDR1 containing the amino acid sequence shown in SEQ ID NO: 19, LCDR2 containing the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 23, or (ii) comprising LCDR1 having an amino acid sequence as shown in SEQ ID NO: 20, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 22, and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 23, and / or The aforementioned second VH is, (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 24, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32 (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 25, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 29, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32 (iii) HCDR1 containing the amino acid sequence shown in SEQ ID NO: 27, HCDR2 containing the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 32, or (iv) A bispecific antibody according to any one of claims 1 to 11, comprising HCDR1 having an amino acid sequence as shown in SEQ ID NO: 26, HCDR2 having an amino acid sequence as shown in SEQ ID NO: 30, and HCDR3 having an amino acid sequence as shown in SEQ ID NO:

33.

13. The bispecific antibody according to any one of claims 1 to 12, wherein the second VL comprises an amino acid sequence as shown in SEQ ID NO: 15, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO:

16.

14. A bispecific antibody according to any one of claims 1 to 13, wherein the first antigen-binding domain is Fab and the second antigen-binding domain is scFv.

15. The bispecific antibody according to claim 14, wherein the bispecific antibody comprises peptide chain I-A, peptide chain I-B, and peptide chain I-C, wherein peptide chain I-A comprises the first VL and the light chain constant region, peptide chain I-B comprises the VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and 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 according to claim 15, wherein the peptide chain I-A comprises the first VL and the light chain constant region from the N-terminus to the C-terminus, the peptide chain I-B comprises the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or the peptide chain I-C comprises (i) the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus, or (ii) the second VH, the second VL and the second Fc domain monomer (or the first Fc domain monomer).

17. The bispecific antibody according to claim 15 or 16, wherein the adjacent domain of peptide chain I-A is arbitrarily linked with or without a linker, the adjacent domain of peptide chain I-B is arbitrarily linked with or without a linker, and / or the adjacent domain of peptide chain I-C is arbitrarily linked with or without a linker.

18. The linkers are either the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers), or the peptide linkers contain one or more glycine (G) and / or serine (S), for example, (GGGGS). ｎ The bispecific antibody according to claim 17, wherein each peptide linker is independently selected from the group consisting of peptide linkers having a structure represented as (SEQ ID NOs: 55-58 or 63-68) (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), or the peptide linker independently comprises the amino acid sequence represented by SEQ ID NOs: 55-58, 61 or 63-68.

19. The bispecific antibody according to any one of claims 15 to 18, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

20. A bispecific antibody according to any one of claims 15 to 19, wherein the peptide chain I-A comprises an amino acid sequence as shown in SEQ ID NO: 1, the peptide chain I-B comprises an amino acid sequence as shown in SEQ ID NO: 2 or 9, and / or the peptide chain I-C comprises an amino acid sequence as shown in SEQ ID NO: 3 or 10.

21. A bispecific antibody according to any one of claims 1 to 13, wherein the first antigen-binding domain is scFv and the second antigen-binding domain is Fab.

22. The bispecific antibody according to claim 21, wherein the bispecific antibody comprises peptide chain II-A, peptide chain II-B, and peptide chain II-C, wherein peptide chain II-A comprises the second VL and the light chain constant region, peptide chain II-B comprises the second VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and 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 according to claim 22, wherein the peptide chain II-A comprises the second VL and the light chain constant region from the N-terminus to the C-terminus, the peptide chain II-B comprises the second VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or the peptide chain II-C comprises (i) the first VL, the first VH and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus, or (ii) the first VH, the first VL and the second Fc domain monomer (or the first Fc domain monomer).

24. The bispecific antibody according to claim 22 or 23, wherein the adjacent domain of peptide chain II-A is arbitrarily linked with or without a linker, the adjacent domain of peptide chain II-B is arbitrarily linked with or without a linker, and / or the adjacent domain of peptide chain II-C is arbitrarily linked with or without a linker.

25. The linkers are either the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers), or the peptide linkers contain one or more glycine (G) and / or serine (S), for example, (GGGGS). ｎ The bispecific antibody according to claim 24, wherein each peptide linker is independently selected from those having the structure shown in (SEQ ID NOs: 55-58 or 63-68) (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), or the peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs: 55-58, 61 or 63-68.

26. The bispecific antibody according to any one of claims 22 to 25, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

27. A bispecific antibody according to any one of claims 22 to 26, wherein the peptide chain II-A comprises an amino acid sequence as shown in SEQ ID NO: 4, the peptide chain II-B comprises an amino acid sequence as shown in SEQ ID NO: 5 or 7, and / or the peptide chain II-C comprises an amino acid sequence as shown in SEQ ID NO: 6 or 8.

28. A bispecific antibody according to any one of claims 1 to 13, wherein the first antigen-binding domain is Fab and the second antigen-binding domain is scFab.

29. The bispecific antibody according to claim 28, wherein the bispecific antibody comprises peptide chain III-A, peptide chain III-B, and peptide chain III-C, wherein peptide chain III-A comprises the first VL and the light chain constant region, peptide chain III-B comprises the first VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and peptide chain III-C comprises 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).

30. The bispecific antibody according to claim 29, wherein the peptide chain III-A comprises the first VL and the light chain constant region from the N-terminus to the C-terminus, the peptide chain III-B comprises the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or the peptide chain III-C comprises (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) from the N-terminus to the C-terminus, 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 according to claim 29 or 30, wherein the adjacent domain of peptide chain III-A is arbitrarily linked with or without a linker, the adjacent domain of peptide chain III-B is arbitrarily linked with or without a linker, and / or the adjacent domain of peptide chain III-C is arbitrarily linked with or without a linker.

32. The linkers are either the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers), or the peptide linkers contain one or more glycine (G) and / or serine (S), for example, (GGGGS). ｎ The bispecific antibody according to claim 31, wherein each peptide linker is independently selected from those having the structure shown in (SEQ ID NOs: 55-58 or 63-68) (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), or the peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs: 55-58, 61 or 63-68.

33. The bispecific antibody according to any one of claims 29 to 32, wherein the constant region of the light chain comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the CH1 region of the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:

54.

34. A bispecific antibody according to any one of claims 1 to 13, wherein the first antigen-binding domain is scFab and the second antigen-binding domain is Fab.

35. The bispecific antibody according to claim 34, wherein the bispecific antibody comprises peptide chain IV-A, peptide chain IV-B, and peptide chain IV-C, wherein peptide chain IV-A comprises the second VL and the light chain constant region, peptide chain IV-B comprises the second VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and peptide chain IV-C comprises 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).

36. The bispecific antibody according to claim 35, wherein the peptide chain IV-A includes the second VL and the light chain constant region from the N-terminus to the C-terminus, the peptide chain IV-B includes the second VH, the CH1 region of the heavy chain and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or the peptide chain IV-C includes (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) from the N-terminus to the C-terminus, 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 according to claim 35 or 36, wherein the adjacent domain of peptide chain IV-A is arbitrarily linked with or without a linker, the adjacent domain of peptide chain IV-B is arbitrarily linked with or without a linker, and / or the adjacent domain of peptide chain IV-C is arbitrarily linked with or without a linker.

38. The linkers are either the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers), or the peptide linkers contain one or more glycine (G) and / or serine (S), for example, (GGGGS). ｎ The bispecific antibody according to claim 37, wherein each peptide linker is independently selected from those having the structure shown in (SEQ ID NOs: 55-58 or 63-68) (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), or the peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs: 55-58, 61 or 63-68.

39. The bispecific antibody according to any one of claims 35 to 38, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

40. A bispecific antibody according to any one of claims 35 to 39, wherein the peptide chain IV-A comprises an amino acid sequence as shown in SEQ ID NO: 4, the peptide chain IV-B comprises an amino acid sequence as shown in SEQ ID NO: 7, and / or the peptide chain IV-C comprises an amino acid sequence as shown in SEQ ID NO:

11.

41. A bispecific antibody according to any one of claims 1 to 13, wherein the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the second antigen-binding domain includes a domain swap in the form of a CrossMab.

42. The bispecific antibody according to claim 41, wherein the bispecific antibody comprises peptide chain V-A, peptide chain V-B, peptide chain V-C, and peptide chain V-D, wherein peptide chain V-A comprises the first VL and the light chain constant region, peptide chain V-B comprises the first VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), peptide chain V-C comprises the second VH, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer), and peptide chain V-D comprises the second VL and the heavy chain CH1 region.

43. The bispecific antibody according to claim 42, wherein the peptide chain V-A includes the first VL and the light chain constant region from the N-terminus to the C-terminus, the peptide chain V-B includes the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, the peptide chain V-C includes the second VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus, and / or the peptide chain V-D includes the second VL and the heavy chain CH1 region from the N-terminus to the C-terminus.

44. The bispecific antibody according to claim 42 or 43, wherein the adjacent domain of peptide chain V-A is arbitrarily linked with or without a linker, the adjacent domain of peptide chain V-B is arbitrarily linked with or without a linker, the adjacent domain of peptide chain V-C is arbitrarily linked with or without a linker, and / or the adjacent domain of peptide chain V-D is arbitrarily linked with or without a linker.

45. The linkers are either the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers), or the peptide linkers contain one or more glycine (G) and / or serine (S), for example, (GGGGS). ｎ The bispecific antibody according to claim 44, wherein each peptide linker is independently selected from those having the structure shown in (SEQ ID NOs: 55-58 or 63-68) (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), or the peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs: 55-58, 61 or 63-68.

46. The bispecific antibody according to any one of claims 42 to 45, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

47. A bispecific antibody according to any one of claims 42 to 46, wherein the peptide chain V-A comprises an amino acid sequence as shown in SEQ ID NO: 1, the peptide chain V-B comprises an amino acid sequence as shown in SEQ ID NO: 9, the peptide chain V-C comprises an amino acid sequence as shown in SEQ ID NO: 12, and / or the sequence of the peptide chain V-D is as shown in SEQ ID NO:

13.

48. A bispecific antibody according to any one of claims 1 to 13, wherein the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the first antigen-binding domain includes a domain swap in the form of a CrossMab.

49. The bispecific antibody according to claim 48, wherein the bispecific antibody comprises peptide chain VI-A, peptide chain VI-B, peptide chain VI-C, and peptide chain VI-D, wherein peptide chain VI-A comprises the second VL and the light chain constant region, peptide chain VI-B comprises the second VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), peptide chain VI-C comprises the first VH, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer), and peptide chain VI-D comprises the first VL and the heavy chain CH1 region.

50. The bispecific antibody according to claim 49, wherein the peptide chain VI-A includes the second VL and the light chain constant region from the N-terminus to the C-terminus, the peptide chain VI-B includes the second VH, the CH1 region of the heavy chain, and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, the peptide chain VI-C includes the first VH, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus, and / or the peptide chain VI-D includes the first VL and the heavy chain CH1 region from the N-terminus to the C-terminus.

51. The bispecific antibody according to claim 49 or 50, wherein the adjacent domain of peptide chain VI-A is arbitrarily linked with or without a linker, the adjacent domain of peptide chain VI-B is arbitrarily linked with or without a linker, the adjacent domain of peptide chain VI-C is arbitrarily linked with or without a linker, and / or the adjacent domain of peptide chain VI-D is arbitrarily linked with or without a linker.

52. The linkers are either the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers), or the peptide linkers contain one or more glycine (G) and / or serine (S), for example, (GGGGS). ｎ The bispecific antibody according to claim 51, wherein each peptide linker is independently selected from those having the structure shown in (SEQ ID NOs: 55-58 or 63-68) (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), or the peptide linker independently comprises the amino acid sequence shown in SEQ ID NOs: 55-58, 61 or 63-68.

53. The bispecific antibody according to any one of claims 49 to 52, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

54. The bispecific antibody according to any one of claims 1 to 13, wherein both the first antigen-binding domain and the second antigen-binding domain are scFv.

55. The bispecific antibody according to claim 54, wherein the bispecific antibody comprises peptide chain VII-A and peptide chain VII-B, wherein peptide chain VII-A comprises the first VL, the first VH, and the first Fc domain monomer (or the second Fc domain monomer), and 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 according to claim 55, wherein the peptide chain VII-A comprises (i) the first VL, the first VH, and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, 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 (i) the second VL, the second VH, and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus.

57. The bispecific antibody according to claim 55 or 56, wherein the adjacent domain of peptide chain VII-A is arbitrarily linked with or without a linker, and / or the adjacent domain of peptide chain VII-B is arbitrarily linked with or without a linker.

58. The linkers are either the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers), or the peptide linkers contain one or more glycine (G) and / or serine (S), for example, (GGGGS). ｎ A bispecific antibody according to claim 57, each independently selected from peptide linkers having the structure shown in (SEQ ID NOs: 55-58 or 63-68) (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

59. The bispecific antibody according to claim 58, wherein each peptide linker independently comprises an amino acid sequence shown in SEQ ID NOs. 55-58, 61, or 63-68.

60. The bispecific antibody according to any one of claims 55 to 59, wherein the peptide chain VII-A comprises an amino acid sequence as shown in SEQ ID NO: 14, and / or the peptide chain VII-B comprises an amino acid sequence as shown in SEQ ID NO:

8.

61. (1) Peptide chain I-A containing the amino acid sequence shown in SEQ ID NO: 1, peptide chain I-B containing the amino acid sequence shown in SEQ ID NO: 2, and peptide chain I-C containing the amino acid sequence shown in SEQ ID NO: 3 (2) Peptide chain I-A containing the amino acid sequence shown in SEQ ID NO: 1, peptide chain I-B containing the amino acid sequence shown in SEQ ID NO: 9, and peptide chain I-C containing the amino acid sequence shown in SEQ ID NO:

10. (3) Peptide chain II-A containing the amino acid sequence shown in SEQ ID NO: 4, Peptide chain II-B containing the amino acid sequence shown in SEQ ID NO: 5, and Peptide chain II-C containing the amino acid sequence shown in SEQ ID NO: 6 (4) Peptide chain II-A containing the amino acid sequence shown in SEQ ID NO: 4, Peptide chain II-B containing the amino acid sequence shown in SEQ ID NO: 7, and Peptide chain II-C containing the amino acid sequence shown in SEQ ID NO: 8 (5) Peptide chain IV-A containing the amino acid sequence shown in SEQ ID NO: 4, peptide chain IV-B containing the amino acid sequence shown in SEQ ID NO: 7, and peptide chain IV-C containing the amino acid sequence shown in SEQ ID NO:

11. (6) Peptide chain V-A containing the amino acid sequence shown in SEQ ID NO: 1, peptide chain V-B containing the amino acid sequence shown in SEQ ID NO: 9, peptide chain V-C containing the amino acid sequence shown in SEQ ID NO: 12, and peptide chain V-D containing the amino acid sequence shown in SEQ ID NO: 13, or (7) Peptide chain VII-A containing the amino acid sequence shown in SEQ ID NO: 14, and peptide chain VII-B containing the amino acid sequence shown in SEQ ID NO: 8 A bispecific antibody according to any one of claims 1 to 60, comprising:

62. A bispecific antibody according to any one of claims 1 to 61, having an enhanced tumor suppressor effect compared to a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody, wherein the amino acid sequence of the CDR of the monospecific anti-c-MET antibody is the same as the amino acid sequence of the CDR of the first antigen-binding domain, and the amino acid sequence of the CDR of the monospecific anti-EGFR antibody is the same as the amino acid sequence of the CDR of the second antigen-binding domain.

63. The bispecific antibody according to claim 62, wherein the tumor suppressor effect comprises inhibition of the 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 a bispecific antibody according to any one of claims 1 to 63.

65. One or more vectors comprising an isolated nucleic acid molecule or a set of nucleic acid molecules as described in claim 64.

66. The one or more vectors according to claim 65, wherein the nucleotide sequences encoding different peptide chains of the bispecific antibody are located within different vector molecules.

67. The one or more vectors according to claim 65 or 66, wherein the one or more vectors are cloning vectors or expression vectors.

68. A host cell comprising an isolated nucleic acid molecule or set of nucleic acid molecules as described in claim 64, or one or more vectors as described in any one of claims 65 to 67.

69. A method for preparing a bispecific antibody according to any one of claims 1 to 63, comprising culturing the host cells according to claim 68 under conditions that enable the expression of the bispecific antibody, and recovering the bispecific antibody from the culture of the host cells.

70. A complex comprising a bispecific antibody according to any one of claims 1 to 63 and a complexing portion linked thereto, wherein the complexing portion is optionally selected from therapeutic agents (such as cytotoxic agents, cytokines, toxins, or radionuclides).

71. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 63, an isolated nucleic acid molecule or set of nucleic acid molecules according to claim 64, one or more vectors according to any one of claims 65 to 67, a host cell according to claim 68, or a complex according to claim 70, and a pharmaceutically acceptable carrier and / or excipient.

72. The pharmaceutical composition according to claim 71, wherein the pharmaceutical composition further comprises an additional pharmaceutically active agent.

73. The pharmaceutical composition according to claim 72, wherein the additional pharmacoactive agent is a drug having antitumor activity.

74. The pharmaceutical composition according to claim 72, wherein the additional pharmacoactive 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 according to claim 72, wherein the bispecific antibody and the additional pharmaceutical activator are provided as separate components or as mixed components.

76. The use of a bispecific antibody according to any one of claims 1 to 63, an isolated nucleic acid molecule or set of nucleic acid molecules according to claim 64, one or more vectors according to any one of claims 65 to 67, a host cell according to claim 68, a complex according to claim 70, or a pharmaceutical composition according to any one of claims 71 to 75, wherein the pharmaceutical is used for the prevention and / or treatment of c-MET and / or EGFR-related diseases in a subject, and / or as an adjuvant in the treatment thereof, and / or for the inhibition of c-MET and / or EGFR activity in vitro or in a subject.

77. The use according to claim 76, wherein the disease associated with c-MET and / or EGFR is cancer.

78. The use according to claim 77, wherein the cancer is associated with an EGFR activating mutation, EGFR gene amplification, elevated circulating HGF levels, a c-MET activating mutation and / or c-MET gene amplification.

79. The use according to claim 77 or 78, wherein 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 cavity cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, gastric cancer, thymic 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 according to claim 76, wherein the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, one or more vectors, host cells, complex, or pharmaceutical composition is administered in combination with another pharmaceutical active agent, for example, simultaneously, separately, or sequentially.

81. The use according to claim 80, wherein the other pharmacoactive agent is a drug having antitumor activity.

82. The use according to claim 80, wherein the additional pharmacoactive 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 according to any one of claims 76 to 82, wherein the subject is resistant to erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab treatment.

84. A method for inhibiting intracellular c-MET and / or EGFR activity, comprising contacting the cells with a bispecific antibody according to any one of claims 1 to 63, an isolated nucleic acid molecule or set of nucleic acid molecules according to claim 64, one or more vectors according to any one of claims 65 to 67, a host cell according to claim 68, a complex according to claim 70, or a pharmaceutical composition according to any one of claims 71 to 75.

85. The method according to claim 84, wherein the cells are c-MET and / or EGFR-expressing cells such as tumor cells.

86. A method for preventing and / or treating a disease associated with c-MET and / or EGFR in a subject, and / or a method for acting as an adjuvant in the treatment thereof, wherein the method comprises administering to the subject in need thereof an effective amount of a bispecific antibody according to any one of claims 1 to 63, an isolated nucleic acid molecule or set of nucleic acid molecules according to claim 64, one or more vectors according to any one of claims 65 to 67, a host cell according to claim 68, a complex according to claim 70, or a pharmaceutical composition according to any one of claims 71 to 75.

87. The method according to claim 86, wherein the disease associated with c-MET and / or EGFR is cancer.

88. The method according to claim 87, wherein the cancer is associated with an EGFR activating mutation, EGFR gene amplification, elevated circulating HGF levels, a c-MET activating mutation and / or c-MET gene amplification.

89. The method according to 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 cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cavity cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, gastric cancer, thymic 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 according to claim 86, further comprising 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, wherein the second therapy may optionally be applied simultaneously, separately, or sequentially.

91. The method according to any one of claims 86 to 90, wherein the subject is resistant to treatment with erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or cetuximab.

Images