Bispecific antibodies against human c-Met and human EGFR and uses thereof

Abstract

The present application provides bispecific antibodies comprising a first antigen-binding fragment directed against human mesenchymal-epithelial transformation factor (c-Met) and a second antigen-binding fragment directed against human epidermal growth factor receptor (EGFR). In addition, the invention also provides medical and biological uses of the bispecific antibody.

Description

Invention Field

[0001] This invention generally relates to the fields of genetic engineering and antibody drugs; more specifically, this application relates to bispecific antibodies against human c-Met and human EGFR and their uses. Background of the Invention

[0003] Epidermal growth factor receptor (EGFR), also known as HER-1 or ERBB-1, is a transmembrane glycoprotein and a member of the protein kinase superfamily. It participates in cell proliferation, division, mitosis, and cancer development, and plays a crucial role in the development of drug resistance. EGFR consists of an extracellular ligand-binding domain, a hydrophobic transmembrane region, an intracellular receptor tyrosine kinase (RTK) domain, and a C-terminal domain. EGFR has multiple ligands, such as AR, β-cytokinin, epidermal growth factor (EGF), heparin-binding EGF-like growth factor, and transforming growth factor (TGF)-α. These ligands can induce receptor dimerization and tyrosine autophosphorylation by binding to EGFR, promoting a signaling cascade and leading to cell proliferation. EGFR is overexpressed in most solid tumors, such as breast cancer, colon cancer, head and neck cancer, kidney cancer, ovarian cancer, and non-small cell lung cancer. Its overexpression or enhanced activity increases tumor invasiveness, growth rate, and drug resistance, promoting cancer progression. Therefore, EGFR is a promising target for anticancer drug design and development.

[0004] Mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase belonging to the MET family, with hepatocyte growth factor (HGF) as its ligand. c-Met is synthesized by mesenchymal cells, fibroblasts, and smooth muscle cells. It activates HGF / c-Met signaling via paracrine mechanisms. Under normal conditions, HGF / c-Met mediates embryogenesis, tissue regeneration, wound healing, and nerve and muscle formation. Abnormal activation of c-Met can promote the occurrence and progression of various cancers, such as liver cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, and gastric cancer, as well as nervous system cancers such as glioblastoma. The HGF / c-Met axis can interact and cooperate with other types of tyrosine kinases, stimulating various downstream signaling pathways in tumor cells, such as PI3K / AKT, JAK / STAT, Ras / MAPK, SRC, and Wnt / β-catenin, regulating multiple biological processes, such as tumor proliferation, invasion, metastasis, anti-apoptosis, EMT, and angiogenesis. Meanwhile, high c-Met expression is closely associated with poor prognosis in cancer patients. Studies have shown that abnormal c-Met activation is crucial for resistance to targeted therapy. Therefore, abnormal c-Met function increases the difficulty of tumor treatment, and targeted c-Met therapy for tumors is considered to have broad clinical significance.

[0005] Lung cancer is a leading cause of cancer death in both men and women, accounting for one-third of all cancer deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases, with a low 5-year survival rate of approximately 18%. Based on the discovery of EGFR activating mutations, EGFR inhibitors such as gefitinib and erlotinib have made significant contributions to the fight against lung cancer, providing patients with significant clinical benefits. However, they have also led to acquired resistance, limiting the long-term efficacy of these drugs.

[0006] c-MET gene amplification is also a significant cause of acquired resistance in NSCLC. Mechanistically, MET amplification activates ErbB3, leading to EGFR-independent phosphorylation, and activation of the PI3K / AKT pathway results in EGFR-TKI resistance. Therefore, simultaneous inhibition of both EGFR and c-MET is necessary to overcome resistance to EGFR inhibitors. To overcome EGFR TKI resistance, numerous clinical trials testing novel EGFR and MET receptors have been designed and initiated worldwide.

[0007] Signal transduction between the c-Met and EGFR pathways is closely related in tumors such as breast cancer, lung cancer, and brain cancer. In non-small cell lung cancer (NSCLC), 70% of EGFR activating mutations are associated with initial positive responses to EGFR inhibitors, and the vast majority of tumors that respond to EGFR inhibitors develop acquired resistance. Studies have shown that in NSCLC patients, c-Met expression and activation are associated with both initial and acquired resistance to EGFR inhibitors. In lung cancer, simultaneous activation of the c-Met and EGFR pathways may lead to initial resistance; inhibiting their downstream signaling pathways can maximize tumor suppression. Therefore, simultaneously targeting and inhibiting c-Met and EGFR may be an effective way to overcome inhibitor resistance in lung cancer.

[0008] Amivantamab is the world's first approved EGFR / cMET bispecific antibody. In May 2021, based on Phase Ib clinical data, it received FDA approval for EGFR exon 20 insertion mutation NSCLC that has progressed after platinum-based chemotherapy. On November 20, 2023, Johnson & Johnson announced that it had submitted a supplemental Biologics License Application (sBLA) to the FDA for a new indication for the drug: in combination with chemotherapy for patients with EGFR-mutant NSCLC whose disease has progressed during or after osimertinib treatment.

[0009] Therefore, developing novel bispecific antibodies against c-Met and EGFR is of great significance for the treatment of cancer, especially non-small cell lung cancer. Invention Overview

[0011] In a first aspect, this application provides a bispecific antibody comprising a first antigen-binding fragment against human mesenchymal-epithelial transforming factor (c-Met) and a second antigen-binding fragment against human epidermal growth factor receptor (EGFR);

[0012] Wherein, the first antigen-binding fragment comprises:

[0013] HCDR1, as shown in SEQ ID NO:1,

[0014] HCDR2, as shown in SEQ ID NO:2,

[0015] HCDR3, as shown in SEQ ID NO:3,

[0016] LCDR1, as shown in SEQ ID NO:4,

[0017] LCDR2, as shown in SEQ ID NO:5, and

[0018] As shown in SEQ ID NO:6, LCDR3; or

[0019] HCDR1, as shown in SEQ ID NO:7,

[0020] HCDR2, as shown in SEQ ID NO:8,

[0021] HCDR3, as shown in SEQ ID NO:9,

[0022] LCDR1, as shown in SEQ ID NO:10,

[0023] LCDR2, as shown in SEQ ID NO:11, and

[0024] As shown in SEQ ID NO:12, LCDR3; or

[0025] HCDR1, as shown in SEQ ID NO:13,

[0026] HCDR2, as shown in SEQ ID NO:14,

[0027] HCDR3, as shown in SEQ ID NO:15,

[0028] LCDR1, as shown in SEQ ID NO:16,

[0029] LCDR2, as shown in SEQ ID NO:17, and

[0030] LCDR3 as shown in SEQ ID NO:18; and

[0031] The second antigen-binding fragment includes:

[0032] As shown in SEQ ID NO:19, HCDR1,

[0033] HCDR2, as shown in SEQ ID NO:20,

[0034] HCDR3, as shown in SEQ ID NO:21,

[0035] LCDR1, as shown in SEQ ID NO:22,

[0036] LCDR2, as shown in SEQ ID NO:23, and

[0037] As shown in SEQ ID NO:24, LCDR3; or

[0038] As shown in SEQ ID NO:19, HCDR1,

[0039] HCDR2, as shown in SEQ ID NO:25,

[0040] HCDR3, as shown in SEQ ID NO:21,

[0041] LCDR1, as shown in SEQ ID NO:22,

[0042] LCDR2, as shown in SEQ ID NO:26, and

[0043] LCDR3 as shown in SEQ ID NO:27;

[0044] HCDR and LCDR are defined according to Kabat.

[0045] Secondly, this application provides a bispecific antibody comprising a first antigen-binding fragment against human mesenchymal-epithelial transforming factor (c-Met) and a second antigen-binding fragment against human epidermal growth factor receptor (EGFR);

[0046] Wherein, the first antigen-binding fragment comprises:

[0047] HCDR1, as shown in SEQ ID NO:28,

[0048] HCDR2, as shown in SEQ ID NO:29,

[0049] HCDR3, as shown in SEQ ID NO:3,

[0050] LCDR1, as shown in SEQ ID NO:4,

[0051] LCDR2, as shown in SEQ ID NO:5, and

[0052] As shown in SEQ ID NO:6, LCDR3; or

[0053] HCDR1, as shown in SEQ ID NO:30,

[0054] HCDR2, as shown in SEQ ID NO:31,

[0055] HCDR3, as shown in SEQ ID NO:9,

[0056] LCDR1, as shown in SEQ ID NO:10,

[0057] LCDR2, as shown in SEQ ID NO:11, and

[0058] As shown in SEQ ID NO:12, LCDR3; or

[0059] As shown in SEQ ID NO:32, HCDR1,

[0060] HCDR2, as shown in SEQ ID NO:33,

[0061] HCDR3, as shown in SEQ ID NO:15,

[0062] LCDR1, as shown in SEQ ID NO:16,

[0063] LCDR2, as shown in SEQ ID NO:17, and

[0064] LCDR3 as shown in SEQ ID NO:18; and

[0065] The second antigen-binding fragment includes:

[0066] HCDR1, as shown in SEQ ID NO:34,

[0067] HCDR2, as shown in SEQ ID NO:35,

[0068] HCDR3, as shown in SEQ ID NO:21,

[0069] LCDR1, as shown in SEQ ID NO:22,

[0070] LCDR2, as shown in SEQ ID NO:23, and

[0071] As shown in SEQ ID NO:24, LCDR3; or

[0072] HCDR1, as shown in SEQ ID NO:36,

[0073] HCDR2, as shown in SEQ ID NO:37,

[0074] HCDR3, as shown in SEQ ID NO:21,

[0075] LCDR1, as shown in SEQ ID NO:22,

[0076] LCDR2, as shown in SEQ ID NO:26, and

[0077] LCDR3 as shown in SEQ ID NO:27;

[0078] HCDR and LCDR are defined according to Chothia.

[0079] In some embodiments of the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:39; or

[0080] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:40 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:41 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:41; or

[0081] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:42 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:43 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:43.

[0082] In some embodiments of the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or

[0083] The amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:46 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:47 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47.

[0084] In some embodiments of the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39 .... The sequence shown in NO:39 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45 ... or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45. The sequence shown in NO:45 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; or

[0085] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:40 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:41 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:41, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:40. The sequence shown in SEQ ID NO:44 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:45 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or

[0086] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:42 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:43 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:43, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:42. The sequence shown in SEQ ID NO:44 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:45 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or

[0087] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:39, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:38. The sequence shown in SEQ ID NO:46 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:47 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47; or

[0088] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:40 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:41 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:41, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:40. The sequence shown in SEQ ID NO:46 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:47 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47; or

[0089] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:42 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:43 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:43, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:42. The sequence shown in SEQ ID NO:46 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment as shown in SEQ ID NO:47 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47.

[0090] In some embodiments of the first or second aspect, the bispecific antibody is an IgG1 antibody comprising a first heavy chain constant region and a second heavy chain constant region, wherein the amino acid at position 405 of the first heavy chain constant region is L and the amino acid at position 409 of the second heavy chain constant region is R; or the amino acid at position 409 of the first heavy chain constant region is R and the amino acid at position 405 of the second heavy chain constant region is L; the amino acid positions of the antibody constant region are determined according to EU numbering.

[0091] In some embodiments of the first or second aspect, the bispecific antibody comprises a first light chain constant region and a second light chain constant region, wherein the first light chain constant region and / or the second light chain constant region is a κ subtype or a λ subtype.

[0092] In some embodiments of the first or second aspect, the first antigen-binding fragment and the second antigen-binding fragment are independently selected from single-chain antibodies (scFv) or Fab fragments.

[0093] In some embodiments of the first or second aspect, the bispecific antibody is in the form of Doubody, Diabody, CrossMab, DVD-ig, Knob-in-Hole, BiTE, DART, YBODY, or FIT-Ig.

[0094] Thirdly, this application provides a pharmaceutical composition comprising the bispecific antibody described in the first or second aspect, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0095] Fourthly, this application provides the use of the bispecific antibody described in the first or second aspect or the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention or treatment of cancer in a subject of need.

[0096] Fifthly, this application provides a method for preventing or treating cancer in a subject in need, comprising administering to the subject an effective amount of the bispecific antibody described in the first or second aspect or the pharmaceutical composition described in the third aspect. Brief description of the attached diagram

[0098] Figure 1 The diagram shows the structure of the bispecific antibody against human c-Met and human EGFR of this application.

[0099] Figure 2 The results of the antibody affinity ELISA assay against huMet-Fc(IgG1) are shown -1; where IPI is an abbreviation for ipilimumab.

[0100] Figure 3 The results of the antibody affinity ELISA assay against huMet-Fc(IgG1) are shown in Figure 2; where NC represents the negative control and BC represents the blank control; and IPI is an abbreviation for ipilimumab.

[0101] Figure 4 The results show the blocking effect of the maturation candidate molecule of the maternal antibody B181 on hu-met-HEK293 cells; where sec is an abbreviation for secondary antibody, and "cells + sec" serves as a negative control.

[0102] Figure 5 The binding and dissociation curves of candidate molecule B181_116_2_10_B181_116 are displayed. The curves showing the antigen-antibody binding before 120 seconds and the antigen-antibody dissociation curves after 120 seconds represent different antibody concentrations.

[0103] Figure 6The results show the affinity activity of the maturation candidate molecule of the maternal antibody C98 to the antigen; where NC represents the negative control and BC represents the blank control; IPI is an abbreviation for ipilimumab.

[0104] Figure 7 The binding and dissociation curves of the candidate molecule C98-002-C98-WT are shown. The curves before 120 seconds represent the antigen-antibody binding, and the curves after 120 seconds represent the antigen-antibody dissociation. Each curve represents a different antibody concentration.

[0105] Figure 8 The results show the binding ability of the maturation candidate molecule of the maternal antibody C85-WT to NCI-H292 cells; where sec is an abbreviation for secondary antibody, and "cells + sec" serves as a negative control.

[0106] Figure 9 The binding and dissociation curves of the candidate molecule C85-mix-4-C85-16 are shown. The curves before 120 seconds represent the antigen-antibody binding, and the curves after 120 seconds represent the antigen-antibody dissociation. Each curve represents a different antibody concentration.

[0107] Figure 10 The flowchart for constructing the EGFR antibody library is shown.

[0108] Figure 11 The results show the affinity activity of the maturation candidate molecule of the maternal antibody Panitumumab-WT to the antigen, where NC represents the negative control and BC represents the blank control; IPI is an abbreviation for ipilimumab.

[0109] Figure 12 The results show the binding ability of the maturation candidate molecule of the maternal antibody Panitumumab-WT to A549 cells; where sec is an abbreviation for secondary antibody, and "cells + sec" serves as a negative control.

[0110] Figure 13 The binding and dissociation curves of the candidate molecule Panitumumab-68 are shown. The curves before 120 seconds represent the antigen-antibody binding, and the curves after 120 seconds represent the antigen-antibody dissociation. Each curve represents a different antibody concentration.

[0111] Figure 14 The binding and dissociation curves of the candidate molecule Panitumumab-WT-113 are shown. The curves before 120 seconds represent the antigen-antibody binding curves, and the curves after 120 seconds represent the antigen-antibody dissociation curves. Each curve represents a different antibody concentration.

[0112] Figure 15The HIC results for the anti-c-Met and EGFR bispecific antibodies are shown.

[0113] Figure 16 The binding and dissociation curves of anti-c-Met and EGFR bispecific antibodies with c-Met antigen are shown. The curves before 120 seconds represent antigen-antibody binding, and the curves after 120 seconds represent antigen-antibody dissociation. Each curve represents a different antibody concentration.

[0114] Figure 17 The binding and dissociation curves of anti-c-Met and anti-EGFR bispecific antibodies with EGFR antigen are shown. The curves before 120 seconds represent antigen-antibody binding, and the curves after 120 seconds represent antigen-antibody dissociation. Each curve represents a different antibody concentration.

[0115] Figure 18 The results show the affinity activities of anti-c-Met and EGFR bispecific antibodies to c-Met antigen detected by ELISA, where NC represents the negative control and BC represents the blank control; IPI is an abbreviation for ipilimumab.

[0116] Figure 19 The results of ELISA detection of the affinity activities of anti-c-Met and EGFR bispecific antibodies with EGFR antigen are shown, where NC represents the negative control and BC represents the blank control; IPI is an abbreviation for ipilimumab.

[0117] Sequence Description

[0118] SEQ ID NO:1-2 show the amino acid sequences of HCDR1 and HCDR2 of the anti-c-Met antibody C85-mix-4-C85-016 as defined by Kabat.

[0119] SEQ ID NO:3-6 show the amino acid sequences of HCDR3, LCDR1, LCDR2 and LCDR3 of the anti-c-Met antibody C85-mix-4-C85-016 as defined by Kabat or Chothia, respectively.

[0120] SEQ ID NO:7-8 show the amino acid sequences of HCDR1 and HCDR2 of the anti-c-Met antibody B181_116_2_10_B181_116 as defined by Kabat.

[0121] SEQ ID NO:9-12 show the amino acid sequences of HCDR3, LCDR1, LCDR2 and LCDR3 of anti-c-Met antibody B181_116_2_10_B181_116 according to the definition of Kabat or Chothia.

[0122] SEQ ID NO:13-14 show the amino acid sequences of HCDR1 and HCDR2 of the anti-c-Met antibody C98-002-C98-WT as defined by Kabat.

[0123] SEQ ID NO:15-18 show the amino acid sequences of HCDR3, LCDR1, LCDR2 and LCDR3 of the anti-c-Met antibody C98-002-C98-WT according to the definition of Kabat or Chothia.

[0124] SEQ ID NO:19 shows the HCDR1 amino acid sequence of the anti-EGFR antibodies Panitumumab-WT-113 and Panitumumab-68 as defined by Kabat.

[0125] SEQ ID NO:20 shows the HCDR2 amino acid sequence of the anti-EGFR antibody Panitumumab-WT-113 as defined by Kabat.

[0126] SEQ ID NO:21-22 show the amino acid sequences of HCDR3 and LCDR1 of the anti-EGFR antibodies Panitumumab-WT-113 and Panitumumab-68, respectively, according to the Kabat definition or the Chothia definition.

[0127] SEQ ID NO:23-24 show the amino acid sequences of LCDR2 and LCDR3 of the anti-EGFR antibody Panitumumab-WT-113 according to the definition of Kabat or Chothia.

[0128] SEQ ID NO:25 shows the amino acid sequence of HCDR2 of the anti-EGFR antibody Panitumumab-68 as defined by Kabat.

[0129] SEQ ID NO:26-27 show the amino acid sequences of LCDR2 and LCDR3 of the anti-EGFR antibody Panitumumab-68 according to the definition of Kabat or Chothia, respectively.

[0130] SEQ ID NO:28-29 show the HCDR1 and HCDR2 amino acid sequences of the anti-c-Met antibody C85-mix-4-C85-16 as defined by Chothia.

[0131] SEQ ID NO:30-31 show the amino acid sequences of HCDR1 and HCDR2 of anti-c-Met antibody B181_116_2_10_B181_116 as defined by Chothia.

[0132] SEQ ID NO:32-33 show the amino acid sequences of HCDR1 and HCDR2 of the anti-c-Met antibody C98-002-C98-WT as defined by Chothia.

[0133] SEQ ID NO:34-35 show the amino acid sequences of HCDR1 and HCDR2 of the anti-EGFR antibody Panitumumab-WT-113 as defined by Chothia.

[0134] SEQ ID NO:36-37 show the amino acid sequences of HCDR1 and HCDR2 of the anti-EGFR antibody Panitumumab-68 as defined by Chothia.

[0135] SEQ ID NO:38-39 show the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-c-Met antibody C85-mix-4-C85-016, respectively.

[0136] SEQ ID NO:40-41 show the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-c-Met antibody B181_116_2_10_B181_116, respectively.

[0137] SEQ ID NO:42-43 show the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-c-Met antibody C98-002-C98-WT, respectively.

[0138] SEQ ID NO:44-45 show the amino acid sequences of the heavy chain variable region and light chain variable region of the anti-EGFR antibody Panitumumab-WT-113, respectively.

[0139] SEQ ID NO:46-47 show the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-EGFR antibody Panitumumab-68, respectively.

[0140] SEQ ID NO:48 shows the amino acid sequence of GSV0-YBR003-C85-016-LC(Kappa).

[0141] SEQ ID NO:49 shows the amino acid sequence of YBR003_H_PM_B181_116_2_1_230321.

[0142] SEQ ID NO:50 shows the amino acid sequence of GSV0-YBR003-C98-WT-LC(hkappa).

[0143] SEQ ID NO:51 shows the amino acid sequence of GSV0-YBR003-Panitumumab-WT-113-LC(hkappa).

[0144] SEQ ID NO:52 shows the amino acid sequence of GSV0-YBR003-Panitumumab-68-LC(kappa).

[0145] SEQ ID NO:53 shows the amino acid sequence of GSV0-YBR003-C85-Mix-4-HC(IgG1-REM-DouBody-F405L).

[0146] SEQ ID NO:54 shows the amino acid sequence of GSV0-YBR003-B181-116-HC(IgG1-REM-DouBody-F405L).

[0147] SEQ ID NO:55 shows the amino acid sequence of GSV0-YBR003-C98-002-HC(IgG1-REM-DouBody-F405L).

[0148] SEQ ID NO:56 shows the amino acid sequence of GSV0-YBR003-Panitumumab-WT-113-HC(IgG1-REM-DouBody-K409R).

[0149] SEQ ID NO:57 shows the amino acid sequence of GSV0-YBR003-Panitumumab-68-HC(IgG1-REM-DouBody-K409R).

[0150] SEQ ID NO:58 shows the nucleotide sequence encoding GSV0-YBR003-C85-016-LC(Kappa).

[0151] SEQ ID NO:59 shows the nucleotide sequence encoding YBR003_H_PM_B181_116_2_1_230321.

[0152] SEQ ID NO:60 shows the nucleotide sequence encoding GSV0-YBR003-C98-WT-LC(hkappa).

[0153] SEQ ID NO:61 shows the nucleotide sequence encoding GSV0-YBR003-Panitumumab-WT-113-LC(hkappa).

[0154] SEQ ID NO:62 shows the nucleotide sequence encoding GSV0-YBR003-Panitumumab-68-LC(kappa).

[0155] SEQ ID NO:63 shows the nucleotide sequence encoding GSV0-YBR003-C85-Mix-4-HC(IgG1-REM-DouBody-F405L).

[0156] SEQ ID NO:64 shows the nucleotide sequence encoding GSV0-YBR003-B181-116-HC(IgG1-REM-DouBody-F405L).

[0157] SEQ ID NO:65 shows the nucleotide sequence encoding GSV0-YBR003-C98-002-HC(IgG1-REM-DouBody-F405L).

[0158] SEQ ID NO:66 shows the nucleotide sequence encoding GSV0-YBR003-Panitumumab-WT-113-HC(IgG1-REM-DouBody-K409R).

[0159] SEQ ID NO:67 shows the nucleotide sequence encoding GSV0-YBR003-Panitumumab-68-HC(IgG1-REM-DouBody-K409R).

[0160] Detailed description of the invention

[0161] definition

[0162] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel).

[0163] The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0164] Although the broad scope of this application refers to the numerical ranges and parameter approximations shown, the values ​​described in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained herein. Additionally, any references marked "incorporated herein" should be understood to be incorporated herein in their entirety.

[0165] The term “object” or “individual” as used in this article refers to mammals, such as humans, but can also refer to other animals, such as wild animals, livestock, or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0166] As used herein, the term "antigen" is a predetermined target to which an antibody can selectively bind. Examples of antigens include, but are not limited to, peptides, sugars, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds.

[0167] In a broad sense, an "antibody" can refer to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Therefore, it encompasses complete antibodies / full-length antibodies, single-chain antibodies, or any antigen-binding fragment of an antibody (also known as an "antigen-binding moiety"). When "antibody" and "antigen-binding fragment / antigen-binding moiety" appear in the same context, "antibody" can be understood as the complete entity relative to the "antigen-binding fragment / antigen-binding moiety," and both correspond to the broad concept of antibody.

[0168] A "full-length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain constant region contains one domain: CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant domain of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be designated into different classes based on the antibody amino acid sequence of the constant domain of the heavy chain. Typically, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known. Chimeric or humanized antibodies are also included in the antibodies according to this application. It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of an antibody. There are several common ways to define the CDR amino acid sequence of VH or VL, such as the Kabat definition, the IMGT definition, and the Chothia definition. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the VH and VL amino acid sequences can usually be determined according to different definitions. In the embodiments of this application, the CDR amino acid sequence is defined using Kabat or Chothia. For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the variable region amino acid sequence can be analyzed using various methods.

[0169] For a given antibody's variable region amino acid sequence, the CDR amino acid sequence can be analyzed in various ways, such as using the online software Abysis (http: / / www.abysis.org / ).

[0170] The term "humanized antibody" refers to an antibody obtained by grafting a CDR sequence derived from another mammalian species, such as a mouse line, onto a human framework sequence. To preserve binding affinity, certain residues in the backbone (called FR) segment can be modified. Humanized antibodies or fragments thereof according to this application can be prepared using techniques known to those skilled in the art.

[0171] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. Chimeric antibodies or fragments thereof according to this application can be prepared using gene recombination technology. For example, the chimeric antibody can be produced by cloning recombinant DNA containing a promoter and a sequence encoding the variable region of a non-human, particularly mouse, monoclonal antibody according to this application, and a sequence encoding the constant region of a human antibody. The chimeric antibody of this application encoded by such a recombinant gene will be, for example, a mouse-human chimera, whose specificity is determined by the variable region derived from mouse DNA and whose isotype is determined by the constant region derived from human DNA.

[0172] The term "monoclonal antibody" refers to an antibody obtained from a population of essentially homogeneous antibodies, meaning that the individual antibodies that make up the population are identical, except that naturally occurring mutations may exist in a small number of individuals.

[0173] As used herein, the terms “antigen-binding fragment” or “antigen-binding moiety” or “antigen-binding region” are used interchangeably to refer to a portion of an antibody containing amino acid residues that interact with the antigen and confer specificity and affinity to the antigen, particularly antibody fragments such as Fv, Fab, F(ab')2, or Fab', or any fragment that should be chemically modified or incorporated into liposomes to increase its half-life, such as by adding poly(alkylene) glycols like polyethylene glycol (“PEGylation”) (referred to as PEGylated fragments such as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) (“PEG” stands for polyethylene glycol). Preferably, the functional fragment will consist of or contain a portion of the heavy or light chain variable chain of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, and such functional fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of its source antibody sequence.

[0174] Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which may be divalent fragments having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; (3) Fv fragments having a single arm of antibody with VL and VH domains; (4) single-chain Fv(scFv), which may be a single multipeptide chain consisting of VH and VL domains connected by peptide linkers; and (5) (scFv)2, which may contain two VH domains and two VL domains connected by peptide linkers, the two VL domains being combined with the two VH domains via disulfide bridges.

[0175] The terms "Fc fragment," "Fc region," "Fc domain," "Fc part," or similar terms refer to a portion of the constant region of the antibody heavy chain, including the hinge region, the CH2 segment, and the CH3 segment of the constant region. The Fc region of an antibody can be engineered or modified, including effector-related modifications, to reduce or eliminate antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), for example, by introducing one or more amino acid substitutions / mutations into the Fc region of the antibody.

[0176] As used in this article, "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

[0177] The term "bispecific antibody" refers to an antibody that simultaneously possesses the ability to bind to two antigenic epitopes. These two epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can consist of two Fc fragments and two antigen-binding portions fused to them (similar to natural antibodies, except that the two arms bind to different antigenic targets or epitopes). The antigen-binding portions can be in the form of a single-chain antibody (scFv) or a Fab fragment. Each of the two different binding portions of the bispecific antibody binds to the N-terminus of an Fc fragment. The antigen-binding portions of the two arms can be configured in four ways: scFv + Fab fragment, Fab fragment + scFv, scFv + scFv, and Fab fragment + Fab fragment. In the embodiments of this application, the antigen-binding portions of the two arms of the bispecific antibody are configured as Fab fragment + Fab fragment.

[0178] Fc fragments can contain mutations that ensure heavy chain heteropolymerization. Knob-in-hole (KIH) technology is a strategy to address heavy chain heteropolymerization. Typically, KIH technology involves modifying the amino acid sequence of the CH3 region to create a structure that facilitates the pairing of heterologous half-antibodies, thus enabling the formation of bispecific antibodies while preserving the structure of normal antibodies as much as possible.

[0179] Generally, to prepare monoclonal antibodies or their functional fragments, especially murine monoclonal antibodies or their functional fragments, one can refer to the techniques described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or refer to the techniques described by Kohler and Milstein for preparation from hybridoma cells (Nature, 256: 495-497, 1975).

[0180] The term "conservative variant" or "conservative amino acid substitution" refers to those substitutions that substantially do not affect or reduce the affinity of a protein. For example, an antibody may include up to about 1, up to 2, up to 5, up to 10, or up to 15 conserved substitutions and specifically bind to the target antigen. The term "conservative variant" also includes the use of substituted amino acids in place of unsubstituted parental amino acids, provided that the antibody specifically binds to the target antigen.

[0181] The term "isolated" refers to biological components (e.g., nucleic acids, proteins (including antibodies), or organelles) that have been substantially separated from or purified from other biological components (i.e., other and extra chromosomal DNA and RNA, proteins, and organelles) in their natural environment (e.g., cells). "Isolated" nucleic acids and proteins include those purified using standard purification methods. The term also includes nucleic acids and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0182] As used herein, the term "pharmaceutical composition" refers to at least one pharmaceutical product combined together to achieve a particular purpose, along with optionally pharmaceutically acceptable excipients, diluents, or carriers. In some embodiments, the pharmaceutical composition comprises combinations that are separate in time and / or space, provided they can work together to achieve the purpose of this application. For example, components contained in the pharmaceutical composition (e.g., bispecific antibodies according to this application) may be administered to an individual as a whole or separately. When components contained in the pharmaceutical composition are administered to an individual separately, the components may be administered to the individual simultaneously or sequentially. Pharmaceutically acceptable carriers may also include water, buffered aqueous solutions, isotonic saline solutions such as PBS (phosphate-buffered saline), glucose, mannitol, dextran, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The pharmaceutical composition or pharmaceutical preparation according to this application may be administered via any suitable route, such as oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc. The pharmaceutical composition according to this application may also contain one or more of the following: lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid, and calcium propionate; sweeteners and / or flavoring agents, etc.

[0183] The term "therapeutic effective dose" or "effective dose" as used herein refers to a dose sufficient to demonstrate its benefit to the individual to which it is administered. The actual amount administered, as well as the rate and duration of administration, will depend on the individual's condition and severity. Prescribing treatment (e.g., determining the dosage) is ultimately the responsibility of and depends on the general practitioner and other physicians, who typically consider the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0184] EC 50 EC50 primarily refers to the concentration of a drug, antibody, or toxin that, after a specific exposure time, achieves 50% of its maximum biological effect. In pharmaceutical science, besides characterizing the activating ability of agonists in in vitro experiments, it can also be used to indicate the blood drug concentration required to achieve half of the maximum biological effect in vivo. In some literature, EC50... 50 EC values ​​are also used to characterize the potency of a compound at the cellular level (including agonist and antagonist), and can be measured by methods such as ELISA. 50 value.

[0185] The term "identity / homology / consistency" in relation to amino acid or nucleic acid sequences is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, achieving the maximum percentage of identity if desired. The methods and computer programs used for alignment are well known in the art.

[0186] In a first aspect, this application provides a bispecific antibody comprising a first antigen-binding fragment against human mesenchymal-epithelial transforming factor (c-Met) and a second antigen-binding fragment against human epidermal growth factor receptor (EGFR);

[0187] Wherein, the first antigen-binding fragment comprises:

[0188] HCDR1, as shown in SEQ ID NO:1,

[0189] HCDR2, as shown in SEQ ID NO:2,

[0190] HCDR3, as shown in SEQ ID NO:3,

[0191] LCDR1, as shown in SEQ ID NO:4,

[0192] LCDR2, as shown in SEQ ID NO:5, and

[0193] As shown in SEQ ID NO:6, LCDR3; or

[0194] HCDR1, as shown in SEQ ID NO:7,

[0195] HCDR2, as shown in SEQ ID NO:8,

[0196] HCDR3, as shown in SEQ ID NO:9,

[0197] LCDR1, as shown in SEQ ID NO:10,

[0198] LCDR2, as shown in SEQ ID NO:11, and

[0199] As shown in SEQ ID NO:12, LCDR3; or

[0200] HCDR1, as shown in SEQ ID NO:13,

[0201] HCDR2, as shown in SEQ ID NO:14,

[0202] HCDR3, as shown in SEQ ID NO:15,

[0203] LCDR1, as shown in SEQ ID NO:16,

[0204] LCDR2, as shown in SEQ ID NO:17, and

[0205] LCDR3 as shown in SEQ ID NO:18; and

[0206] The second antigen-binding fragment includes:

[0207] As shown in SEQ ID NO:19, HCDR1,

[0208] HCDR2, as shown in SEQ ID NO:20,

[0209] HCDR3, as shown in SEQ ID NO:21,

[0210] LCDR1, as shown in SEQ ID NO:22,

[0211] LCDR2, as shown in SEQ ID NO:23, and

[0212] As shown in SEQ ID NO:24, LCDR3; or

[0213] As shown in SEQ ID NO:19, HCDR1,

[0214] HCDR2, as shown in SEQ ID NO:25,

[0215] HCDR3, as shown in SEQ ID NO:21,

[0216] LCDR1, as shown in SEQ ID NO:22,

[0217] LCDR2, as shown in SEQ ID NO:26, and

[0218] LCDR3 as shown in SEQ ID NO:27;

[0219] HCDR and LCDR are defined according to Kabat.

[0220] Secondly, this application provides a bispecific antibody comprising a first antigen-binding fragment against human mesenchymal-epithelial transforming factor (c-Met) and a second antigen-binding fragment against human epidermal growth factor receptor (EGFR);

[0221] Wherein, the first antigen-binding fragment comprises:

[0222] HCDR1, as shown in SEQ ID NO:28,

[0223] HCDR2, as shown in SEQ ID NO:29,

[0224] HCDR3, as shown in SEQ ID NO:3,

[0225] LCDR1, as shown in SEQ ID NO:4,

[0226] LCDR2, as shown in SEQ ID NO:5, and

[0227] As shown in SEQ ID NO:6, LCDR3; or

[0228] HCDR1, as shown in SEQ ID NO:30,

[0229] HCDR2, as shown in SEQ ID NO:31,

[0230] HCDR3, as shown in SEQ ID NO:9,

[0231] LCDR1, as shown in SEQ ID NO:10,

[0232] LCDR2, as shown in SEQ ID NO:11, and

[0233] As shown in SEQ ID NO:12, LCDR3; or

[0234] As shown in SEQ ID NO:32, HCDR1,

[0235] HCDR2, as shown in SEQ ID NO:33,

[0236] HCDR3, as shown in SEQ ID NO:15,

[0237] LCDR1, as shown in SEQ ID NO:16,

[0238] LCDR2, as shown in SEQ ID NO:17, and

[0239] LCDR3 as shown in SEQ ID NO:18; and

[0240] The second antigen-binding fragment includes:

[0241] HCDR1, as shown in SEQ ID NO:34,

[0242] HCDR2, as shown in SEQ ID NO:35,

[0243] HCDR3, as shown in SEQ ID NO:21,

[0244] LCDR1, as shown in SEQ ID NO:22,

[0245] LCDR2, as shown in SEQ ID NO:23, and

[0246] As shown in SEQ ID NO:24, LCDR3; or

[0247] HCDR1, as shown in SEQ ID NO:36,

[0248] HCDR2, as shown in SEQ ID NO:37,

[0249] HCDR3, as shown in SEQ ID NO:21,

[0250] LCDR1, as shown in SEQ ID NO:22,

[0251] LCDR2, as shown in SEQ ID NO:26, and

[0252] LCDR3 as shown in SEQ ID NO:27;

[0253] HCDR and LCDR are defined according to Chothia.

[0254] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38, 40 or 42, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO:38, 40 or 42.

[0255] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment differs from the amino acid sequence shown in SEQ ID NO:38, 40 or 42 by substitution, deletion and / or addition of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0256] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence represented by SEQ ID NO:38, 40 or 42 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of a similar heavy chain variable region of the first antigen-binding fragment.

[0257] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 38, 40 or 42, and the resulting amino acid sequence still retains the function of the heavy chain variable region similar to that of the first antigen-binding fragment.

[0258] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 38, 40 or 42, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the first antigen-binding fragment.

[0259] In some embodiments described in the first or second aspect, the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39, 41 or 43, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO:39, 41 or 43.

[0260] In some embodiments described in the first or second aspect, the amino acid sequence of the light chain variable region of the first antigen-binding fragment differs from the amino acid sequence shown in SEQ ID NO:39, 41 or 43 by substitution, deletion and / or addition of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0261] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:39, 41 or 43 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of a similar light chain variable region of the first antigen-binding fragment.

[0262] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 39, 41 or 43, and the resulting amino acid sequence still retains the function of the light chain variable region similar to that of the first antigen-binding fragment.

[0263] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 39, 41 or 43, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the first antigen-binding fragment.

[0264] In some embodiments of the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:39; or

[0265] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:40 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:41 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:41; or

[0266] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:42 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:43 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:43.

[0267] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39; or

[0268] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:41; or

[0269] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:43.

[0270] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 or 46, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44 or 46.

[0271] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment differs from the amino acid sequence shown in SEQ ID NO:44 or 46 by substitution, deletion, and / or addition of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0272] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:44 or 46 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of a similar heavy chain variable region of the second antigen-binding fragment.

[0273] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 44 or 46, and the resulting amino acid sequence still retains the function of the heavy chain variable region similar to that of the second antigen-binding fragment.

[0274] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:44 or 46, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the second antigen-binding fragment.

[0275] In some embodiments described in the first or second aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45 or 47 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45 or 47.

[0276] In some embodiments described in the first or second aspect, the amino acid sequence of the light chain variable region of the second antigen-binding fragment differs from the amino acid sequence shown in SEQ ID NO:45 or 47 by substitution, deletion, and / or addition of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0277] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:45 or 47 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of a similar light chain variable region of the second antigen-binding fragment.

[0278] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 45 or 47, and the resulting amino acid sequence still retains the function of the light chain variable region similar to that of the second antigen-binding fragment.

[0279] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 45 or 47, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the second antigen-binding fragment.

[0280] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or

[0281] The amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:46 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:47 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47.

[0282] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45; or

[0283] The amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:47.

[0284] In some embodiments of the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39 .... The sequence shown in NO:39 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45 ... or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45. The sequence shown in NO:45 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity; or

[0285] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:40 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:41 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:41, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:40. The sequence shown in SEQ ID NO:44 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:45 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or

[0286] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:42 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:43 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:43, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:42. The sequence shown in SEQ ID NO:44 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:45 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or

[0287] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:39, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:38. The sequence shown in SEQ ID NO:46 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:47 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47; or

[0288] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:40 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:41 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:41, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:40. The sequence shown in SEQ ID NO:46 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:47 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47; or

[0289] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:42 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:43 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:43, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:42. The sequence shown in SEQ ID NO:46 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment as shown in SEQ ID NO:47 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47.

[0290] In some embodiments of the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45; or

[0291] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:41; the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:45; or

[0292] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:43; the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:45; or

[0293] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:39; the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:47; or

[0294] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:41; the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:47; or

[0295] The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:42 and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:43, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:46 and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:47.

[0296] In some embodiments described in the first or second aspect, the bispecific antibody is an IgG1 antibody comprising a first heavy chain constant region and a second heavy chain constant region, wherein the amino acid at position 405 of the first heavy chain constant region is L and the amino acid at position 409 of the second heavy chain constant region is R, wherein the amino acid positions of the antibody constant region are determined according to EU numbering.

[0297] In some embodiments described in the first or second aspect, the bispecific antibody is an IgG1 antibody comprising a first heavy chain constant region and a second heavy chain constant region, wherein the amino acid at position 409 of the first heavy chain constant region is R, and the amino acid at position 405 of the second heavy chain constant region is L, wherein the amino acid positions of the antibody constant region are determined according to EU numbering.

[0298] In some embodiments described in the first or second aspect, the bispecific antibody comprises a first light chain constant region and a second light chain constant region, wherein the first light chain constant region and / or the second light chain constant region is a κ subtype or a λ subtype. In some embodiments, the bispecific antibody comprises a first light chain constant region and a second light chain constant region, wherein the first light chain constant region and / or the second light chain constant region is a κ subtype.

[0299] In some embodiments described in the first or second aspect, the first antigen-binding fragment and the second antigen-binding fragment are independently selected from single-chain antibodies (scFv) or Fab fragments. In some embodiments, both the first antigen-binding fragment and the second antigen-binding fragment are Fab fragments.

[0300] In some embodiments described in the first or second aspect, the bispecific antibody is in the form of Doubody, Diabody, CrossMab, DVD-ig, Knob-in-Hole, BiTE, DART, YBODY, or FIT-Ig. In some embodiments, the bispecific antibody is in the form of Doubody. For a description of the form of the bispecific antibody, see AFLabrijn, ML Janmaat, JM Reichert, PWHI Parren, Bispecific antibodies: a mechanism review of the pipeline, Nat Rev Drug Discov. 2019 Aug; 18(8):585-608.

[0301] Thirdly, this application provides a pharmaceutical composition comprising the bispecific antibody described in the first or second aspect, and a pharmaceutically acceptable excipient, diluent, or carrier.

[0302] In some embodiments of the third aspect, the pharmaceutical composition is used to prevent or treat cancer in a subject of need.

[0303] In some implementations of the third aspect, the cancer cells of the cancer express EGFR.

[0304] In some embodiments of the third aspect, the cancer cells of the cancer overexpress EGFR.

[0305] In some implementations of the third aspect, the cancer is selected from: lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), liver cancer, colon cancer, breast cancer, brain cancer, head and neck cancer, pancreatic cancer, ovarian cancer, prostate cancer, stomach cancer, nervous system cancers (e.g. glioblastoma) and metastatic lesions of the above cancers.

[0306] In some embodiments of the third aspect, the pharmaceutical composition may further comprise one or more of the following: lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid, and calcium propionate; sweeteners and / or flavoring agents, etc.

[0307] In some embodiments of the third aspect, the pharmaceutical composition of this application may be formulated as tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.

[0308] In some embodiments of the third aspect, the pharmaceutical composition of this application may be delivered using any physiologically acceptable method of administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.

[0309] In some embodiments of the third aspect, a pharmaceutical composition for therapeutic use can be formulated for storage by mixing a reagent of the desired purity with, as appropriate, a pharmaceutically acceptable carrier, excipient, etc., in the form of a lyophilized formulation or an aqueous solution.

[0310] In some embodiments of the third aspect, the pharmaceutical composition is provided as a sterile fresh formulation or a sterile frozen formulation. In some embodiments, where conditions permit, the pharmaceutical composition may be provided directly to the patient as a sterile fresh formulation. If long-term storage (e.g., for multiple doses) or transportation is required, the pharmaceutical composition may be provided as a sterile frozen formulation, which is thawed and rehydrated before use.

[0311] Fourthly, this application provides the use of the bispecific antibody described in the first or second aspect or the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention or treatment of cancer in a subject of need.

[0312] In some implementations of the fourth aspect, the cancer cells of the cancer express EGFR.

[0313] In some embodiments of the fourth aspect, the cancer cells of the said cancer overexpress EGFR.

[0314] In some implementations of the fourth aspect, the cancer is selected from: lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), liver cancer, colon cancer, breast cancer, brain cancer, head and neck cancer, pancreatic cancer, ovarian cancer, prostate cancer, stomach cancer, nervous system cancers (e.g. glioblastoma) and metastatic lesions of the above cancers.

[0315] Fifthly, this application provides a method for preventing or treating cancer in a subject in need, comprising administering to the subject an effective amount of the bispecific antibody described in the first or second aspect or the pharmaceutical composition described in the third aspect.

[0316] In some embodiments of the fifth aspect, the cancer cells of the cancer express EGFR.

[0317] In some embodiments of the fifth aspect, the cancer cells of the cancer overexpress EGFR.

[0318] In some implementations of the fifth aspect, the cancer is selected from: lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), liver cancer, colon cancer, breast cancer, brain cancer, head and neck cancer, pancreatic cancer, ovarian cancer, prostate cancer, stomach cancer, nervous system cancers (e.g. glioblastoma) and metastatic lesions of the above cancers.

[0319] In other aspects, this application also provides a nucleic acid molecule encoding a light or heavy chain of a bispecific antibody of this application, or a first or second antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, a host cell comprising the nucleic acid molecule or the vector, and a method for producing the bispecific antibody. In some embodiments, the nucleic acid molecule is operatively linked to a regulatory nucleotide sequence that can be recognized by a host cell transformed with the vector. In some embodiments, the method of producing the antibody includes culturing the host cell to express the nucleic acid. In some embodiments, the method of producing the antibody further includes recovering the antibody from the host cell culture medium.

[0320] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments. Example

[0321] The present application will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0322] Example 1: Screening of c-Met antibodies from a humanized recombinant antibody library

[0323] This experiment employed immunosorbent assay (IRISA) tubes and a magnetic bead screening instrument, using solid-phase, liquid-phase, and cell-based cross-screening methods to screen our constructed humanized recombinant antibody library. Specific Fab antibodies against huMet-his, CynoMet-His, and huMet-HEK293 were enriched using trypsin and glycine hydrochloride elution. The enrichment of different output sets was detected by ELISA, yielding several well-enriched output sets at the ELISA level. After initial ELISA screening, a total of 1710 clones were selected, including 425 positive clones binding to humans, 262 positive clones binding to monkeys, and 232 positive clones from the human-monkey cross-scan. Analysis yielded 43 molecules with unique sequences, which were then constructed. A third initial screening (adapted from the second preliminary screening) selected 990 clones, including 597 positive clones binding to humans, 312 positive clones binding to monkeys, and 297 positive clones from the human-monkey cross-scan. The 297 positive clones from the human-monkey cross-scan were sent for testing. 76 molecules with unique sequences were obtained, 58 new molecules were added, and 52 molecules were finally constructed.

[0324] 1.1 Screening of Humanized Recombinant Antibody Libraries (Kingfisher Method)

[0325] The implementation method is as follows: The preserved natural library phage suspension was diluted and blocked with 2.5% BSA, then co-incubated with Dynabeads. Phages incubated after negative screening were collected. Dynabeads were used for blocking and coating, and the magnetic beads were bound and washed according to the Kingfisher magnetic bead screening system method. The magnetic beads were then incubated with 2.5% BSA. The phage suspension collected after negative screening was incubated with Dynabeads coated and blocked with biotin-labeled antigen, and bound and washed according to the Kingfisher magnetic bead screening system method. Phages were eluted with trypsin. After elution, the phage solution was thoroughly mixed with logarithmic SS320 cells and incubated at 37°C for 30 min. The mixture was then plated on 2YT-Car+-Tet+ plates and incubated overnight at 37°C. The eluted phage solution was serially diluted 10-fold with logarithmic-phase SS320 cells, incubated at 37°C for 30 min, mixed thoroughly, and then 2 μL was dropped onto a plate and incubated overnight at 37°C. Phage input and output were calculated, and the input phages were prepared by scraping and screened in three rounds. Each round of screening used a 3-fold gradient of antigen concentration decreasing to obtain antibody clones with high affinity.

[0326] 1.2 Screening of Humanized Recombinant Antibody Library (Immunotube Method)

[0327] The implementation method is as follows: Background subtraction: The preserved natural library phage suspension was blocked with 5% PBSM. The antigen protein was coated with 5% PBSM and blocked. The phage suspension and protein-coated and blocked immunotubes were incubated, and binding and washing were performed according to the immunotube screening system method. The phage was eluted with trypsin. After elution, the phage solution was thoroughly mixed with logarithmic-phase SS320 cells and incubated at 37°C for 30 min. It was plated on 2YT-Car+-Tet+ plates and incubated overnight at 37°C. The eluted phage solution was serially diluted 10-fold with logarithmic-phase SS320 cells, incubated at 37°C for 30 min, mixed well, and 2 μL was dropped onto a plate and incubated overnight at 37°C. Phage input and output were calculated, and the input phage was prepared by scraping and three rounds of screening were performed. Each round of screening used a 3-fold gradient of antigen concentration decreasing to obtain antibody clones with high affinity.

[0328] 1.3 Initial screening of humanized recombinant antibody libraries (cell screening)

[0329] The procedure is as follows: For blocking, dilute the panned phage 4-fold with 5% FBS-PBS. Add 3 mL of 5% FBS to 1 mL of phage and mix and block at room temperature for 1 hour on a four-dimensional rotating incubator. Using a disposable pipette, slowly add the blocked phage to the prepared cell culture flask along the sidewall. Before this, negative sieving is required. First, add the blocked phage to the negative sieve cells, adding from the cell-free side to avoid washing away the cells. Then, mix and bind on a track-mounted shaker at 80 rpm for 1 hour at room temperature. After binding, gently stand the cell culture flask upright and use a disposable pipette to aspirate the negatively sieved phage library and add it to the corresponding positively sieved cell culture flask. Incubate on a track-mounted shaker at 80 rpm for 2 hours at room temperature. Discard the supernatant from the culture flask. Add 5% FBS-PBS along the non-cell side, avoiding blowing the cells away. Then, invert the cell culture plate to cover the cells with the liquid. After each addition of 5% FBS-PBS, shake the plate on a track-type shaker at 80 rpm for 1 minute. Then, slowly stand the cell culture flask upright and carefully aspirate the elution using a disposable pipette. The number of washes depends on the screening round. Add 2 mL of 100 mM glycine-HCl (pH 2.2) and incubate at room temperature on a shaker at 80 rpm for 10 minutes (do not exceed 10 minutes). Add 140 μL of 1 M Trisbase to neutralize to pH 7–7.2 (the volume added needs to be adjusted when preparing the solution). Transfer the eluent to a new 15 mL centrifuge tube.

[0330] By using ELISA to detect the enrichment of different output sets, this screening yielded several output sets with good enrichment at the ELISA level.

[0331] 1.4 Single-clone selection

[0332] After initial ELISA screening, a total of 1710 clones were selected, including 425 positive clones that bound to humans, 262 positive clones that bound to monkeys, and 232 positive clones that crossed human and monkey genes. Analysis yielded 43 molecules with unique sequences, which were then constructed. The third initial screening (adapted from the second preliminary screening) selected 990 clones, including 597 positive clones that bound to humans, 312 positive clones that bound to monkeys, and 297 positive clones that crossed human and monkey genes. The 297 positive clones from the human-monkey cross were sent for testing. This resulted in 76 molecules with unique sequences, with 58 new molecules added, ultimately leading to the construction of 52 molecules.

[0333] Based on a comprehensive evaluation of antigen-binding ability, positive cell binding ability, blocking ability, and affinity kinetics, monoclonal antibodies B181, C85, and C98 were selected.

[0334] Example 2: c-Met antibody affinity maturation

[0335] In Example 1, the selected monoclonal molecules B181, C85, and C98 exhibited inferior affinity activity compared to the control antibody Met-H (the anti-cMet antigen-binding fragment in the bispecific antibody ervantumab). Antibody affinity activity was improved through antibody engineering. A phage display mutant library that passed quality control was screened using huMet-Fc, huMet-His, c-MET Protein human target antigen, and human c-Met CHO-S and huMetHEK293 cells to obtain candidate molecules with affinity activity superior to the parent antibody. This process generally involved three steps:

[0336] 1. Library construction: Four phage display mutant antibody libraries were designed according to the library construction strategy. After electroporation, the library volume was determined by dilution plate method, and the correct insertion rate of antibody genes was verified by single-clone sequencing analysis.

[0337] 2. Library Screening: Phage mutant antibody libraries were screened using three methods: immunotube screening, magnetic bead screening, and cell screening. These methods included solid-phase, liquid-phase, and cell screening. Antibody molecules that specifically bound the target antigen and exhibited superior affinity to the parent antibody were screened at the ELISA level.

[0338] 3. Candidate molecule validation: After eukaryotic expression and purification, candidate molecules are subjected to SDS detection, affinity kinetics detection, ELISA and FACS detection.

[0339] 2.1 Construction of antibody affinity maturation library

[0340] Mutant primers were designed according to the library construction strategy. Using a correctly sequenced maternal plasmid as a template, the target fragment was amplified using molecular cloning technology, and the antibody gene was recombined using in vitro ligation. After obtaining the antibody gene, the vector and gene sequence were digested with enzymes, and the antibody nucleotide sequence was constructed into a phage display vector. The constructed vector was then electroporated into E. coli to obtain a phage display library.

[0341] 2.2 Screening of antibody affinity maturation libraries

[0342] Phage display mutant libraries or obtained output sets are processed through a series of steps including inoculation, helper phage infection, phage amplification, phage precipitation and resuspension to prepare enriched phages for the next round of screening. Biotin-labeled antigens are combined with streptavidin-conjugated magnetic beads, then incubated, washed, and eluted with the prepared phages. After 3-4 rounds of screening, specific monoclonal antibodies against the antigen are enriched. Antigens are coated onto the surface of highly absorbent immunotubes, and the prepared phages are added to the immunotubes for incubation, washing, and elution. After 3-4 rounds of screening, specific monoclonal antibodies against the antigen are enriched. Negatively screened phages and antigen-overexpressing cells are incubated, washed, and eluted. After 3-4 rounds of screening, specific monoclonal antibodies against the antigen are enriched. The enrichment effect is detected by ELISA on the selected output sets. The process involved a series of steps, including plating, blocking, primary antibody incubation, secondary antibody addition, color development, termination, and OD value detection. For the phage supernatant, the titer values ​​of each output set were determined using the dilution plating method. A graph was plotted with titer on the x-axis and OD value on the y-axis, using the parent's EC50 as the reference value. 50 The enrichment level in the pool was evaluated using the target antigen as a reference. The process involved a series of steps, including plate coating, blocking, primary antibody incubation, secondary antibody addition, color development, termination, and OD value detection. Clones with OD values ​​higher than the parent clone were selected as preferred clones and submitted for testing.

[0343] 2.3 Validation of antibody affinity maturation candidate molecules

[0344] The sequences of candidate molecules were constructed into the cDNA-3.4 vector, and plasmids were obtained after PCR, enzyme digestion, ligation, transformation, identification, sequencing, alignment, and extraction. The candidate molecule plasmids were expressed in CHO cells using the ExpiCHOs expression system, and finally purified, aliquoted, and subjected to quality control. Candidate molecules that passed quality control underwent affinity kinetics, ELISA, FACS binding, and FACS blocking tests.

[0345] Through phage display library construction, screening, and selection, 39 B181 candidates with specific binding to the target antigen and superior affinity to the parent antibody were obtained. Affinity kinetics analysis revealed that one full-length antibody (B181_116_2_10_B181_116(P97315)) had an affinity more than 5 times higher than the parent antibody and exhibited superior blocking activity compared to the parent B181-WT and the control antibody Met H. Figure 4 The binding and dissociation curves of B181_116_2_10_B181_116 with the antigen are shown in the figure. Figure 5 .

[0346] ELISA results for the C98 maternal antibody showed that one molecule (C98-002-C98-WT(P89556)) had a higher affinity than the control antibody Met H and the maternal antibody NL-C98. Figure 6 The binding and dissociation curves of C98-002-C98-WT with the antigen are shown below. Figure 7 .

[0347] FACS binding assays of the C85-WT parent antibody showed that all candidate molecules were superior to the parent antibody, and the Top MFI of C85-071-C85-WT was comparable to that of the control antibody Met H. FACS blocking assays showed that, except for C85-008-C85-WT and C85-063-C85-WT, which were weaker than the control antibody (Met H), the remaining candidate molecules were slightly stronger than the control antibody. Affinity kinetics results showed that four molecules (C85-055-C85-WT, C85-053-C85-WT, C85-024-C85-015, and C85-071-C85-WT) had a affinity that was more than 5-fold higher than that of the parent antibody. Following a second round of affinity maturation, functional evaluation was conducted using affinity kinetics and FACS binding / blocking. The four mixed molecules demonstrated superior FACS binding / blocking performance on NCI-H292 cells compared to the parental C85-WT and comparable to the control antibody Met H. Figure 8 Based on comprehensive data, C85-mix-4-C85-016 was selected as the optimal choice. Its binding and dissociation curves with the antigen are shown below. Figure 9 .

[0348] Example 3: EGFR antibody affinity maturation

[0349] Due to the need for bispecific antibody design, the affinity of the Panituzumab-WT molecule was reduced through antibody engineering to decrease its affinity activity. A phage display mutant library that passed quality control was screened using EGFR-P00533-25-645-Fc, EGFR-P00533-25-645-Chis, and EGFR-P00533-25-645-FC (IgGI) target antigens and NCl-H292 cells to obtain candidate molecules with affinity inferior to the parent antibody. This process can be roughly divided into three steps:

[0350] 1. Library construction: Four phage display mutant antibody libraries were designed according to the library construction strategy. The library numbers were AM1237, AM1238, AM1239 and AM1240. After electroporation, the library volume was determined by dilution plate method, and the correct insertion rate of antibody genes was verified by single-clone sequencing analysis.

[0351] 2. Library Screening: Immunotube and cell screening were employed, using both solid-phase and cell-based methods, to screen phage mutant antibody libraries. Antibody molecules that specifically bind to the target antigen and exhibit inferior affinity to the parent antibody were screened at the ELISA level.

[0352] 3. Candidate molecule validation: After eukaryotic expression and purification, candidate molecules are identified by SDS-PAGE, SEC, ELISA, affinity kinetics and FACS.

[0353] 3.1 Construction of antibody affinity maturation library

[0354] Mutant primers were designed according to the library construction strategy. Using a correctly sequenced maternal plasmid as a template, the target fragment was amplified using molecular cloning technology, and the antibody gene was recombined using in vitro ligation. After obtaining the antibody gene, the vector and gene sequence were digested with enzymes, and the antibody nucleotide sequence was constructed into a phage display vector. The constructed vector was then electroporated into E. coli to obtain a phage display library.

[0355] 3.2 Screening of antibody affinity maturation libraries

[0356] Phage display mutant libraries or obtained output sets are processed through a series of steps including inoculation, phage infection assistance, phage amplification, phage precipitation and resuspension to prepare enriched phages for the next round of screening. Biotin-labeled antigens are combined with streptavidin-conjugated magnetic beads, then incubated, washed, and eluted with the prepared phages. After 3-4 rounds of screening, specific monoclonal antibodies against the antigen are enriched. Antigens are coated onto the surface of highly absorbent immunotubes, and the prepared phages are added to the immunotubes for incubation, washing, and elution. After 3-4 rounds of screening, specific monoclonal antibodies against the antigen are enriched. Using non-biotin-targeted antigen incubation, a series of steps are performed including plating, blocking, primary antibody incubation, secondary antibody addition, color development, termination, and OD value detection. Clones with OD values ​​lower than the parent culture are selected as preferred clones and submitted for testing.

[0357] Through ELISA screening, a total of 2,610 positive clones were selected, and 35 candidate molecules with unique sequences and lower affinity than the parent antibody were obtained. Based on the combined screening data and sequence diversity analysis, 35 molecules were selected for full-length construction.

[0358] 3.3 Validation of antibody affinity maturation candidate molecules

[0359] Candidate molecules were constructed into eukaryotic expression vectors. After expression, Koff and FACS tests were performed on the supernatant to select molecules with weaker affinity than the parent antibody. After expression and purification, quality control was performed on the candidate molecules, including SDS-PAGE and SEC identification. Candidate molecules that passed the quality control were further tested for affinity kinetics, ELISA, and FACS to determine the affinity of the Panitumumab candidate antibody at the full-length antibody level.

[0360] Candidate molecules were identified by Koff and FACS in the supernatant, and then subjected to biochemical detection: ELISA results showed that one molecule (Panitumumab-57 (P97334)) had a weaker affinity than the maternal antibody Panituzumab-WT (…). Figure 11 FACS results showed that the three molecules (Panitumumab-57 (P97334), Panitumumab-66 (P97335), and Panitumumab-68 (P97336)) had weaker affinity at the cellular level than the maternal antibody and the control antibody EGFR (IgG1) (the anti-EGFR antibody in the bispecific antibody Amivantamab). Figure 12 Based on the antibody-antigen binding and dissociation curves, Panitumumab-WT-113 is preferred due to its slower antigen-antibody dissociation. Figure 14Based on comprehensive data, Panitumumab-68 and Panitumumab-WT-113 were selected as the preferred choices. Their binding and dissociation curves with the antigen are shown below. Figure 13 and Figure 14 .

[0361] Example 4: Preparation of anti-c-Met and EGFR bispecific antibodies

[0362] 4.1 Protein Replacement

[0363] 1) Remove the two maternal antibodies to be assembled from the -80℃ freezer and place them in an ice box to thaw.

[0364] 2) Transfer the antibody from 1) to an X mL 30KD ultrafiltration centrifuge tube, add 1×PBS buffer (pH 7.4) to X mL, centrifuge at 3500g to concentrate to Y mL, add buffer again to X mL, and repeat ultrafiltration 4 times (20mg>concentrated antibody amount>2mg, X=15, Y=1.5; concentrated antibody amount<2mg, X=0.5, Y=0.1).

[0365] 3) After ultrafiltration, the absorbance of the sample at 280 nm was detected using a Nano-300. The results were repeated three times and recorded. The protein concentration after replacement was usually greater than 2 mg / mL.

[0366] 4.2 Protein Reduction

[0367] 1) Heat the protein and 2-MEA solution together in a water bath to 31°C after changing the solution.

[0368] 2) Protein addition: Add equal amounts of the two parent molecules to the centrifuge tube according to the required amount.

[0369] 3) 2-MEA addition: Add 750mM of 2-MEA solution to make the final concentration of 2-MEA 75mM.

[0370] 4) Incubation: Place the solution from step 3) in a 31°C hot water bath and incubate for 5 hours without shaking.

[0371] 4.3 Reducing agent removal

[0372] 1) After incubation, add the sample to a 10kD dialysis bag using a sterile syringe, place it in 5L of 1×PBS buffer (pH 7.4) for dialysis for 4 hours, and then transfer it to a new 5L of 1×PBS buffer (pH 7.4) for dialysis overnight.

[0373] 2) The next morning, replace with 5L of 100mM Pro buffer (pH 5.0) (the last dialysis should avoid salt ions to facilitate CIEF detection) and dialyze for 4 hours.

[0374] 4.4 Repackaging and Testing

[0375] 1) Disassemble the assembled samples.

[0376] 2) Reserve approximately 150 μg of the assembled product and dilute it to 1–2 mg / mL for HIC, SDS, SEC, and iCIEF detection.

[0377] Example 5: Physicochemical characterization of anti-c-Met and EGFR bispecific antibodies

[0378] Several monospecific EGFR and c-Met antibodies were expressed as IgG1 antibodies with Fc substitutions of K409R or F405L (numbered according to the EU index) in their Fc region. The two parent antibodies to be assembled were removed from a -80°C freezer and thawed in an ice box. The antibodies were transferred to X mL 30KD ultrafiltration centrifuge tubes, and 1×PBS (pH 7.4) was added to X mL. The tubes were centrifuged at 3500g to concentrate to Y mL, and buffer was added again to X mL. Ultrafiltration was repeated four times (20 mg > concentrated antibody > 2 mg, X = 15, Y = 1.5; concentrated antibody < 2 mg, X = 0.5, Y = 0.1). After ultrafiltration, the absorbance of the sample at 280 nm was measured using a Nano-300 microscope. This was repeated three times and the results were recorded. The protein concentration after replacement was typically greater than 2 mg / mL. The replaced protein and 2-MEA solution were heated together in a water bath to 31°C. The two parent molecules were added to the centrifuge tubes in equal amounts as needed. Add 750 mM 2-MEA solution to bring the final 2-MEA concentration to 75 mM. Incubate the solution in a 31°C hot water bath for 5 hours without shaking. After incubation, add the sample to a 10 kDa dialysis bag using a sterile syringe, and dialyze in 5 L of 1×PBS (pH 7.4) buffer for 4 hours. Then, transfer to a fresh 5 L of 1×PBS buffer (pH 7.4) and dialyze overnight. The next morning, replace the buffer with 5 L of 100 mM Pro buffer (pH 5.0) (the final dialysis should avoid salt ions to facilitate CIEF detection) and dialyze for 4 hours.

[0379] HIC testing:

[0380] 1) Preparation of mobile phase: Mobile phase A: 20mM sodium phosphate, 1.5M ammonium sulfate, pH 7.0; Mobile phase D: 20mM sodium phosphate, pH 7.0, 25% isopropanol (v / v).

[0381] 2) Column conditions: TSKgel Butyl-NPR column (2.5) 4.6mm×35mm was used, column temperature was set to 25℃, and the detection baseline was stable.

[0382] 3) Parameter settings: Flow rate is set to 1 mL / min; sample injection volume is 50 μg; detection wavelength is 280 nm.

[0383] 4) Gradient elution

[0384] iCIEF detection:

[0385] 1) Preparation of electrophoresis buffer: Add 2 μL of pI marker 5.85, 2 μL of pI marker 9.99, 70 μL of 1% methylcellulose, 4 μL of 500mM Arg, 8 μL of Pharmalyte 3-10 and 80 μL of 10M urea respectively and mix well.

[0386] 2) Sample preparation: Add 34 μL of 1 mg / mL sample to 166 μL of electrophoresis buffer and mix well.

[0387] 3) Parameter settings: Pre-focusing: 1500V, 1.0min; Focusing: 3000V, 8.0min; Detection: UV, 0.005s; Sample tray temperature: 15℃; Sample loading time: 55s.

[0388] Table 1. Summary of Physicochemical Property Test Results

[0389]

[0390] Example 6: In vitro activity evaluation of bispecific antibodies

[0391] Candidate bispecific antibody molecules that pass quality control will be subjected to affinity kinetics, ELISA, FACS, and endocytosis assays for candidate molecule evaluation.

[0392] 6.1 Affinity Dynamics:

[0393] 6.1.1 Methods and Procedures - Gator Detection

[0394] 1) Select mode: Open the GATOR instrument and related software, and select the Kientics experimental mode.

[0395] 2) On-computer: The analysis procedure is shown in the table below.

[0396]

[0397] 6.1.2 System adaptability: K detected by the label-free biomolecule analyzer (Gator) D The limit was 1E-06M. Experimental results showed that the correlation coefficient R² of all antibodies in the Global fitting mode was greater than 0.95.

[0398] 6.1.3 Methods and Procedures - Biacore Detection: The device used was Biacore T200 (Cytiva), using a Protein A chip to capture bispecific antibody molecules onto the chip, with a serially diluted antigen as the mobile phase.

[0399] 6.2 FACS Detection

[0400] 6.2.1 Cell Plating

[0401] NCI-H292 cells were prepared into a cell suspension and the density was adjusted to 1×10⁻⁶. 6 Cells / mL. Take a 96-well round-bottom plate and use a 100μL pipette to add the cell suspension to each well. Centrifuge at 300g for 5 min. Discard the supernatant.

[0402] 6.2.2 Add antibody diluent

[0403] The antibody was diluted with FACS buffer to prepare eight concentration gradients: 150.0000, 37.5000, 9.3750, 2.3438, 0.5859, 0.1465, 0.0366, and 0.0092 nM. The antibody was also diluted with FACS buffer to prepare eight concentration gradients: 50.0000, 12.5000, 3.1250, 0.78125, 0.195313, 0.048828, 0.012207, and 0.001221 nM. Using a 100 μL pipette, 100 μL of the antibody dilution was added to each well of a plate and incubated at 4 °C for 60 min.

[0404] Wash plate: Wash cells twice with FACS buffer.

[0405] 6.2.3 Add secondary antibody

[0406] The secondary antibody PE-labeled anti-human IgG Fc was diluted 1:300 with FACS buffer and added to the corresponding well at a rate of 100 μL / well. The plate was incubated at 4°C for 30 min. The plate was washed twice with FACS buffer.

[0407] 6.2.4 Hands-on Practice

[0408] Turn on the flow cytometer and software. After the startup process is complete, create a folder for this experiment and perform sample testing in that folder.

[0409] 6.3 Internalization Detection

[0410] 6.3.1 Cell Plating

[0411] NCI-H292 cells (human lung cancer cells (lymph node metastasis)) in the culture flask were digested, transferred to centrifuge tubes, centrifuged to remove the supernatant, resuspended in 1640 medium containing 10% FBS, counted, and the cell density was adjusted to 6 × 10⁻⁶. 4 Cells / mL. Take four 96-well cell culture plates and add 50 μL of cells to each well using a 100 μL pipette. Incubate the cell culture plates at 37°C in a 5% CO2 incubator for 12-14 hours.

[0412] 6.3.2 Addition of antibody diluent

[0413] Fab-ZAP has a molecular weight of 106 kDa and a concentration of 2.2 mg / mL. It was diluted to a final concentration of 1.00000 nM using 1640 medium containing 10% FBS. The antibody was then diluted with the prepared Fab-ZAP diluent to nine concentration gradients: 0.30000, 0.10000, 0.03333, 0.01111, 0.00370, 0.00123, 0.00041, 0.00014, and 0.00005 nM. After dilution, the antibody was incubated at room temperature for 30 min. Then, 50 μL of the diluted antibody solution was added to each well of a cell culture plate using a 100 μL 12-line pipette.

[0414] 6.3.3 Incubation

[0415] Gently tap to mix, then place the cell plate in a 37°C, 5% CO2 incubator for 72 hours.

[0416] 6.3.4 Add the detection solution

[0417] Before assay, remove the cell culture plate from the incubator and allow it to equilibrate to room temperature for 10 min. Add 7.5 μL of Triton X-100 solution to each well of the cell + Triton plate, gently tap to mix, and incubate the cell culture plate at 37°C for 30 min. Using a 12-channel pipette, add 20 μL of MTS to each well and incubate at 37°C for 1 h to 4 h (until the color deepens).

[0418] 6.3.5 Data Acquisition

[0419] Turn on the microplate reader, wait for the instrument to complete its self-test, create a folder for this experiment, set the detection wavelength to 492nm, shake for 5 minutes, read the data, and save the data.

[0420] ELISA analysis showed that the assembled bispecific antibody had similar affinity for both antigens to the parent monoclonal antibody. FACS analysis showed that the assembled bispecific antibody exhibited predominantly anti-EGFR parental affinity for cell lines with high EGFR expression; and for cells with comparable c-Met and EGFR expression levels, it showed affinity comparable to the control bispecific antibody Met-H-EGFR (i.e., Amivantamab).

[0421] Table 2. Results of Affinity Dynamics Detection

[0422]

[0423] KD: Binding dissociation constant; Ka: Binding constant; Kd: Dissociation constant;

[0424] R2: Linear fit number; Rmax: Highest response value of curve fitting.

[0425] Table 3. Summary of ELISA Results for Bispecific Antibody Activity - EC 50 value

[0426]

[0427] Table 4. Summary of FACS Detection Results of Bispecific Antibody Activity

[0428]

[0429]

[0430] Table 5. Summary of Fab-ZAP Detection Results

[0431]

[0432] The bispecific antibody Bimab-Met-H-EGFR was constructed using anti-c-Met antibody Met-H and anti-EGFR antibody Panitumumab-WT as parent antibodies. Details of the construction plasmids for anti-c-Met antibodies C85-Mix-4-C85-016, C98-002-C98-WT, and B181_116_2_10_B181_116, as well as anti-EGFR antibodies Panitumumab-WT-113 and Panitumumab-68, are shown below:

[0433] Five light chain plasmids were Kappa type; three heavy chain plasmids were of type HC (IgG1-REM-DouBody-F405L), and two plasmids were of type HC (IgG1-REM-DouBody-K409R).

[0434]

[0435] The use of any and all embodiments or exemplary language (e.g., "such as" or "for example") provided herein is intended only to better illustrate the application and does not constitute a limitation on the scope of the application, unless otherwise required. The language in the specification should not be construed as indicating that any unclaimed element is necessary for carrying out the application.

[0436] All publications and patent applications cited in this specification are incorporated herein by reference, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Furthermore, any theories, mechanisms, proofs, or findings described herein are intended to further enhance the understanding of this application and are not intended to limit this application in any way to such theories, mechanisms, proofs, or findings. Although this application has been shown and described in detail in the accompanying drawings and the foregoing description, this application should be considered illustrative rather than restrictive.

[0437] Sequence information

[0438] SEQ ID NO:1

[0439] PYYWS

[0440] SEQ ID NO:2

[0441] YIESHRRTEYNPSLSG

[0442] SEQ ID NO:3

[0443] GVYTAYFDT

[0444] SEQ ID NO:4

[0445] RASQGISSWLA

[0446] SEQ ID NO:5

[0447] AASSLRS

[0448] SEQ ID NO:6

[0449] QQANSFPLT

[0450] SEQ ID NO:7

[0451] SYGMH

[0452] SEQ ID NO:8

[0453] VISYDGSNKYYADSVKG

[0454] SEQ ID NO:9

[0455] DRWMGATFPDY

[0456] SEQ ID NO:10

[0457] RASQYISSLLN

[0458] SEQ ID NO:11

[0459] AASSLQS

[0460] SEQ ID NO:12

[0461] LQDYTYPPM

[0462] SEQ ID NO:13

[0463] SSNWWS

[0464] SEQ ID NO:14

[0465] EIYHSGSTNYNPSLKS

[0466] SEQ ID NO:15

[0467] TMYYDFWSGYYPSYYYYMDV

[0468] SEQ ID NO:16

[0469] RASQSINNWLA

[0470] SEQ ID NO:17

[0471] KASGLEG

[0472] SEQ ID NO:18

[0473] QQYDDYPLT

[0474] SEQ ID NO:19

[0475] SGDYYWT

[0476] SEQ ID NO:20

[0477] HIYYSGNTNYNPSLKS

[0478] SEQ ID NO:21

[0479] DRVTGAFDI

[0480] SEQ ID NO:22

[0481] QASQDISNYLN

[0482] SEQ ID NO:23

[0483] DASNLET

[0484] SEQ ID NO:24

[0485] QHFDSTPLA

[0486] SEQ ID NO:25

[0487] HIYYSYNTNYNPSLKS

[0488] SEQ ID NO:26

[0489] SASNLET

[0490] SEQ ID NO:27

[0491] QHFDHLPLA

[0492] SEQ ID NO:28

[0493] GGSLAPY

[0494] SEQ ID NO:29

[0495] ESHRR

[0496] SEQ ID NO:30

[0497] GYTFSSY

[0498] SEQ ID NO:31

[0499] SYDGSN

[0500] SEQ ID NO:32

[0501] GGSISSSN

[0502] SEQ ID NO:33

[0503] YHSGS

[0504] SEQ ID NO:34

[0505] GGSVSSGDY

[0506] SEQ ID NO:35

[0507] YYSGN

[0508] SEQ ID NO:36

[0509] RGSVSSGDY

[0510] SEQ ID NO:37

[0511] YYSYN

[0512] SEQ ID NO:38

[0513] QVQLQDSGPGLVKPSETLTLTCSVSGGSLAPYYWSWVRQPPGQRLQWIGYIESHRRTEYNPSLSGRVAISL

[0514] DTSNSRFNLYLKSLTAADTANYYCARGVYTAYFDTWGRGTLVTVSS

[0515] SEQ ID NO:39

[0516] DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLRSGVPSRFSGSGSGTDFT

[0517] LTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK

[0518] SEQ ID NO:40

[0519] QVQLVQSGAEVVKPGASVKVSCKASGYTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGR

[0520] FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRWMGATFPDYWGQGTLVTVSS

[0521] SEQ ID NO:41

[0522] DIQLTQSPSSLSASVGDRVTITCRASQYISSLLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSASGSGTDFT

[0523] LAISSLQPEDFATYYCLQDYTYPPMFGQGTKVEIK

[0524] SEQ ID NO:42

[0525] QLQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTIS

[0526] VDKSKNQFSLKLSSVTAADTAVYYCARTMYYDFWSGYYPSYYYYMDVWGKGTTVTVSS

[0527] SEQ ID NO:43

[0528] DIQMIQSPSTLSASVGDRVTITCRASQSINNWLAWYQQKPGKAPKLLISKASGLEGGVPSRFSGSGSGTEF

[0529] TLTISSLQPDDSATYYCQQYDDYPLTFGPGTRLEIK

[0530] SEQ ID NO:44

[0531] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTI

[0532] SIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS

[0533] SEQ ID NO:45DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDSTPLAFGGGTKVEIK

[0534] SEQ ID NO:46

[0535] QVQLQESGPGLVKPSETLSLTCTVSRGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSYNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS

[0536] SEQ ID NO:47

[0537] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYSASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK

[0538] SEQ ID NO:48

[0539] DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0540] SEQ ID NO:49

[0541] DIQLTQSPSSLSASVGDRVTITCRASQYISSLLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSASGSGTDFTLAISSLQPEDFATYYCLQDYTYPPMFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0542] SEQ ID NO:50

[0543] DIQMIQSPSTLSASVGDRVTITCRASQSINNWLAWYQQKPGKAPKLLISKASGLEGGVPSRFSGSGSGTEFTLTISSLQPDDSATYYCQQYDDYPLTFGPGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0544] SEQ ID NO:51

[0545] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDSTPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0546] SEQ ID NO:52

[0547] DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYSASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0548] SEQ ID NO:53

[0549] QVQLQDSGPGLVKPSETLTLTCSVSGGSLAPYYWSWVRQPPGQRLQWIGYIESHRRTEYNPSLSGRVAISLDTSNSRFNLYLKSLTAADTANYYCARGVYTAYFDTWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0550] SEQ ID NO:54

[0551] QVQLVQSGAEVVKPGASVKVSCKASGYTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRWMGATFPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0552] SEQ ID NO:55

[0553] QLQLQESGPGLVKPSGTLSLTCAVSGGISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARTMYDFWSGYYPSYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0554] SEQ ID NO:56

[0555] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0556] SEQ ID NO:57

[0557] QVQLQESGPGLVKPSETLSLTCTVSRGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSYNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0558] SEQ ID NO:58

[0559] GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCGGAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAAGGACCGTGGCTGCCCCCAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGTCTGCTGAATAACTTCTATCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAACTCCCAGGAGTCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCTCCACCCTGACCCTGTCCAAGGCTGATTATGAGAAGCACAAGGTGTATGCTTGCGAGGTGACACACCAGGGCCTGTCCAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGTGC

[0560] SEQ ID NO:59

[0561] GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGTACATTAGCAGCTTGTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGCCAGTGGATCTGGCACAGATTTCACTCTCGCCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGGATTACACTTACCCTCCGATGTTCGGCCAAGGGACCAAGGTGGAAATCAAAAGGACCGTGGCTGCCCCCAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGTCTGCTGAATAACTTCTATCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAACTCCCAGGAGTCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCTCCACCCTGACCCTGTCCAAGGCTGATTATGAGAAGCACAAGGTGTATGCTTGCGAGGTGACACACCAGGGCCTGTCCAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGTGC

[0562] SEQ ID NO:60

[0563] GACATCCAGATGATCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAATAACTGGTTGGCCTGGTATCAGCAAAAGCCAGGGAAGGCCCCTAAACTCCTGATCTCTAAGGCGTCTGGTTTAGAAGGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTCTGCAACTTATTACTGCCAACAGTATGATGATTATCCGCTCACCTTCGGCCCAGGGACACGACTGGAGATTAAAAGGACCGTGGCTGCCCCCAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGTCTGCTGAATAACTTCTATCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAACTCCCAGGAGTCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCTCCACCCTGACCCTGTCCAAGGCTGATTATGAGAAGCACAAGGTGTATGCTTGCGAGGTGACACACCAGGGCCTGTCCAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGTGC

[0564] SEQ ID NO:61

[0565] GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGGGCGACAGAGTGACCATTACCTGTCAGGCCAGCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCCTCCAATCTGGAAACCGGCGTGCCCTCTAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCTTTACAATCTCCAGCCTGCAGCCTGAGGATATCGCCACCTACTTTTGCCAGCACTTCGACAGTACTCCTCTGGCCTTTGGCGGCGGAACAAAGGTGGAAATCAAGAGGACCGTGGCTGCCCCCAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGTCTGCTGAATAACTTCTATCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAACTCCCAGGAGTCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCTCCACCCTGACCCTGTCCAAGGCTGATTATGAGAAGCACAAGGTGTATGCTTGCGAGGTGACACACCAGGGCCTGTCCAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGTGC

[0566] SEQ ID NO:62

[0567] GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCCGCCTCTGTGGGCGACAGAGTGACCATTACCTGTCAGGCCAGCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACAGTGCCTCCAATCTGGAAACCGGCGTGCCCTCTAGATTCTCCGGCTCTGGCTCTGGCACCGACTTTACCTTTACAATCTCCAGCCTGCAGCCTGAGGATATCGCCACCTACTTTTGCCAGCACTTCGACCATCTGCCTCTGGCCTTTGGCGGCGGAACAAAGGTGGAAATCAAGAGGACCGTGGCTGCCCCCAGCGTGTTCATCTTCCCTCCTAGCGACGAGCAGCTGAAGAGCGGCACCGCTAGCGTGGTGTGTCTGCTGAATAACTTCTATCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGCGGCAACTCCCAGGAGTCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGAGCTCCACCCTGACCCTGTCCAAGGCTGATTATGAGAAGCACAAGGTGTATGCTTGCGAGGTGACACACCAGGGCCTGTCCAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGTGC

[0568] SEQ ID NO:63

[0569]

[0570] SEQ ID NO:64

[0571]

[0572] SEQ ID NO:65

[0573]

[0574] SEQ ID NO:66

[0575]

[0576] SEQ ID NO:67

[0577]

Claims

1. A bispecific antibody comprising a first antigen-binding fragment against human mesenchymal-epithelial transforming factor (c-Met) and a second antigen-binding fragment against human epidermal growth factor receptor (EGFR); in, The first antigen-binding fragment includes: HCDR1, as shown in SEQ ID NO:1, HCDR2, as shown in SEQ ID NO:2, HCDR3, as shown in SEQ ID NO:3, LCDR1, as shown in SEQ ID NO:4, LCDR2, as shown in SEQ ID NO:5, and As shown in SEQ ID NO:6, LCDR3; or As shown in SEQ ID NO:7, HCDR1, HCDR2, as shown in SEQ ID NO:8, HCDR3, as shown in SEQ ID NO:9, LCDR1, as shown in SEQ ID NO:10, LCDR2, as shown in SEQ ID NO:11, and As shown in SEQ ID NO:12, LCDR3; or HCDR1, as shown in SEQ ID NO:13, HCDR2, as shown in SEQ ID NO:14, HCDR3, as shown in SEQ ID NO:15, LCDR1, as shown in SEQ ID NO:16, LCDR2, as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18; and The second antigen-binding fragment includes: As shown in SEQ ID NO:19, HCDR1, HCDR2, as shown in SEQ ID NO:20, HCDR3, as shown in SEQ ID NO:21, LCDR1, as shown in SEQ ID NO:22, LCDR2, as shown in SEQ ID NO:23, and As shown in SEQ ID NO:24, LCDR3; or As shown in SEQ ID NO:19, HCDR1, HCDR2, as shown in SEQ ID NO:25, HCDR3, as shown in SEQ ID NO:21, LCDR1, as shown in SEQ ID NO:22, LCDR2, as shown in SEQ ID NO:26, and LCDR3 as shown in SEQ ID NO:27; HCDR and LCDR are defined according to Kabat.

2. A bispecific antibody comprising a first antigen-binding fragment against human mesenchymal-epithelial transforming factor (c-Met) and a second antigen-binding fragment against human epidermal growth factor receptor (EGFR); in, The first antigen-binding fragment includes: As shown in SEQ ID NO:28, HCDR1, HCDR2, as shown in SEQ ID NO:29, HCDR3, as shown in SEQ ID NO:3, LCDR1, as shown in SEQ ID NO:4, LCDR2, as shown in SEQ ID NO:5, and As shown in SEQ ID NO:6, LCDR3; or HCDR1, as shown in SEQ ID NO:30, HCDR2, as shown in SEQ ID NO:31, HCDR3, as shown in SEQ ID NO:9, LCDR1, as shown in SEQ ID NO:10, LCDR2, as shown in SEQ ID NO:11, and As shown in SEQ ID NO:12, LCDR3; or As shown in SEQ ID NO:32, HCDR1, HCDR2, as shown in SEQ ID NO:33, HCDR3, as shown in SEQ ID NO:15, LCDR1, as shown in SEQ ID NO:16, LCDR2, as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18; and The second antigen-binding fragment includes: HCDR1, as shown in SEQ ID NO:34, HCDR2, as shown in SEQ ID NO:35, HCDR3, as shown in SEQ ID NO:21, LCDR1, as shown in SEQ ID NO:22, LCDR2, as shown in SEQ ID NO:23, and As shown in SEQ ID NO:24, LCDR3; or HCDR1, as shown in SEQ ID NO:36, HCDR2, as shown in SEQ ID NO:37, HCDR3, as shown in SEQ ID NO:21, LCDR1, as shown in SEQ ID NO:22, LCDR2, as shown in SEQ ID NO:26, and LCDR3 as shown in SEQ ID NO:27; HCDR and LCDR are defined according to Chothia.

3. The bispecific antibody as described in claim 1 or 2, wherein... The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:39; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:40 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:41 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:41; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:42 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:43 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:43; Preferably, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:41; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:

43.

4. The bispecific antibody as described in claim 1 or 2, wherein... The amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or The amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:46 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46; and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:47 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:

47. Preferably, the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45; or The amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:

47.

5. The bispecific antibody as described in claim 1 or 2, wherein... The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:39, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:

39. The sequence shown in SEQ ID NO:44 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:45 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:40 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:41 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:41, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:

40. The sequence shown in SEQ ID NO:44 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:45 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:42 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:43 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:43, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:

42. The sequence shown in SEQ ID NO:44 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:45 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:45; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:39, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:

39. The sequence shown in SEQ ID NO:46 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:47 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:40 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:41 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:41, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:

40. The sequence shown in SEQ ID NO:46 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment being as shown in SEQ ID NO:47 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:42 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:43 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:43, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:

42. The sequence shown in SEQ ID NO:46 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment as shown in SEQ ID NO:47 or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO:47; Preferably, the amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:38 and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is as shown in SEQ ID NO:39, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:44 and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is as shown in SEQ ID NO:45; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:41; the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:45; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:42, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:43; the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:45; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:38, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:39; the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:47; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:41; the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:46, and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:47; or The amino acid sequence of the heavy chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:42 and the amino acid sequence of the light chain variable region of the first antigen-binding fragment is shown in SEQ ID NO:43, and the amino acid sequence of the heavy chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:46 and the amino acid sequence of the light chain variable region of the second antigen-binding fragment is shown in SEQ ID NO:

47.

6. The bispecific antibody according to any one of claims 1-5, wherein The bispecific antibody is an IgG1 antibody, which includes a first heavy chain constant region and a second heavy chain constant region, wherein... The amino acid at position 405 of the first heavy chain constant region is L, and the amino acid at position 409 of the second heavy chain constant region is R; or The amino acid at position 409 of the first heavy chain constant region is R, and the amino acid at position 405 of the second heavy chain constant region is L; The amino acid positions in the antibody constant region are determined according to EU numbering.

7. The bispecific antibody according to any one of claims 1-6, wherein The bispecific antibody comprises a first light chain constant region and a second light chain constant region, wherein the first light chain constant region and / or the second light chain constant region are κ or λ subtypes; preferably, the first light chain constant region and / or the second light chain constant region are κ subtypes; and / or The first antigen-binding fragment and the second antigen-binding fragment are independently selected from single-chain antibodies (scFv) or Fab fragments; preferably, both the first antigen-binding fragment and the second antigen-binding fragment are Fab fragments.

8. The bispecific antibody according to any one of claims 1-7, wherein the bispecific antibody is in the form of Doubody, Diabody, CrossMab, DVD-ig, Knob-in-Hole, BiTE, DART, YBODY or FIT-Ig; preferably, the bispecific antibody is in the form of Doubody.

9. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-8 and a pharmaceutically acceptable excipient, diluent or carrier.

10. Use of the bispecific antibody of any one of claims 1-8 or the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention or treatment of cancer in a subject of need; preferably, the cancer cells of said cancer express EGFR; more preferably, the cancer cells of said cancer overexpress EGFR; most preferably, said cancer is selected from: lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), liver cancer, colon cancer, breast cancer, colon cancer, brain cancer, head and neck cancer, pancreatic cancer, ovarian cancer, prostate cancer, gastric cancer, nervous system cancers (e.g., glioblastoma) and metastatic lesions of the above cancers.

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