Anti-EGFR monoclonal antibody, bispecific antibody thereof, pharmaceutical composition and application

Abstract

The invention belongs to the field of biological medicine, and relates to an anti-EGFR monoclonal antibody, a bispecific antibody thereof, a pharmaceutical composition and application. Specifically, the invention relates to an anti-EGFR antibody or an antigen binding fragment thereof. The invention also relates to an anti-EGFR (Epidermal Growth Factor Receptor) and anti-MET (Methyl The anti-EGFR monoclonal antibody and the bispecific antibody provided by the invention both have good anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to an anti-EGFR monoclonal antibody, its bispecific antibody, pharmaceutical composition and use. The present invention also relates to an anti-EGFR-anti-MET bispecific antibody. Background Art

[0002] EGFR (Epidermal Growth Factor Receptor) is the receptor of epidermal growth factor (EGF) and belongs to the ErbB receptor family. EGFR is a transmembrane glycoprotein with a molecular weight of 170KDa and belongs to the receptor-type tyrosine kinase. Under the action of related ligands such as epidermal growth factor (EGF) and transforming growth factor-α (TGFα), EGFR is activated by converting from a monomer to a dimer, and further activates downstream signal transduction pathways, such as phosphorylation of kinases such as Akt and ERK, to regulate cell proliferation (Jorissen RN, Walker F, Pouliot N, et al. Epidermal growth factor receptor: mechanisms of activation and signaling. Exp Cell Res 2003; 284: 31-53.). A large number of studies have shown that there is high expression or abnormal expression of EGFR in most tumors such as glioblastoma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, etc. (Modjtahedi H, and Dean C. The receptor for EGF and its ligands Expression, prognostic value and target for therapy in cancer. International Journal of Oncology 1994; (4): 277-96.). The abnormal function of EGFR is related to the proliferation, angiogenesis, tumor invasion, metastasis and inhibition of apoptosis of tumor cells (Castillo L, Etienne Grimaldi MC, Fischel JL, et al. Pharmacological background of EGFR targeting. Ann Oncol 2004; 15: 1007-12.). The abnormal function is mainly manifested in two aspects: one is the excessive abnormal expression in tumor tissues, and the other is the persistent activation of EGFR mutants in tumor cells (without ligand stimulation or formation of a self-circulating stimulation pathway).

[0003] c-Mesenchymal-epithelial transition factor (abbreviated as c-MET, cMET or MET) is a member of the receptor tyrosine kinase family. The MET receptor binds to its ligand, hepatocyte growth factor (HGF), which can induce MET dimerization and cause it to enter an activated state, thereby activating downstream signaling pathways, such as the phosphorylation of kinases like Akt and ERK. MET plays an important role in embryonic development, organ growth, and wound healing, and is usually only expressed in stem cells and progenitor cells. In cancer, abnormal activation of MET caused by MET mutations promotes angiogenesis and cancer metastasis.

[0004] Lung cancer is the most common cancer with the highest incidence and mortality rate, and non-small cell lung cancer (NSCLC) accounts for 90% of lung cancer patients. EGFR mutations are the main driving factors in NSCLC, reaching more than 40% in Asian NSCLC patients. EGFR kinase inhibitors (EGFR-TKIs) are the main treatment methods. However, the proportion of patients developing drug resistance after treatment is high and the mechanisms are complex, among which 7-15% of patients have MET amplification. In addition, exon 14 skipping mutations, MET fusions, MET amplifications, and overexpressions of MET are also primary driving factors in NSCLC. Bispecific antibodies targeting EGFR and MET can simultaneously inhibit two signaling pathways and are expected to be used in patients who are ineffective or resistant to EGFR-TKIs.

[0005] Currently, several EGFR×MET bispecific antibodies have been publicly reported. US9328173B2 (Eli Lilly and Company), WO2018221969A1 (Chong Kun Dang BioPharma Co., Ltd.), and WO2022104236A2 (Ab theraputics) disclose the construction method of the "2+2" type EGFR×MET bispecific antibody, that is, both the anti-EGFR end and the anti-MET end are in a bivalent form. Since EGFR antibodies have skin toxicity, the "2+2" type EGFR×MET bispecific antibody has potentially higher safety risks. Amivantamab (US2017275367A1) from Johnson & Johnson discloses the construction method of the "1+1" type EGFR×MET bispecific antibody, that is, both the anti-EGFR end and the anti-MET end are in a monovalent form. This antibody has been approved for marketing and has good safety.

[0006] The construction of the "1+1" type EGFR×MET bispecific antibody poses huge technical challenges, and problems such as correct pairing of heavy and light chains and heterodimerization of heavy chains need to be solved. As reported in US2017275367A1, Amivantamab uses the Fab arm exchange technology. However, this method requires the preparation of anti-EGFR antibody and anti-MET antibody separately, and then a bispecific antibody is obtained by an in vitro recombination method, increasing the production cost. The common light chain technology is one of the methods for constructing bispecific antibodies. Since there is no problem of heavy and light chain mismatch, it greatly facilitates the preparation of bispecific antibodies.

[0007] Currently, there is still a need to develop new anti-EGFR antibodies and anti-EGFR-anti-MET bispecific antibodies. Summary of the Invention

[0008] Through in-depth research and creative work, the inventor has obtained bispecific antibodies, especially anti-EGFR-anti-MET bispecific antibodies. The present invention constructs an EGFR×MET bispecific antibody using the common light chain technology, which can inhibit the phosphorylation of Akt and ERK induced by EGF and HGF; promote the endocytosis of EGFR and MET; and kill tumor cells through the ADCC effect. The bispecific antibody of the present invention has good anti-tumor activity. Thus, the following invention is provided:

[0009] One aspect of the present invention relates to an anti-EGFR antibody or an antigen-binding fragment thereof, the anti-EGFR antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 to HCDR3, and the light chain variable region comprising LCDR1 to LCDR3, wherein:

[0010] The amino acid sequence of HCDR1 is as shown in SEQ ID NO:25 or SEQ ID NO:26, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:27, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:37; and

[0011] The amino acid sequence of LCDR1 is as shown in SEQ ID NO:38, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:39, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:76.

[0012] In some embodiments of the present invention, the anti-EGFR antibody or an antigen-binding fragment thereof, wherein,

[0013] The amino acid sequence of HCDR3 is as shown in any one of SEQ ID NO:28 to SEQ ID NO:36.

[0014] In some embodiments of the present invention, the anti-EGFR antibody or its antigen-binding fragment, wherein,

[0015] the amino acid sequence of the heavy chain variable region of the anti-EGFR antibody is selected from any one of SEQ ID NO: 1 to SEQ ID NO: 10; and

[0016] the amino acid sequence of the light chain variable region of the anti-EGFR antibody is selected from SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 77.

[0017] In some embodiments of the present invention, the anti-EGFR or its antigen-binding fragment, wherein,

[0018] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 42;

[0019] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 42;

[0020] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 42;

[0021] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 42;

[0022] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 42;

[0023] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 42;

[0024] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 42;

[0025] the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 42;

[0026] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:42;

[0027] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:42;

[0028] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0029] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0030] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0031] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0032] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0033] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0034] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0035] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0036] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0037] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43;

[0038] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0039] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0040] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0041] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0042] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0043] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0044] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0045] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0046] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0047] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:44;

[0048] Or

[0049] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:77.

[0050] In some embodiments of the present invention, the anti-EGFR antibody or its antigen-binding fragment, wherein,

[0051] The anti-EGFR antibody described above includes non-CDR regions, and the non-CDR regions are from a species other than murine, such as from a human antibody.

[0052] In some embodiments of the present invention, the anti-EGFR antibody or its antigen-binding fragment, wherein the constant region of the anti-EGFR antibody is selected from the constant regions of human IgG1, IgG2, IgG3, or IgG4.

[0053] In some embodiments of the present invention, the anti-EGFR antibody or its antigen-binding fragment, wherein the heavy chain constant region of the anti-EGFR antibody is Ig gamma-1 chain C region or Ig gamma-4 chain C region; the light chain constant region is Ig kappa chain C region or Ig lambda chain C region.

[0054] In some embodiments of the present invention, the anti-EGFR antibody or its antigen-binding fragment, wherein the heavy chain constant region sequence of the anti-EGFR antibody is selected from SEQ ID NO: 79, 80, and 81.

[0055] In some embodiments of the present invention, the heavy chain of the anti-EGFR antibody is selected from SEQ ID NO: 17, 18, 19, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75; and

[0056] the light chain of the anti-EGFR antibody is selected from SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

[0057] In some embodiments of the present invention, the anti-EGFR antibody or its antigen-binding fragment, wherein:

[0058] the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 11;

[0059] the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 12;

[0060] the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 13;

[0061] the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 11;

[0062] The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 12;

[0063] The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 13;

[0064] The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 11;

[0065] The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 12;

[0066] The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 13;

[0067] Or

[0068] The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 17, 18, 19, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 57.

[0069] In some embodiments of the present invention, the anti-EGFR antibody or its antigen-binding fragment, wherein the anti-EGFR antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.

[0070] In some embodiments of the present invention, the heavy chain constant region is Ig gamma-1 chain C region (e.g., NCBI ACCESSION: P01857) or Ig gamma-4 chain C region (e.g., NCBI ACCESSION: P01861.1); the light chain constant region is Ig kappa chain C region (e.g., NCBI ACCESSION: P01834)

[0071] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the anti-EGFR antibody or its antigen-binding fragment according to any one of the present invention.

[0072] The present invention also relates to a recombinant vector, which comprises the isolated nucleic acid molecule of the present invention.

[0073] Another aspect of the present invention relates to a host cell, which comprises the isolated nucleic acid molecule of the present invention, or the recombinant vector of the present invention.

[0074] Another aspect of the present invention relates to an antibody-drug conjugate, which comprises an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or the antigen-binding fragment thereof is the anti-EGFR antibody or the antigen-binding fragment thereof as described in any one of the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug;

[0075] Preferably, the antibody or the antigen-binding fragment thereof is linked to the small molecule drug through a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond;

[0076] Preferably, the molar ratio of the antibody or the antigen-binding fragment thereof to the small molecule drug is 1:(2-8);

[0077] Preferably, the molar ratio of the antibody or the antigen-binding fragment thereof to the small molecule drug is 1:(2-4).

[0078] Another aspect of the present invention relates to a pharmaceutical composition, which comprises an effective amount of the anti-EGFR antibody or the antigen-binding fragment thereof as described in any one of the present invention, and one or more pharmaceutically acceptable excipients.

[0079] In some embodiments of the present invention, the pharmaceutical composition further comprises an effective amount of an anti-MET antibody or the antigen-binding fragment thereof;

[0080] Preferably, the anti-MET antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

[0081] The amino acid sequence of HCDR1 is as shown in SEQ ID NO:45, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:46, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:47; or the amino acid sequence of HCDR1 is as shown in SEQ ID NO:48, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:49, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:50;

[0082] And

[0083] The amino acid sequence of LCDR1 is as shown in SEQ ID NO:51, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:52, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:53.

[0084] In some embodiments of the present invention, the pharmaceutical composition, wherein,

[0085] The amino acid sequence of the heavy chain variable region of the anti-MET antibody is selected from SEQ ID NO:54 and SEQ ID NO:55; and

[0086] The amino acid sequence of the light chain variable region of the anti-MET antibody is as shown in SEQ ID NO:56;

[0087] Preferably, the anti-MET antibody:

[0088] The amino acid sequence of the heavy chain is as shown in SEQ ID NO:16 or SEQ ID NO:21, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14.

[0089] Another aspect of the present invention relates to a combination drug product, comprising a first drug product and a second drug product in separate packages, wherein:

[0090] The first drug product contains an effective amount of the anti-EGFR antibody or its antigen-binding fragment described in any one of the present invention, and one or more pharmaceutically acceptable excipients;

[0091] The second drug product contains an effective amount of an anti-MET antibody or its antigen-binding fragment, and one or more pharmaceutically acceptable excipients;

[0092] Optionally, the combination drug product further contains a product instruction.

[0093] In some embodiments of the present invention, the combination drug product, wherein,

[0094] The anti-MET antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

[0095] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 45, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 46, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 47; or the amino acid sequence of HCDR1 is shown in SEQ ID NO: 48, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 49, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 50;

[0096] and

[0097] The amino acid sequence of LCDR1 is shown in SEQ ID NO: 51, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 52, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 53.

[0098] In some embodiments of the present invention, the combined pharmaceutical product, wherein,

[0099] the amino acid sequence of the heavy chain variable region of the anti-MET antibody is selected from SEQ ID NO: 54 and SEQ ID NO: 55; and

[0100] the amino acid sequence of the light chain variable region of the anti-MET antibody is shown in SEQ ID NO: 56;

[0101] Preferably, the anti-MET antibody:

[0102] The amino acid sequence of the heavy chain is shown in SEQ ID NO: 16 or SEQ ID NO: 21, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14.

[0103] Another aspect of the present invention relates to a bispecific antibody, which comprises:

[0104] a first protein functional region targeting EGFR, and

[0105] a second protein functional region targeting a target different from EGFR (such as MET);

[0106] wherein, the first protein functional region comprises the heavy chain variable region of the anti-EGFR antibody or its antigen-binding fragment described in any one of the present invention, or comprises the heavy chain variable region and the light chain variable region of the anti-EGFR antibody or its antigen-binding fragment described in any one of the present invention.

[0107] In some embodiments of the present invention, the bispecific antibody is an anti-EGFR-anti-MET bispecific antibody, also known as an EGFR×MET bispecific antibody.

[0108] In some embodiments of the present invention, the bispecific antibody is a "1+1" type EGFR×MET bispecific antibody.

[0109] In some embodiments of the present invention, the bispecific antibody, wherein,

[0110] the second protein functional region comprises the heavy chain variable region of an anti-MET antibody or its antigen-binding fragment; or comprises the heavy chain variable region and the light chain variable region of an anti-MET antibody or its antigen-binding fragment;

[0111] wherein,

[0112] the anti-MET antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

[0113] the amino acid sequence of HCDR1 is as shown in SEQ ID NO:45, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:46, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:47; or the amino acid sequence of HCDR1 is as shown in SEQ ID NO:48, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:49, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:50;

[0114] and

[0115] the amino acid sequence of LCDR1 is as shown in SEQ ID NO:51, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:52, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:53.

[0116] In some embodiments of the present invention, the bispecific antibody, wherein,

[0117] the amino acid sequence of the heavy chain variable region of the anti-MET antibody is selected from SEQ ID NO:54 and SEQ ID NO:55; and

[0118] the amino acid sequence of the light chain variable region of the anti-MET antibody is as shown in SEQ ID NO:56;

[0119] Preferably, the anti-MET antibody:

[0120] has the amino acid sequence of the heavy chain as shown in SEQ ID NO:16 or SEQ ID NO:21, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:14.

[0121] In some embodiments of the present invention, the bispecific antibody, wherein,

[0122] wherein, the first protein functional region and the second protein functional region are independently a fusion protein of single-chain antibodies or a half-molecule monovalent antibody (IgG half molecule, IgG-HM).

[0123] In some embodiments of the present invention, the bispecific antibody, wherein,

[0124] the first protein functional region is a half-molecule monovalent antibody, and

[0125] the second protein functional region is a half-molecule monovalent antibody.

[0126] In some embodiments of the present invention, the bispecific antibody, wherein,

[0127] the first protein functional region is a half-molecule monovalent antibody targeting EGFR, and

[0128] the second protein functional region is a half-molecule monovalent antibody targeting MET.

[0129] In some embodiments of the present invention, the bispecific antibody, wherein,

[0130] the heavy chain constant regions of the two half-molecule monovalent antibodies respectively contain a first CH3 region and a second CH3 region, the sequences of the first CH3 region and the second CH3 region are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than the homodimer interaction of each of the first CH3 region and the second CH3 region.

[0131] In some embodiments of the present invention, the bispecific antibody, wherein,

[0132] the heavy chain constant regions of the two half-molecule monovalent antibodies respectively contain a first CH3 region and a second CH3 region, and according to the EU Numbering System, the 405th position of the first CH3 region is mutated to Ala, Asp, Glu, His, Ile, Met, Asn, Gln, Thr, Val, Tyr, Leu, Lys, Ser or Trp; the 409th position of the second CH3 region is mutated to the amino acid Ala, Asp, Glu, Phe, Gly, His, Ile, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr.

[0133] In some embodiments of the present invention, the bispecific antibody, wherein,

[0134] The heavy chain constant regions of the two half-molecule monovalent antibodies it contains respectively contain a first CH3 region and a second CH3 region, and according to the EU Numbering System, the 405th position of the first CH3 region is mutated to Leu; the 409th position of the second CH3 region is mutated to the amino acid Arg.

[0135] In some embodiments of the present invention, the bispecific antibody, wherein,

[0136] The heavy chain constant region of the half-molecule monovalent antibody is the heavy chain constant region of human IgG1.

[0137] In some embodiments of the present invention, the bispecific antibody, wherein,

[0138] The heavy chain constant region of the half-molecule monovalent antibody is the heavy chain constant region of human IgG1 and has a Knob mutation (such as S354C and T366W mutations); and

[0139] The heavy chain constant region of the half-molecule monovalent antibody is the heavy chain constant region of human IgG1 and has a Hole mutation (such as Y349C, T366S, L368A and Y407V mutations).

[0140] In the present invention, the mutation positions of Knob and hole are numbered according to the EU numbering system. In some embodiments of the present invention, the Knob mutation refers to S354C and T366W mutations. In some embodiments of the present invention, the Hole mutation refers to Y349C, T366S, L368A and Y407V mutations.

[0141] In some embodiments of the present invention, the bispecific antibody, wherein the heavy chain constant region of the half-molecule monovalent antibody is the heavy chain constant region of human IgG1, and according to the EU Numbering System, the 435th position of the heavy chain constant region of one of the half-molecule monovalent antibodies is mutated to the amino acid Arg (R), and the 436th position is mutated to the amino acid Phe (F).

[0142] In some embodiments of the present invention, the bispecific antibody is in the IgG form, preferably in the IgG1 form;

[0143] Preferably, the sequences of the light chains in the bispecific antibody are the same;

[0144] Preferably, the bispecific antibody has two light chains with the same sequence;

[0145] Preferably, the bispecific antibody is composed of the following peptide chains:

[0146] (1) Peptide chains selected from SEQ ID NO:17 to SEQ ID NO:19, and SEQ ID NO:58 to SEQ ID NO:75,

[0147] (2) Peptide chains selected from SEQ ID NO:16 and SEQ ID NO:20, and

[0148] (3) Peptide chains selected from SEQ ID NO:11 to SEQ ID NO:13, and peptide chains in SEQ ID NO:57,

[0149] wherein the peptide chains in (3) are two identical copies;

[0150] Preferably, the peptide chains in (1) and (2), the peptide chains in (2) and (3), and the peptide chains in (1) and (3) are linked by one or more disulfide bonds (such as 2 or 3 disulfide bonds).

[0151] In some embodiments of the present invention, the bispecific antibody is composed of the following peptide chains:

[0152] The peptide chain shown in SEQ ID NO:17, the peptide chain shown in SEQ ID NO:16, and the peptide chain shown in SEQ ID NO:12, wherein the peptide chain shown in SEQ ID NO:12 is two identical copies;

[0153] The peptide chain shown in SEQ ID NO:17, the peptide chain shown in SEQ ID NO:16, and the peptide chain shown in SEQ ID NO:13, wherein the peptide chain shown in SEQ ID NO:13 is two identical copies;

[0154] The peptide chain shown in SEQ ID NO:18, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:12, wherein the peptide chain shown in SEQ ID NO:12 is two identical copies;

[0155] The peptide chain shown in SEQ ID NO:18, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:13, wherein the peptide chain shown in SEQ ID NO:13 is two identical copies;

[0156] The peptide chain shown in SEQ ID NO:19, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:12, wherein the peptide chain shown in SEQ ID NO:12 is two identical copies;

[0157] The peptide chain shown in SEQ ID NO:19, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:13, where the peptide chain shown in SEQ ID NO:13 is two identical copies;

[0158] The peptide chain shown by any one of SEQ ID NO:17, 18, 19, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75, the peptide chain shown in SEQ ID NO:16, and the peptide chain shown in SEQ ID NO:57, where the peptide chain shown in SEQ ID NO:57 is two identical copies;

[0159] Or

[0160] The peptide chain shown by any one of SEQ ID NO:17, 18, 19, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:57, where the peptide chain shown in SEQ ID NO:57 is two identical copies.

[0161] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the bispecific antibody described in any one of the present invention.

[0162] Another aspect of the present invention relates to a recombinant expression vector comprising the isolated nucleic acid molecule of the present invention.

[0163] Another aspect of the present invention relates to a recombinant host cell comprising the recombinant expression vector of the present invention. Preferably, the recombinant host cell is a recombinant CHO-K1 cell.

[0164] Another aspect of the present invention relates to a conjugate comprising a bispecific antibody and a small molecule drug, wherein the bispecific antibody is the bispecific antibody described in any one of the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug;

[0165] Preferably, the bispecific antibody is linked to the small molecule drug through a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond;

[0166] Preferably, the molar ratio of the bispecific antibody to the small molecule drug is 1:(2 - 8);

[0167] Preferably, the molar ratio of the bispecific antibody to the small molecule drug is 1:(2 - 4).

[0168] Another aspect of the present invention relates to a pharmaceutical composition comprising the bispecific antibody described in any one of the present invention, and one or more pharmaceutically acceptable excipients.

[0169] Another aspect of the present invention relates to the use of the anti-EGFR antibody or its antigen-binding fragment described in any one of the present invention or the bispecific antibody described in any one of the present invention in the preparation of a medicament for treating or preventing tumors;

[0170] Preferably, the tumor is a tumor with high expression of EGFR and / or MET;

[0171] Preferably, the tumor is one or more selected from glioma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological tumor, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, brain cancer, thyroid cancer and head and neck cancer;

[0172] Preferably, the lung cancer is small cell lung cancer or non-small cell lung cancer.

[0173] The anti-EGFR antibody or its antigen-binding fragment described in any one of the present invention or the bispecific antibody described in any one of the present invention is used for treating or preventing tumors;

[0174] Preferably, the tumor is a tumor with high expression of EGFR and / or MET;

[0175] Preferably, the tumor is one or more selected from glioma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological tumor, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, brain cancer, thyroid cancer and head and neck cancer;

[0176] Preferably, the lung cancer is small cell lung cancer or non-small cell lung cancer.

[0177] Another aspect of the present invention relates to a method for treating or preventing tumors, comprising the step of administering to a subject in need an effective amount of the anti-EGFR antibody or its antigen-binding fragment described in any one of the present invention or the bispecific antibody described in any one of the present invention;

[0178] Preferably, the tumor is a tumor with high expression of EGFR and / or MET;

[0179] Preferably, the tumor is one or more selected from glioma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological tumor, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, brain cancer, thyroid cancer and head and neck cancer;

[0180] Preferably, the lung cancer is small cell lung cancer or non-small cell lung cancer.

[0181] In some embodiments of the present invention, the method for treating or preventing a tumor, wherein the drug is administered before or after surgery, and / or before or after radiotherapy.

[0182] In some embodiments of the present invention, the method for treating or preventing a tumor, wherein

[0183] The single-dose of the anti-EGFR antibody or its antigen-binding fragment or the bispecific antibody is 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0184] Preferably, the drug is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every 1 week, every 2 weeks or every 3 weeks;

[0185] Preferably, the administration method is intravenous drip or intravenous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0186] Figure 1 : Schematic diagram of the structure of the bispecific antibody E2mut34-91A×M5-91A-FAE-LF.

[0187] Figure 2 : Schematic diagram of the structure of the bispecific antibody E2mut34-69×M5-69-FAE-LF.

[0188] Figure 3 : Schematic diagram of the structure of the bispecific antibody JNJ-372.

[0189] Figure 4 : Results of EGFR ELISA binding experiment.

[0190] Figure 5 : Results of Met ELISA binding experiment.

[0191] Figure 6 : Results of EGFR ELISA blocking experiment.

[0192] Figure 7 : Results of Met ELISA blocking experiment.

[0193] Figures 8A to 8C : The antibodies E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 all have binding activity with recombinant human EGFR. Among them: Figure 8A: Dissociation curve of the binding of antibody E2-mut34-91A×M5-91A-FAE-LF to human EGFR; Figure 8B : Dissociation curve of the binding of antibody E2-mut34-69×M5-69-FAE-LF to human EGFR; Figure 8C : Dissociation curve of the binding of antibody JNJ-372 to human EGFR.

[0194] Figures 9A to 9C : Antibodies E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 all have binding activity to recombinant human Met. Among them: Figure 9A : Dissociation curve of the binding of antibody E2-mut34-91A×M5-91A-FAE-LF to human Met; Figure 9B : Dissociation curve of the binding of antibody E2-mut34-69×M5-69-FAE-LF to human Met; Figure 9C : Dissociation curve of the binding of antibody JNJ-372 to human Met.

[0195] Figures 10A to 10G : Binding experiment of the test substance at the cellular level.

[0196] Figures 11A to 11M : Detection results of the phosphorylation of AKT or ERK by the test substance.

[0197] Figure 12A : Results of the ADCC experiment detected by the reporter gene method.

[0198] Figure 12B : Results of the ADCC experiment detected by the reporter gene method.

[0199] Figure 12C : Results of the ADCC experiment detected by the LDH method.

[0200] Figure 12D : Results of the ADCC experiment detected by the reporter gene method.

[0201] Figure 12E : Results of the ADCC experiment detected by the LDH method.

[0202] Figure 13 : Inhibitory effect of the test substance on the NCI-H1975 human lung cancer model transplanted into CB-17 SCID mice.

[0203] Figure 14 : Inhibitory effect of the test substance on the NCI-H1975 human lung cancer subcutaneous tumor model.

[0204] Figure 15:Inhibitory effect of the test substance on the H1975 (L858R / T790M / C797S) human lung cancer subcutaneous tumor model.

[0205] Figure 16 :Inhibitory effect of the test substance on the H292 human lung cancer subcutaneous tumor model. Detailed implementation manners

[0206] Scientific and technical terms

[0207] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are all conventional steps widely used in the corresponding fields. Meanwhile, to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0208] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximal effect.

[0209] As used herein, the term "antibody" refers to an immunoglobulin molecule typically consisting of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The VH and VL regions can also be further divided into regions of high variability (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 carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site. The assignment of amino acids to each region or domain follows the Bethesda, M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or the definitions of Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF [J]. Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0210] The term "antibody" is not limited by any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0211] As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for possible naturally occurring spontaneous mutations. A mAb has high specificity for a single epitope on an antigen. A polyclonal antibody, in contrast to a monoclonal antibody, generally contains at least two or more different antibodies, which usually recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained by the hybridoma technique first reported by Kohler et al. ([ G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. nature, 1975; 256(5517): 495), but can also be obtained by recombinant DNA techniques (see, e.g., U.S. Patent 4,816,567).

[0212] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment in which all or part of the CDR regions of a human immunoglobulin (recipient antibody) have been replaced with the CDR regions of a non-human antibody (donor antibody), where the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework regions (FRs) of the recipient antibody can also be replaced with the amino acid residues of the corresponding non-human antibody or with the amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, e.g., Jones et al., Nature 1986; 321: 522 525; Reichmann et al., Nature, 1988; 332: 323 329; Presta, Curr. Op. Struct. Biol. 1992; 2: 593 - 596; and Clark, Immunol. Today 2000; 21: 397 402. In some cases, the antigen-binding fragment of the antibody is a diabody, in which V H and V LThe domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and generating two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448 and Poljak R.J. et al., Structure 1994; 2:1121-1123).

[0213] As used herein, the term "single chain fragment variable (ScFv)" refers to a molecule comprising the variable region of the antibody heavy chain (V H ) and the variable region of the antibody light chain (V L ) linked by a linker. Wherein V L and V H domains are paired by a linker that enables them to be produced as a single polypeptide chain to form a monovalent molecule (see, e.g., Bird et al, Science 1988; 242:423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883). Such scFv molecules can have the general structure: NH2-V L -linker fragment-V H -COOH or NH2-V H -linker fragment-V L -COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker with the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al, Protein Eng. 1995; 8:725-731, Choi et al, Eur. J. Immunol. 2001; 31:94-106, Hu et al, Cancer Res. 1996; 56:3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293:41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

[0214] As used herein, the term "isolated" or "separated" refers to being obtained by artificial means from its natural state. If a "separated" substance or component occurs in nature, it may be that its natural environment has changed, or the substance has been separated from its natural environment, or both. For example, a certain polynucleotide or polypeptide that naturally exists in a living animal in an unseparated state, and the highly purified same polynucleotide or polypeptide separated from this natural state is called an isolated one. The term "isolated" or "separated" does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0215] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic element it carries can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector can contain various elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can also contain an origin of replication.

[0216] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, and it includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0217] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) means that the antibody binds with a dissociation constant of less than about 10 -5 M, for example less than about 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M or 10 -10 M or less affinity (K D ) binds to the antigen.

[0218] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, an antibody binds to an antigen (e.g., EGFR protein) with a dissociation equilibrium constant less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or less dissociation equilibrium constant (K D ). K D can be determined by methods known to those skilled in the art, such as using a Fortebio molecular interaction instrument.

[0219] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0220] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers. For example, pH regulators include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.

[0221] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (such as a tumor) is an amount sufficient to prevent, arrest, or delay the onset of the disease (such as a tumor); an effective amount for treating a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the general condition of the patient such as age, weight, and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0222] As used herein, when referring to the amino acid sequence of the EGFR protein, it includes the full length of the EGFR protein, fragments containing the EGFR ECD; it also includes fusion proteins of the full length of the EGFR protein or fusion proteins of the EGFR ECD, such as fragments fused with the Fc protein fragment (mFc or hFc) of murine or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can occur naturally or be introduced artificially in the amino acid sequence of the EGFR protein without affecting its biological function. Therefore, in the present invention, the term "protein" should include all such sequences, including their natural or artificial variants. And when describing a sequence fragment of the EGFR protein, it also includes the corresponding sequence fragment in its natural or artificial variants.

[0223] As used herein, when referring to the amino acid sequence of the MET protein, it includes the full length of the MET protein, fragments containing the MET ECD; it also includes fusion proteins of the full length of the MET protein or fusion proteins of the MET ECD, such as fragments fused with the Fc protein fragment (mFc or hFc) of murine or human IgG. However, those skilled in the art understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can occur naturally or be introduced artificially in the amino acid sequence of the MET protein without affecting its biological function. Therefore, in the present invention, the term "protein" should include all such sequences, including their natural or artificial variants. And when describing a sequence fragment of the MET protein, it also includes the corresponding sequence fragment in its natural or artificial variants.

[0224] In the present invention, the term "ADCC" refers to antibody-dependent cell-mediated cytotoxicity. The Fab segment of the antibody binds to the antigenic epitope of virus-infected cells or tumor cells, and its Fc segment binds to the Fc receptor (Fc Receptor, FcR) on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by the killer cells.

[0225] In the present invention, unless otherwise specified, the "first" (such as the first protein functional region or the first drug product) and the "second" (such as the second protein functional region or the second drug product) are for the purpose of distinction in reference or clarity in expression, and do not have the typical meaning of order.

[0226] In the present invention, the term "IgG half molecule (IgG-HM)" refers to an antibody molecule composed of one heavy chain and one light chain of an IgG antibody (such as IgG1, IgG2, IgG3 or IgG4), which is a monovalent antibody (for example, see Feng Yifan et al., Construction and activity analysis of the monovalent antibody of HIV-1 specific monoclonal antibody 2G12, Chinese Journal of Viral Diseases, Vol. 5, No. 3, May 2015, p171-175).

[0227] Some sequences involved in the present invention are as follows (the underlined part is the CDR of the Kabat numbering scheme):

[0228] (1) E2-M1-VH

[0229] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSIYKDSGFDY WGQGTLVTVSS (SEQ ID NO:1)

[0230] (2) E2-M2-VH

[0231] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSVYKDSGFDY WGQGTLVTVSS (SEQ ID NO:2)

[0232] (3) E2-M5-VH

[0233] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSVYQDSGFDY WGQGTLVTVSS(SEQ ID NO:3)

[0234] (4)E2-M12-VH

[0235] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSVYEDSRFDY WGQGTLVTVSS(SEQ ID NO:4)

[0236] (5)E2-M13-VH

[0237] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSVYSDSMFDY WGQGTLVTVSS(SEQ ID NO:5)

[0238] (6)E2-M16-VH

[0239] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS NGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSLYKDSRFDY WGQGTLVTVSS(SEQ ID NO:6)

[0240] (7)E2-M17-VH

[0241] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSLYKDSRFDY WGQGTLVTVSS(SEQ ID NO:7)

[0242] (8)E2-M25-VH

[0243] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSVYKDSRFDY WGQGTLVTVSS(SEQ ID NO:8)

[0244] (9)E2-M34-VH

[0245] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSIYEDSGFDY WGQGTLVTVSS(SEQ ID NO:9)

[0246] (10)E2-M38-VH

[0247] QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYC ARVSLYEDSGFDY WGQGTLVTVSS(SEQ ID NO:10)

[0248] (11)E2-LC

[0249] EIVMTQSPATLSLSPGERATLSC RASQSVSSWLA WYQQKPGQAPRLLIY GASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC LQVGSTPLT FGGGTKAEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:11)

[0250] The sequences of three light chain CDRs are as follows:

[0251] LCDR1: RASQSVSSWLA(SEQ ID NO:38)

[0252] LCDR2: GASNRAT (SEQ ID NO:39)

[0253] LCDR3: QVGSTPLT (SEQ ID NO:40)

[0254] The light chain variable region VL sequence is as follows:

[0255] EIVMTQSPATLSLSPGERATLSC RASQSVSSWLA WYQQKPGQAPRLLIY GASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC LQVGSTPLT FGGGTKAEIK (SEQ ID NO:42)

[0256] (12) E2-10-LC-13-91A

[0257] EIVMTQSPATLSLSPGERATLSC RASQSVSSWLA WYQQKPGQAPRLLIY GASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC LQAGSTPLT FGGGTKAEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:12)

[0258] Among them, the sequences of 3 light chain CDRs are as follows:

[0259] LCDR1: RASQSVSSWLA (SEQ ID NO:38)

[0260] LCDR2: GASNRAT (SEQ ID NO:39)

[0261] LCDR3: LQAGSTPLT (SEQ ID NO:41)

[0262] The light chain variable region VL sequence is as follows:

[0263] EIVMTQSPATLSLSPGERATLSC RASQSVSSWLA WYQQKPGQAPRLLIY GASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC LQAGSTPLT FGGGTKAEIK (SEQ ID NO:43)

[0264] (13)E2-10-LC-69

[0265] EIVMTQSPATLSLSPGERATLSC RASQSVSSWLA WYQQKPGQAPRLLIY GASNLAK GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC LQAGSTPLT FGGGTKAEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:13)

[0266] The sequences of the three light chain CDRs are as follows:

[0267] LCDR1: RASQSVSSWLA (SEQ ID NO:38)

[0268] LCDR2: GASNRAT (SEQ ID NO:39)

[0269] LCDR3: LQAGSTPLT (SEQ ID NO:41)

[0270] The sequence of the light chain variable region VL is as follows:

[0271] EIVMTQSPATLSLSPGERATLSC RASQSVSSWLA WYQQKPGQAPRLLIY GASNLAK GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC LQAGSTPLT FGGGTKAEIK (SEQ ID NO:44)

[0272] (14)JNJ-372-ML (the italic part is the light chain variable region)

[0273]

[0274] The sequences of the three light chain CDRs are as follows:

[0275] RASQGISNWLA (SEQ ID NO:51)

[0276] AASSLLS (SEQ ID NO:52)

[0277] QQANSFPIT (SEQ ID NO:53)

[0278] The sequence of the light chain variable region VL is as follows:

[0279] DIQMTQSPSSVSASVGDRVTITC RASQGISNWLA WFQHKPGKAPKLLIY AASSLLS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFPIT FGQGTRLEIK(SEQ ID NO:56)

[0280] (15)JNJ-372-EL (where the italic part is the light chain variable region)

[0281]

[0282] (16)M5-HC-K409R (where the italic part is the heavy chain variable region)

[0283]

[0284] The sequences of the three heavy chain CDRs are as follows:

[0285] HCDR1: SSVYYWS(SEQ ID NO:45)

[0286] HCDR2: VIYPSGNTYYSPSLKS(SEQ ID NO:46)

[0287] HCDR3: TIYDLFDI(SEQ ID NO:47)

[0288] The sequence of the heavy chain variable region VH is as follows:

[0289] QLQLQESGPGLVKPSETLSLTCTVSGGSIS SSVYYWS WIRQPPGKGLEWIG VIYPSGNTYYSPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR TIYDLFDI WGQGTMVTVSS(SEQ ID NO:54)

[0290] The sequence of the heavy chain constant region is as follows:

[0291] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:78)

[0292] (17)E2mut34-HC-F405L (italic part is the heavy chain variable region)

[0293]

[0294] The heavy chain constant region sequence is as follows:

[0295] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 79)

[0296] (18)E2mut34-HC-hole (italic part is the heavy chain variable region)

[0297]

[0298] The constant region sequences are as follows:

[0299] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 80)

[0300] (19)E2mut34-HC-holeRF (where the italic part is the heavy chain variable region)

[0301]

[0302]

[0303] The constant region sequences are as follows:

[0304] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK(SEQ ID NO:81)

[0305] (20)M5-HC-knob (where the italic part is the heavy chain variable region)

[0306]

[0307] The constant region sequences are as follows:

[0308] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:82)

[0309] (21) JNJ-372-MH (the italic part is the heavy chain variable region)

[0310]

[0311]

[0312] The sequences of the three heavy chain CDRs are as follows:

[0313] SYGIS (SEQ ID NO:48)

[0314] WISAYNGYTNYAQKLQG (SEQ ID NO:49)

[0315] DLRGTNYFDY (SEQ ID NO:50)

[0316] The heavy chain variable region VH is as follows:

[0317] QVQLVQSGAEVKKPGASVKVSCETSGYTFT SYGIS WVRQAPGHGLEWMG WISAYNGYTNYAQKLQG RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR DLRGTNYFDY WGQGTLVTVSS (SEQ ID NO:55)

[0318] (22) JNJ-372-EH (where the italic part is the heavy chain variable region)

[0319]

[0320] (23) EGFR-his-tag

[0321] MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSHHHHHH (SEQ ID NO:23)

[0322] (24) MET-his-tag

[0323] MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTHHHHHH(SEQ ID NO:24)

[0324] (25)E2-10-LC-13-91Q

[0325] EIVMTQSPATLSLSPGERATLSC RASQSVSSWLA WYQQKPGQAPRLLIY GASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC LQQGSTPLT FGGGTKAEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:57)

[0326] The sequences of the three light - chain CDRs are as follows:

[0327] LCDR1: RASQSVSSWLA(SEQ ID NO:38)

[0328] LCDR2: GASNRAT(SEQ ID NO:39)

[0329] LCDR3: LQQGSTPLT (SEQ ID NO:76)

[0330] The sequence of the light - chain variable region VL is as follows:

[0331] EIVMTQSPATLSLSPGERATLSC RASQSVSSWLA WYQQKPGQAPRLLIY GASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC LQQGSTPLT FGGGTKAEIK(SEQ ID NO:77)

[0332] (26)E2mut1 - HC - F405L (where the italic part is the heavy - chain variable region)

[0333]

[0334] (27)E2mut2 - HC - F405L (where the italic part is the heavy - chain variable region)

[0335]

[0336] (28)E2mut5 - HC - F405L (where the italic part is the heavy - chain variable region)

[0337]

[0338]

[0339] (29) E2mut12-HC-F405L (where the italic part is the heavy chain variable region)

[0340]

[0341] (30) E2mut13-HC-F405L (where the italic part is the heavy chain variable region)

[0342]

[0343] (31) E2mut16-HC-F405L (where the italic part is the heavy chain variable region)

[0344]

[0345]

[0346] (32) E2mut17-HC-F405L (where the italic part is the heavy chain variable region)

[0347]

[0348] (33) E2mut25-HC-F405L (where the italic part is the heavy chain variable region)

[0349]

[0350] (34) E2mut38-HC-F405L (where the italic part is the heavy chain variable region)

[0351]

[0352]

[0353] (35) E2mut1-HC-hole (where the italic part is the heavy chain variable region)

[0354]

[0355] (36) E2mut2-HC-hole (where the italic part is the heavy chain variable region)

[0356]

[0357] (37) E2mut5-HC-hole (where the italic part is the heavy chain variable region)

[0358]

[0359] (38)E2mut12-HC-hole (where the italic part is the heavy chain variable region)

[0360]

[0361] (39)E2mut13-HC-hole (where the italic part is the heavy chain variable region)

[0362]

[0363] (40)E2mut16-HC-hole (where the italic part is the heavy chain variable region)

[0364]

[0365] (41)E2mut17-HC-hole (where the italic part is the heavy chain variable region)

[0366]

[0367]

[0368] (42)E2mut25-HC-hole (where the italic part is the heavy chain variable region)

[0369]

[0370] (43)E2mut38-HC-hole (where the italic part is the heavy chain variable region)

[0371]

[0372] Advantages of the Invention

[0373] The bispecific antibody of the present invention has achieved one or more of the following technical effects (1)-(5):

[0374] (1) It has good affinity and / or specificity for the targets EGFR and / or MET;

[0375] (2) It has high ADCC activity;

[0376] (3) It has a high endocytosis rate;

[0377] (4) It has good anti-tumor activity;

[0378] (5) The production process is simple and the cost is low.

[0379] The embodiments of the present invention will be described in detail below in conjunction with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those conditions not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0380] Preparation Example 1: Sequence Design and Preparation of Anti-EGFR Monoclonal Antibody

[0381] Ten anti-EGFR monoclonal antibodies were designed and screened, and were named E2-M1, E2-M2, E2-M5, E2-M12, E2-M13, E2-M16, E2-M17, E2-M25, E2-M34 and E2-M38, respectively. The amino acid sequences of the heavy chain variable regions of these 10 anti-EGFR monoclonal antibodies are shown in SEQ ID NO: 1-10 respectively, and the heavy chain constant regions are selected from the amino acid sequences shown in SEQ ID NO: 79, 80, 81. The amino acid sequences of the light chains are all shown in SEQ ID NO: 11. Among them, HCDR and LCDR are determined by the kabat numbering system and are underlined as shown in Table A below.

[0382] Table A

[0383]

[0384]

[0385] In the above antibodies, the structure of HCDR3 is shown in SEQ ID NO: 37 below: ARVSX1YX2DSX3FDY (SEQ ID NO: 37); wherein, X1 is selected from amino acids I, V and L, X2 is selected from amino acids K, Q, E and S, and X3 is selected from amino acids G, R and M.

[0386] Example 1: Determination of the Affinity of E2 Mutant for EGFR by Octet Red 96e

[0387] The kinetic parameters of the 10 anti-EGFR monoclonal antibodies prepared above binding to the antigen EGFR-his-tag were measured using the protein A capture method. The monoclonal antibody with a concentration of 1 μg / ml and the control antibody JNJ-372-E-LF (prepared in Preparation Example 2 below) were bound to the Protein A probe (Cat No: 18-5010; lot: 2001131), and the antigen EGFR-his-tag was serially diluted 2-fold from 50 nM downwards with 1X Fortebio working solution (1X PBS + 0.05% Tween 20) to set 4 concentration gradients to bind to the antibody and dissociated in 1X Fortebio working solution.

[0388] The kinetic parameters of the E2 mutant binding to EGFR-his-tag are shown in Table B.

[0389] Table B

[0390] Antibody Name <![CDATA[K D (M)]]> ka (1 / Ms) kd (1 / s) JNJ-372-E-LF 4.34E-09 2.47E+05 1.07E-03 E2-M1 2.16E-09 4.11E+05 8.88E-04 E2-M2 1.92E-09 4.19E+05 8.05E-04 E2-M5 8.86E-09 6.25E+04 5.54E-04 E2-M12 8.01E-09 4.75E+04 3.80E-04 E2-M13 8.64E-09 5.24E+04 4.53E-04 E2-M16 9.31E-09 4.88E+04 4.54E-04 E2-M17 9.04E-09 5.02E+04 4.54E-04 E2-M25 1.52E-09 2.79E+05 4.24E-04 E2-M34 1.13E-09 4.06E+05 4.59E-04 E2-M38 2.15E-09 4.04E+05 8.66E-04

[0391] The results showed that the affinities of all 10 E2 mutants reached the nM level. Among them, the affinities of E2-M1, E2-M2, E2-M25, E2-M34, and E2-M38 were better than those of the control antibody JNJ-372-E-LF, and the affinity of E2-M34 was the highest.

[0392] Preparation Example 2: Design and Preparation of the First Batch of EGFR×MET Bispecific Antibodies

[0393] 1. Construction of Bispecific Antibody Molecules

[0394] The heavy chain of the parental antibody at the EGFR end of the EGFR×MET bispecific antibody was constructed by selecting E2mut34 from the 10 high-affinity EGFR antibody heavy chain variable regions prepared in Preparation Example 1 (see SEQ ID NOs: 17-19). In addition, referring to the heavy chain of the publicly available MET antibody (SEQ ID NO: 410 in CN105705519A), after making corresponding modifications in the constant region according to the needs of preparing the bispecific antibody, it was used as the heavy chain of the parental antibody at the MET end of the EGFR×MET bispecific antibody (see SEQ ID NOs: 16 and 20). E2-10-LC-13-91A or E2-10-LC-69 was used as the common light chain.

[0395] GenScript was entrusted to synthesize the DNA sequences encoding the 4 light chains E2-10-LC-13-91A, E2-10-LC-69, JNJ-372-ML, and JNJ-372-EL in Table 1. After digestion with SapI (purchased from NEB, catalog number: R0569L) and ligation to the HXT2 vector (a vector independently modified by Junshi Biosciences, derived from pTT5), 4 expression vectors were obtained, named HXT2-E2-10-LC-13-91A, HXT2-E2-10-LC-69, HXT2-JNJ-372-ML, and HXT2-JNJ-372-EL, respectively.

[0396] Among them, HXT2-JNJ-372-ML is the light chain of the MET end antibody of Amivantamab, and HXT2-JNJ-372-EL is the light chain of the EGFR end antibody of Amivantamab (sequence source: IMGT official website).

[0397] Commissioned Qingke Biotech to synthesize the DNA sequences of the 7 heavy chains in Table 1, namely M5-HC-K409R, E2mut34-HC-F405L, E2mut34-HC-hole, E2mut34-HC-holeRF, M5-HC-knob, JNJ-372-MH, and JNJ-372-EH. Digest with SapI (purchased from NEB, catalog number: R0569L) and ligate to the HXT1S vector (a vector independently modified by Junshi Biosciences, derived from pTT5) to obtain 7 expression vectors, named HXT1S-E2mut34-HC-F405L, HXT1S-M5-HC-K409R, HXT1S-E2mut34-HC-hole, HXT1S-E2mut34-HC-holeRF, HXT1S-M5-HC-knob, HXT1S-JNJ-372-MH, and HXT1S-JNJ-372-EH respectively.

[0398] Among them, HXT1S-JNJ-372-MH is the heavy chain of the MET end antibody of Amivantamab, and HXT1S-JNJ-372-EH is the heavy chain of the EGFR end antibody of Amivantamab (sequence source: IMGT official website).

[0399] Table 1: Heavy chain and light chain names

[0400]

[0401]

[0402] 2. Transient Protein Expression and Purification

[0403] The required heavy chains, light chains, and expression vectors are shown in Table 2. Among them:

[0404] E2mut34-91A-F405L-LF and M5-91A-K409R-LF are used for the preparation of subsequent bispecific antibodies (in vitro recombination method);

[0405] E2mut34-69-F405L-LF and M5-69-K409R-LF are used for the preparation of subsequent bispecific antibodies (in vitro recombination method);

[0406] JNJ-372-M-LF and JNJ-372-E-LF are used for the preparation of subsequent bispecific antibodies (in vitro recombination method).

[0407] ALK101-2 and ALK101-4 use the common light chain and the "knob-into-hole" technology (refer to WO1996027011A1) to prepare bispecific antibodies.

[0408] Table 2: Combinations of heavy and light chains and corresponding expression vectors

[0409]

[0410] 2.1 Expression of bispecific antibody molecules

[0411] Count the CHO-K1 cells (owned by Suzhou Junmeng) that are being cultured. When the cell density is 2 - 6×10 6 / ml, passage and amplify them using CD CHO medium (purchased from Thermofisher, catalog number: 12490-001). Dilute the cell density to 1.8 - 2.5×10 6 / ml one day before transfection. The next day, when the cell density reaches approximately 3.5 - 5.0×10 6 / ml, perform transfection. First, add one-tenth of the transfection volume of CD CHO medium, add 1 - 2 μg / ml of plasmid (self-made by the company) according to the combination table shown in Table 2, and finally add 3 - 14 μg / ml of PEI (purchased from Polysciences, catalog number: 24765-1). Mix well and incubate at room temperature. Finally, slowly add the transfection mixture to the pre-treated cells while mixing. Place the transfected mixture in a shaker for culture. Add 6% of the glycoform regulator (purchased from OPMI, catalog number: R170026) on the first day after transfection, supplement 4% of Cell Boost 7a (purchased from Hyclone, catalog number: SH31026.05) and 0.4% of Cell Boost 7b (purchased from Hyclone, catalog number: SH31027.04CN), and then supplement once every two days. Sample collection is carried out 5 - 9 days after transfection.

[0412] 3. In Vitro Recombination and Purification

[0413] 3.1 Affinity capture of bispecific antibody molecules

[0414] After the culture was completed, the cells were centrifuged at 1000 g for 5 min using a benchtop centrifuge (purchased from ThermoFisher, R404A) to discard the precipitate, and then centrifuged at 8000 g for 30 min to collect the cell supernatant, which was sterile filtered using a 0.22 μm filter cup (purchased from jet, FPE-214-000). Purification was performed using a protein purification instrument (purchased from GE, AKTA avant). The Mabselect sure LX column (Cytiva, 17547403) was equilibrated with PBS equilibration buffer (purchased from Wuxi OriGene Technologies Co., Ltd., ZLI-9061). After sample loading was completed, it was first washed with affinity chromatography wash buffer A (pH 5.5, 45 mM acetic acid-sodium acetate + 1 M sodium chloride system), then washed with wash buffer B (pH 5.5, 45 mM acetic acid-sodium acetate system), and finally the target protein was eluted with affinity elution buffer (pH 3.6, 10 mM acetic acid-sodium acetate buffer), and the sample was neutralized with 1 M Tris (purchased from Merck, product number: E300016981946) buffer to adjust the pH to 5.5 - 6 for the next in vitro recombination.

[0415] 3.2 In Vitro Recombination of Bispecific Antibody Molecules

[0416] Referring to the technical solution disclosed in WO2011131746, the bispecific antibody was prepared using the Fab arm exchange technology as follows:

[0417] The two parental antibodies after affinity purification were concentrated and buffer exchanged into PBS equilibration buffer (purchased from Wuxi OriGene Technologies Co., Ltd., ZLI-9061), and the protein concentration was set at 1 ± 0.05 mg / ml.

[0418] Preparation of 750 mM Cysteamine hydrochloride (purchased from VETEC, V900342-25G) stock solution: Add 2.556 g of Cysteamine hydrochloride to 14 ml of PBS, make up to 30 ml with PBS, then filter through a 0.22 μm filter (purchased from sartorius, 16541-K), and finally wrap with aluminum foil and store in the dark.

[0419] Construct an incubation system: Take the maternal antibody processed in the above steps. As shown in Table 3, add 2 ml of maternal antibody M, 2.4 ml of maternal antibody E, and 0.489 ml of 750 mM Cysteamine hydrochloridel into a 15 ml centrifuge tube (purchased from Genemore, G3210015) according to the molar ratio M:E = 1:1.2. Seal the constructed incubation system with aluminum foil and place it in a 31 °C water bath (Shanghai Jinghong Experimental Equipment Co., Ltd., DK-S28) in the dark for 3 h of incubation. After incubation, concentrate and change the solution with PBS balance buffer, and place it in the dark at room temperature for 16 h - 24 h. Finally, the molecular structure of the bispecific antibody prepared by the in vitro recombination method is as Figures 1 to 3 shown.

[0420] Table 3: In vitro recombination combination table of bispecific antibodies

[0421]

[0422] 3.3 Purification of bispecific antibody molecules

[0423] Select CaptoTM MMC Impres (Cytiva, 17371602) packing material for purification. Pre-equilibrate with the washing buffer (pH 7.5, 20 mM Tris-HCl + 1 M NaCl buffer system), and equilibrate the system with the equilibration solution (pH 7.5, 20 mM Tris-HCl system). After sample loading, equilibrate with the equilibration solution for 3 - 6 column volumes, and finally perform linear elution with the elution buffer (pH 7.5, 20 mM Tris-HCl + 1 M NaCl buffer system) to collect the target protein. Thus, the 3 bispecific antibodies in Table 3 are obtained.

[0424] Example 2: ELISA Binding Experiment

[0425] 4.1 EGFR ELISA binding experiment

[0426] Dilute EGFR-his-tag (purchased from Suzhou Junmeng, batch number: 20210803. Sequence source: nuiprot official website) to 5.0 μg / ml with PBS (purchased from Hyclone, product number: SH30256.01), add 100 μl / well to the ELISA plate, and let it stand and coat in a constant temperature incubator at 37 °C for 60 minutes; wash the plate; add 200 μl / well of 2% BSA (purchased from Sigma, product number: B2064) to the plate, incubate in a constant temperature incubator at 37 °C for 60 minutes, wash the plate; dilute the sample to 10 μg / ml with 2% BSA, perform 3-fold serial dilution to 0.056 ng / ml, add 100 μl / well to the ELISA plate, incubate in a constant temperature incubator at 37 °C for 60 minutes, wash the plate; dilute the goat anti-human IgG (Fc specific) antibody conjugated with horseradish peroxidase (HRP) (purchased from Sigma, product number: A0170) 5000-fold with 2% BSA, add 100 μl / well to the ELISA plate, incubate in a constant temperature incubator at 37 °C for 60 minutes, wash the plate; add chromogenic solution TMB (purchased from Sigma, product number: T2885) at 0.1 mg / ml, 100 μl / well, avoid air bubbles, develop color at 37 °C in the dark for 10 minutes; finally add 2M hydrochloric acid solution to terminate the reaction, 100 μl / well, avoid air bubbles, complete the ELISA reader reading within 10 minutes (detection wavelength 450 nm; reference wavelength 620 nm), and use a four-parameter logistic regression (4PL) model to fit the EC 50 As Figure 4 shown, the EC 50 values of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 binding to EGFR are 13.57, 11.71, and 33.90 ng / ml respectively. The binding affinity of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF to EGFR is better than that of JNJ-372.

[0427] 4.2 MET ELISA Binding Experiment

[0428] Dilute MET-his-tag (purchased from Suzhou Junmeng, batch number: 20220404. Sequence source: nuiprot official website) to 2.0 μg / ml with PBS (purchased from Hyclone, product number: SH30256.01), add 100 μl / well to the ELISA plate, and let it stand and coat in a 37°C incubator for 60 minutes; wash the plate; add 200 μl / well of 2% BSA (purchased from Sigma, product number: B2064) to the plate, incubate in a 37°C incubator for 60 minutes, wash the plate; dilute the sample to 10 μg / ml with 2% BSA, perform 3-fold serial dilution to 0.056 ng / ml, add 100 μl / well to the ELISA plate, and incubate in a 37°C incubator for 60 minutes. Wash the plate; dilute the goat anti-human IgG (Fc specific) antibody conjugated with horseradish peroxidase (HRP) (purchased from Sigma, product number: A0170) 5000-fold with 2% BSA, add 100 μl / well to the ELISA plate, incubate in a 37°C incubator for 60 minutes, wash the plate; add chromogenic solution TMB (purchased from Sigma, product number: T2885) at 0.1 mg / ml, 100 μl / well, avoid air bubbles, and develop color in the dark at 37°C for 10 minutes; finally add 2M hydrochloric acid solution to terminate the reaction, 100 μl / well, avoid air bubbles, and complete the ELISA reader reading within 10 minutes (detection wavelength 450 nm; reference wavelength 620 nm), and use a four-parameter logistic regression (4PL) model to fit the EC 50 . As Figure 5 shown, the EC 50 values of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 binding to Met are 7.461, 6.558, and 8.416 ng / ml respectively. The binding affinity of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF to Met is comparable to that of JNJ-372.

[0429] Example 3: ELISA Blocking Experiment

[0430] 5.1 EGFR ELISA Blocking Experiment

[0431] Dilute EGFR-his-tag (purchased from Suzhou Junmeng, batch number: 20210803) to 2.0 μg / ml with PBS (purchased from Hyclone, product number: SH30256.01), add 100 μl / well to the ELISA plate, and let it stand and coat in a constant temperature incubator at 37°C for 60 minutes; wash the plate; add 200 μl / well of 2% BSA (purchased from Sigma, product number: B2064) to the plate, and incubate in a constant temperature incubator at 37°C for 60 minutes; wash the plate; dilute EGF mFc (purchased from Acro, product number: EGF-H525b) with 2% BSA to 3.0 μg / ml, then dilute the sample with 3.0 μg / ml of EGF mFc to 400 μg / ml, and perform 3-fold serial dilution to 0.0023 μg / ml, add 100 μl / well to the ELISA plate, and incubate in a constant temperature incubator at 37°C for 60 minutes, wash the plate; dilute the horseradish peroxidase (HRP)-conjugated anti-mouse Fc antibody (purchased from Sigma, product number: A2554) 5000-fold with 2% BSA, add 100 μl / well to the ELISA plate, and incubate in a constant temperature incubator at 37°C for 60 minutes, wash the plate; add chromogenic solution TMB (purchased from Sigma, product number: T2885) at 0.1 mg / ml, 100 μl / well, avoid air bubbles, and develop color at 37°C in the dark for 10 minutes; finally add 2M hydrochloric acid solution to terminate the reaction, 100 μl / well, avoid air bubbles, and complete the ELISA reader reading within 10 minutes (detection wavelength 450 nm; reference wavelength 620 nm), and use a four-parameter logistic regression (4PL) model to fit the IC 50 . As Figure 6 shown, the IC 50 values of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 blocking EGFR are 1749, 1668, and 3356 ng / ml respectively. The blocking ability of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF against EGFR is slightly better than that of JNJ-372.

[0432] 5.2 MET ELISA Blocking Experiment

[0433] Dilute HGF-his-tag (purchased from Acro, lot number: HGF-H52H3) with PBS (purchased from Hyclone, product number: SH30256.01) to 4.0 μg / ml, add 100 μl / well to the ELISA plate, and coat overnight at 4°C; wash the plate; add 200 μl / well of 2% BSA (purchased from Sigma, product number: B2064) to the plate, incubate at room temperature for 90 minutes, wash the plate; dilute MET biotinylated (purchased from Suzhou Junmeng, lot number: 20220526) with 2% BSA to 0.1 μg / ml, then dilute the sample with 0.1 μg / ml of MET biotinylated to 20 μg / ml, and perform 2.5-fold serial dilution to 0.84 ng / ml, add 100 μl / well to the ELISA plate, incubate at room temperature for 60 minutes, wash the plate: Dilute Streptavidin conjugated with horseradish peroxidase (HRP) (purchased from Jackson ImmunoResearch, product number: 016-030-084) 5000-fold with 2% BSA, add 100 μl / well to the ELISA plate, incubate at room temperature for 60 minutes, wash the plate; add chromogenic solution TMB (purchased from Sigma, product number: T2885) at 0.1 mg / ml, 100 μl / well, avoid air bubbles, develop color at room temperature in the dark for 10 minutes; finally add 2 M hydrochloric acid solution to terminate the reaction, 100 μl / well, avoid air bubbles, complete the ELISA reader reading within 10 minutes (detection wavelength 450 nm; reference wavelength 620 nm), and use a four-parameter logistic regression (4PL) model to fit the IC 50 . As Figure 7 shown, the IC 50 values of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 blocking Met are 216.7, 185.2, and 165 ng / ml respectively. The ability of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF to block Met is comparable to that of JNJ-372.

[0434] Example 4: Biacore Affinity Measurement

[0435] Use a Biacore T200 molecular interaction analyzer (GE Healthcare Life Sciences) to detect the binding affinities of antibodies E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 to recombinant human EGFR and Met respectively.

[0436] The method for determining the binding affinity of the antibody to EGFR is as follows: Coupling 40 μg / ml goat anti-human IgG-Fc fragment antibody to the surface of a CM5 chip (Cytiva, catalog number BR-1005-30) for capturing the antibody. Capture 1 μg / ml of E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 antibodies on the surface of the CM5 chip. Inject 20 nM and 5 nM of EGFR to bind with E2-mut34-91A×M5-91A-FAE-LF and E2-mut34-69×M5-69-FAE-LF, and inject 80 nM and 20 nM of EGFR to bind with JNJ-372. Use a Biacore T200 system (GE Healthcare) to detect the binding and dissociation kinetic signals. Use the software Biacore T200 Evaluation Software 3.0 to fit the binding and dissociation curves to calculate the affinity K D value.

[0437] The method for determining the binding affinity of the antibody to Met is as follows: Coupling 40 μg / ml goat anti-human IgG-Fc fragment antibody (Jackson ImmunoResearch) to the surface of a CM5 chip (Cytiva, catalog number BR-1005-30) for capturing the antibody. Capture 1 μg / ml of E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 antibodies on the surface of the CM5 chip. Inject 50 nM and 12.5 nM of Met to bind with the three antibodies. Use a Biacore T200 system (GE Healthcare) to detect the binding and dissociation kinetic signals. Use the software Biacore T200 Evaluation Software 3.0 to fit the binding and dissociation curves to calculate the affinity K D value.

[0438] The Biacore data are shown in Table 4, Figures 8A to 8C , Figures 9A to 9C as shown.

[0439] Table 4: Statistical table of antibody affinity determined by Biacore

[0440]

[0441] The results showed that the antibodies E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 all had binding activities to human EGFR and Met. The EGFR affinities of E2-mut34-91A×M5-91A-FAE-LF and E2-mut34-69×M5-69-FAE-LF were similar, and were approximately 5 times stronger than that of JNJ-372. The Met affinities of E2-mut34-91A×M5-91A-FAE-LF and E2-mut34-69×M5-69-FAE-LF were similar, and were approximately 2 times weaker than that of JNJ-372.

[0442] Example 5: Cell Binding Experiment

[0443] 293F-EGFR and 293-cMet cells overexpressing human EGFR and human cMET respectively (the 293F cells were purchased from the ATCC cell bank, catalog number: CRL-1573. Based on these cells, stable cell lines overexpressing human EGFR and human cMET were independently constructed. These 2 cell lines were used to detect the respective binding affinities of the EGFR and cMet ends in the bispecific antibody. The amino acid sequence of human EGFR was from NCBI accession number NP_005219.2, and the amino acid sequence of human cMET was from NCBI accession number NP_000236.2) were incubated with different concentrations of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, JNJ-372, and anti-KLH IgG1 antibody (starting concentration of 25 μg / ml, diluted 4-fold, a total of 10 concentration gradients) at 4°C for 30 min. Then, the unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS), and incubated with a fluorescent secondary antibody goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) containing 5‰ (v / v) prepared with staining buffer in the dark at 4°C for 30 minutes. Finally, the cells were collected with a flow cytometer (BD company, C6 PLUS model) to detect the fluorescent antibodies bound to the cell surface. The original data was analyzed with FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted with GraphPad and the EC 50 。

[0444] The NCI-H1975 cells (purchased from Shanghai Novobio Biotech Co., Ltd., catalog number: C01-HE, which endogenously co-express human EGFR and human cMet and are used to detect the cooperative binding affinity of bispecific antibodies) were incubated with different concentrations of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, JNJ-372, and anti-KLH IgG1 antibody (starting concentration 100 μg / ml, 4-fold dilution for the first 7 concentration gradients; 10-fold dilution for the last 5 concentration gradients) at 4 °C for 30 min. Then, the unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS), and incubated with a fluorescent secondary antibody goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) prepared with staining buffer containing 5‰ (v / v) in the dark at 4 °C for 30 minutes. Finally, the cells were collected using a flow cytometer (BD, model C6 PLUS), and the fluorescent antibodies bound to the cell surface were detected. The original data was analyzed using FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted using GraphPad and the EC 50 。

[0445] As Figures 10A to 10C shown, the binding EC 50 of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 to 293F-EGFR cells were 100.7 ng / ml, 85.02 ng / ml, and 57.40 ng / ml, respectively; the binding EC 50 to 293F-cMet cells were 127.7 ng / ml, 87.39 ng / ml, and 46.65 ng / ml, respectively; the binding EC 50 to H1975 cells were 19.33 ng / ml, 25.19 ng / ml, and 180.3 ng / ml, respectively. The results showed that the binding affinity of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF to single antigens EGFR or cMet was slightly weaker than that of the positive control JNJ-372, but the binding affinity to the dual antigens EGFR&cMet was significantly better than that of the positive control JNJ-372.

[0446] Example 6: Phosphorylation Inhibition Experiment

[0447] The NCI-H1975 cells were digested, centrifuged, and resuspended in complete medium (RPMI-1640 medium + 10 v / v% FBS), and seeded at 2×10 4Cells were seeded at a density of [number] cells per well in a 96-well flat-bottom plate (Corning, catalog number: 3599) and incubated overnight in a CO₂ incubator. The next day, the medium in the 96-well plate was replaced with serum-free RPMI-1640 medium and incubated overnight in the CO₂ incubator. On the third day, antibodies E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 were prepared at 2× the analytical concentration in serum-free RPMI-1640 medium. For EGF (R&D, catalog number: 236-EG-200) activation conditions, the starting concentration was 6 μM, diluted 3-fold, with a total of 11 concentration gradients; for HGF (R&D, catalog number: 294-HG-100 / CF) activation conditions, the starting concentration was 660 nM, diluted 3-fold, with a total of 12 concentration gradients. After discarding the medium in the 96-well plate, 50 μl of the above-diluted antibody was added and pre-incubated at 37 °C for 30 min. Serum-free RPMI-1640 medium was used to prepare 2× the analytical concentration of the agonist EGF (concentration 8 ng / ml) or HGF (concentration 140 ng / ml), and after preparation, it was placed in the incubator for standby. After the pre-incubation was completed, the 96-well flat-bottom plate was taken out of the incubator, and 50 μl of the diluted EGF or HGF agonist solution was added to each well and incubated at 37 °C for another 30 min. After the incubation was completed, the 96-well flat-bottom plate was taken out, the supernatant was discarded, and 50 μl of 1× lysis buffer was added according to the instructions of the KITs (phospho-AKT(Ser473)kit (Cisbio, catalog number: 64AKSPEH) & Advanced phospho-ERK1 / 2(Thr202 / Tyr204) (Cisbio, catalog number: 64AERPEH)). After shaking at 350 rpm at room temperature for 30 min, 16 μl of the cell lysate and 4 μl of the mix antibody in the KITs were added to a HTRF 96well lowvolume plate (Cisbio, catalog number: 66PL96025), gently mixed with a pipette tip, the plate was sealed with a film, and centrifuged at 1000 rpm for 30 s in a centrifuge. Finally, the plate was incubated in the dark at room temperature (20 °C - 25 °C) for 4 - 24 h, and the absorbance values (A) at wavelengths 655 nm and 620 nm were measured with a microplate reader (BioTek, Synergy H1 model), and the ratio (Ratio 665 / 620) was calculated. The calculation formula was Ratio665 / 620 = A655 / A620 × 10000, and the data analysis of Ratio 665 / 620 was processed using GraphPad Prism software.

[0448] Such as Figures 11A to 11EAs shown, under the condition of EGF activation, the IC values of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF and JNJ-372 in inhibiting AKT phosphorylation are 4.370 nM, 2.837 nM and 22.41 nM respectively; the IC values in inhibiting ERK phosphorylation are 15.51 nM, 10.49 nM and 53.58 nM respectively. It can be seen from the results that under the condition of EGF activation, the activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF in inhibiting AKT and ERK phosphorylation are better than those of the positive control JNJ-372. Under the condition of HGF activation, the IC values of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF and JNJ-372 in inhibiting AKT phosphorylation are 0.5869 nM, 0.3944 nM and 0.8861 nM respectively; the IC values in inhibiting ERK phosphorylation are 0.9124 nM, 0.9498 nM and 1.617 nM respectively. It can be seen from the results that under the condition of HGF activation, the activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF in inhibiting AKT and ERK phosphorylation are comparable to those of the positive control JNJ-372. 50 values for inhibiting AKT phosphorylation are 4.370 nM, 2.837 nM, 22.41 nM respectively; the IC values for inhibiting ERK phosphorylation are 15.51 nM, 10.49 nM, 53.58 nM respectively. As can be seen from the results, under the condition of EGF activation, the activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF in inhibiting AKT and ERK phosphorylation are better than those of the positive control JNJ-372. Under the condition of HGF activation, the IC values of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF and JNJ-372 in inhibiting AKT phosphorylation are 0.5869 nM, 0.3944 nM, 0.8861 nM respectively; the IC values in inhibiting ERK phosphorylation are 0.9124 nM, 0.9498 nM, 1.617 nM respectively. As can be seen from the results, under the condition of HGF activation, the activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF in inhibiting AKT and ERK phosphorylation are comparable to those of the positive control JNJ-372. 50 values are 15.51 nM, 10.49 nM, 53.58 nM respectively. As can be seen from the results, under the condition of EGF activation, the activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF in inhibiting AKT and ERK phosphorylation are better than those of the positive control JNJ-372. Under the condition of HGF activation, the IC values of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF and JNJ-372 in inhibiting AKT phosphorylation are 0.5869 nM, 0.3944 nM, 0.8861 nM respectively; the IC values in inhibiting ERK phosphorylation are 0.9124 nM, 0.9498 nM, 1.617 nM respectively. As can be seen from the results, under the condition of HGF activation, the activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF in inhibiting AKT and ERK phosphorylation are comparable to those of the positive control JNJ-372. 50 values for inhibiting AKT phosphorylation are 0.5869 nM, 0.3944 nM, 0.8861 nM respectively; the IC values for inhibiting ERK phosphorylation are 0.9124 nM, 0.9498 nM, 1.617 nM respectively. As can be seen from the results, under the condition of HGF activation, the activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF in inhibiting AKT and ERK phosphorylation are comparable to those of the positive control JNJ-372. 50 values are 0.9124 nM, 0.9498 nM, 1.617 nM respectively. As can be seen from the results, under the condition of HGF activation, the activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF in inhibiting AKT and ERK phosphorylation are comparable to those of the positive control JNJ-372.

[0449] Example 7: ADCC Activity Experiment

[0450] Reporter gene method: Seed NCI-H1975 cells at 5×10 4 cells per well in a 96-well white plate (Costar, catalog number: 3917) and incubate overnight in a CO2 incubator. The next day, remove the 96-well white plate from the CO2 incubator, discard the culture supernatant, and add 40 μl of antibodies E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF and JNJ-372 (starting concentration 50 μg / ml, 3-fold dilution for the first 3 concentration gradients; 5-fold dilution for the next 7 concentration gradients) diluted with assay buffer (RPMI-1640 medium + 2 v / v% FBS) to each well and pre-incubate with the target cells for 30 min. After the pre-incubation, add 40 μl of effector cells Jurkat ADCC expressing NFAT-Luc and FcγRIIIa resuspended with assay buffer (RPMI-1640 medium + 2 v / v% FBS) (1×10 5The cells were incubated at 37°C for an additional 6 h. Finally, the substrate One-Lite (Vazyme, Catalog No.: DD1203-03) was added, and the luciferase signal was detected using a microplate reader (TECAN, model M1000pro). The higher the fluorescence reading, the stronger the ADCC effect. The ADCC effect curve dependent on the antibody dose was fitted using GraphPad.

[0451] LDH method: First, resuscitate the PBMC effector cells for standby and recover them overnight in a CO2 incubator. After digesting and centrifuging the target cells NCI-H1975, discard the supernatant, and adjust the cell density to 4×10 5 cells / ml using RPMI Medium 1640 without phenol red + 1 v / v% FBS medium. Add 50 μl of the target cells to each well of a 96-well low-attachment plate (Costar, Catalog No.: 7007). Then, dilute the antibodies E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, JNJ-372, and anti-KLH IgG1 using RPMI Medium 1640 without phenol red + 1% FBS medium to prepare 4× the analytical concentration (the initial antibody concentration is 66.67 μg / ml, with 2 concentration gradients before 3-fold dilution; 7 concentration gradients after 5-fold dilution). Add 50 μl of the diluted antibody solution to each well, gently pipette to mix several times, and incubate in the incubator for 30 min. Take out the PBMCs that have been resuscitated overnight and adjust the density of the PBMCs to 5×10 6 cells / ml (effector-to-target ratio of 25:1). Add 100 μl of the PBMCs to each well of the 96-well low-attachment plate and co-culture them in a CO2 incubator for 4 h. Half an hour before the end of the co-culture time, add 20 μl of the cell lysis buffer in the LDH-cytotoxicity kit (BioVision, Catalog No.: K311-400) to each well of the Tmax group, and then put the 96-well low-attachment plate back into the incubator for continued incubation. Take out the 96-well low-attachment plate and centrifuge (1500 rpm × 5 min). Pipette 50 μl of the culture supernatant from each well into a new 96-well flat-bottom plate (Corning, Catalog No.: 3599), add 50 μl of the Dye Solution in the diluted LDH-cytotoxicity kit, incubate in the dark on a shaker for 10 - 30 min, and finally read the plate using a multimode microplate reader (TECAN, model M1000 pro) and perform data analysis and processing using GraphPad Prism software. The killing calculation formula is: ADCC% = [(Sample - Buffer) / (Tmax - Ts)] × 100.

[0452] such as Figures 12A to 12BAs shown, in the reporter gene assay, both E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF have ADCC activity, and their EC 50 values are 22.21 ng / ml and 14.12 ng / ml respectively, and the ADCC activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF are stronger than that of the positive control JNJ-372.

[0453] As Figure 12C shown, when PBMC is used as the effector cell and NCI-H1975 is used as the target cell, E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF and JNJ-372 can induce significant ADCC effects, and their EC 50 values are 1.408 ng / ml, 4.556 ng / ml and 42.60 ng / ml respectively, while the negative control anti-KLH IgG1 cannot induce ADCC effect. The ADCC activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF are stronger than that of the positive control JNJ-372.

[0454] Example 8: Endocytosis Experiment

[0455] After digestion and centrifugation of MKN45 cells (purchased from Shanghai Novobiotech Co., Ltd., catalog number: C01-1C, endogenously expressing human EGFR and human cMet), they were resuspended with staining buffer (PBS + 1 v / v% FBS), and 2×10 5Cells were seeded in a 96-well round bottom plate (Corning, catalog number: 3799), and antibodies E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, JNJ-372, and anti-KLH IgG1 (initial concentration 100 μg / ml, 4-fold dilution, 11 concentration gradients) diluted with staining buffer (PBS + 1 v / v% FBS) were added and incubated at 4°C for 30 min. Then, unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS). After resuspending the cells with 200 μl of complete medium (RPMI-1640 medium + 10 v / v% FBS), the cells were divided into two parts. One part was continuously incubated at 4°C for 24 h, and the other part was incubated at 37°C for 24 h. After the incubation time ended, the supernatant was discarded by centrifugation, and a fluorescence secondary antibody goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) containing 5‰ (v / v) prepared with staining buffer was added and incubated in the dark at 4°C for 30 minutes. Then, unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS). After fixation with PFA (Absin, catalog number: abs9179), cells were collected on a flow cytometer (BD, C6 PLUS model) to detect the fluorescent antibodies bound to the cell surface. The original data was analyzed with FlowJo to obtain the MFI value, and the Top value was obtained by fitting the antibody dose-dependent binding curve with GraphPad. The formula for the endocytosis rate is: Endocytosis rate (%) = (1 - Top_37°C / Top_4°C) × 100.

[0456] The results are shown in Table 5.

[0457] Table 5: Results of the detection of endocytic activity of MKN45 cells

[0458]

[0459]

[0460] The results showed that E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 could all be endocytosed by MKN45 cells, while the negative control anti-KLH IgG1 had almost no endocytic effect. The results showed that the endocytic activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF were comparable, and the endocytosis rate at 24 h was 72%, which was better than that of JNJ-372 (the endocytosis rate at 24 h was 49%).

[0461] Example 9: H1975 Xenograft Model

[0462] This example evaluates the inhibitory effect of the present invention, E2mut34-91A×M5-91A-FAE-LF, on the model of NCI-H1975 human lung adenocarcinoma cells transplanted into CB-17 SCID mice.

[0463] 1. Test procedure

[0464] Female CB-17 SCID mice, 6-8 weeks old (purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd., animal certificate number: 20170012022482), were subcutaneously inoculated with 5×10 6 NCI-H1975 cells on the right side. When the average tumor volume was approximately 164 mm 3 , suitable animals were selected and randomly divided into 5 groups according to tumor volume, with 6 animals in each group. They were respectively:

[0465] G1 Normal saline control group (solvent control group);

[0466] G2 JNJ-372 (5 mg / kg) group (positive control group);

[0467] G3 E2mut34-91A×M5-91A-FAE-LF (5 mg / kg) group (treatment group).

[0468] Intraperitoneal injection was performed, administered 2 times a week for 3 consecutive weeks, and the experiment ended 3 days after the last administration. The tumor volume and body weight were measured 2 times a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the relative tumor proliferation rate was calculated, TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%. (Ti: The average tumor volume of the treatment group on the i-th day of administration, T0: The average tumor volume of the treatment group on the 0-th day of administration; Vi: The average tumor volume of the solvent control group on the i-th day of administration, V0: The average tumor volume of the solvent control group on the 0-th day of administration).

[0469] The results are shown in Table 6 and Figure 13 as follows.

[0470] Table 6: Pharmacodynamic analysis table of each group in the NCI-H1975 human lung cancer subcutaneous tumor model

[0471]

[0472]

[0473] Note: 1. Data are expressed as "mean ± standard error";

[0474] 2. TGI% = [1 - (Ti - T0) / (Vi - V0)] × 100%;

[0475] 3. The P value was obtained by comparing the tumor volumes of each group using the T - test method.

[0476] The results showed that on the 20th day after administration, the average tumor volume of the normal saline control group was 3374 mm 3 . The average tumor volume of the JNJ - 372 (5 mg / kg) group was 708 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 83.0%, significantly inhibiting tumor growth; the average tumor volume of the E2mut34 - 91A×M5 - 91A - FAE - LF (5 mg / kg) group was 711 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 83.0%, significantly inhibiting tumor growth. The results indicated that in the CB - 17 SCID mouse transplanted with the NCI - H1975 model, at the dose level of 5 mg / kg, E2mut34 - 91A×M5 - 91A - FAE - LF had a significant tumor - inhibiting effect.

[0477] Preparation Example 3: Design and Preparation of the Second Batch of EGFR×MET Bispecific Antibody Mutants

[0478] 1. Construction of Bispecific Antibody Molecules

[0479] Ten high - affinity EGFR antibody heavy chains prepared by the previous Preparation Example 1 were used as the heavy chains of the EGFR - terminal parent antibody of the EGFR×MET bispecific antibody. Additionally, referring to the heavy chain of the publicly available MET antibody (SEQ ID NO: 410 in CN105705519A), after making corresponding modifications in the constant region according to the requirements for preparing the bispecific antibody, it was used as the heavy chain of the MET - terminal parent antibody of the EGFR×MET bispecific antibody (see SEQ ID NO: 20). E2 - 10 - LC - 13 - 91Q was used as the common light chain.

[0480] GenScript was commissioned to synthesize the heavy chains and light chains listed in Table 7, and the method was referred to Preparation Example 2.

[0481] Table 7: Names and combinations of mutant heavy chains and light chains

[0482]

[0483]

[0484]

[0485] 2. Transient protein expression and purification

[0486] The required heavy chains and light chains are shown in Table 7. Among them:

[0487] E2mut1-91Q-F405L-LF, E2mut2-91Q-F405L-LF, E2mut5-91Q-F405L-LF, E2mut12-91Q-F405L-LF, E2mut13-91Q-F405L-LF, E2mut16-91Q-F405L-LF, E2mut17-91Q-F405L-LF, E2mut25-91Q-F405L-LF, E2mut34-91Q-F405L-LF, E2mut38-91Q-F405L-LF, and M5-91Q-K409R-LF are used for the preparation of subsequent bispecific antibodies (in vitro recombination method);

[0488] Bispecific antibodies were prepared using the common light chain and "knob-into-hole" technology (refer to WO1996027011A1) for E2mut1-91Q×M5-91Q-KIH-LF, E2mut2-91Q×M5-91Q-KIH-LF, E2mut5-91Q×M5-91Q-KIH-LF, E2mut12-91Q×M5-91Q-KIH-LF, E2mut13-91Q×M5-91Q-KIH-LF, E2mut16-91Q×M5-91Q-KIH-LF, E2mut17-91Q×M5-91Q-KIH-LF, E2mut25-91Q×M5-91Q-KIH-LF, E2mut34-91Q×M5-91Q-KIH-LF, E2mut38-91Q×M5-91Q-KIH-LF.

[0489] 2.1 Expression of bispecific antibody molecules

[0490] Count the CHO-K1 cells (owned by Suzhou Junmeng) that are being cultured. When the cell density is between 2 - 6×10⁶ / ml, passage and expand them using CD CHO medium (purchased from Thermofisher, catalog number: 12490-001). Dilute the cell density to 1.8 - 2.5×10⁶ / ml one day before transfection. The next day, when the cell density reaches approximately 3.5 - 5.0×10⁶ / ml, perform transfection. First, add one-tenth of the transfection volume of CD CHO medium, add 1 - 2 μg / ml of the plasmid (self-made by the company) according to the combination table shown in Table 2, and finally add 3 - 14 μg / ml of PEI (purchased from Polysciences, catalog number: 24765-1). Mix well and incubate at room temperature. Finally, slowly add the transfection mixture to the pre-treated cells, mixing while adding. Place the transfected mixture in a shaker for culture. On the first day after transfection, add 6% of the glycoform regulator (purchased from OPMI, catalog number: R170026), supplement with 4% of Cell Boost 7a (purchased from Hyclone, catalog number: SH31026.05) and 0.4% of Cell Boost 7b (purchased from Hyclone, catalog number: SH31027.04CN), and then supplement once every two days. Sample collection is performed 5 - 9 days after transfection.

[0491] 3. In Vitro Recombination and Purification

[0492] 3.1 Affinity Capture of Bispecific Antibody Molecules

[0493] After the culture is completed, centrifuge at 1000g for 5 min using a floor centrifuge (purchased from ThermoFisher, R404A) to discard the precipitate, and then centrifuge at 8000g for 30 min to collect the cell supernatant and perform sterile filtration using a 0.22 μm filter cup (purchased from jet, FPE-214-000). Purify using a protein purification instrument (purchased from GE, AKTA avant). Equilibrate the Mabselect sure LX column (Cytiva, 17547403) with PBS equilibration buffer (purchased from Wuxi OriGene Technologies Co., Ltd., ZLI-9061). After sample loading is completed, first wash with affinity chromatography wash buffer A (pH 5.5, 45 mM acetic acid - sodium acetate + 1 M sodium chloride system), then wash with wash buffer B (pH 5.5, 45 mM acetic acid - sodium acetate system), and finally elute the target protein with an affinity eluent (pH 3.6, 10 mM acetic acid - sodium acetate buffer). Neutralize the sample with 1M Tris (purchased from Merck, catalog number: E300016981946) buffer and adjust the pH to 5.5 - 6 for the next step of in vitro recombination.

[0494] 3.2 In Vitro Recombination of Bispecific Antibody Molecules

[0495] Referring to the technical solution disclosed in WO2011131746, the bispecific antibody is prepared using the Fab arm exchange technology, and the method is as follows:

[0496] The two parental antibodies after affinity purification are concentrated and buffer-exchanged into PBS equilibration buffer (purchased from Wuxi OriGene Technologies Co., Ltd., ZLI-9061), and the protein concentration is set at 1 ± 0.05 mg / ml.

[0497] Preparation of 750 mM Cysteamine hydrochloride mother liquor (purchased from VETEC, V900342-25G): Add 2.556 g of Cysteamine hydrochloride to 14 ml of PBS, make up the volume to 30 ml with PBS, then filter through a 0.22 μm filter (purchased from sartorius, 16541-K), and finally wrap it with aluminum foil and store it in the dark.

[0498] Construct an incubation system: Take the parental antibodies processed in the above steps. As shown in Table 3, according to the molar ratio M:E = 1:1.2, add 2 ml of M parental antibody, 2.4 ml of E parental antibody, and 0.489 ml of 750 mM Cysteamine hydrochloridel to a 15 ml centrifuge tube (purchased from Genemore, G3210015). Seal the constructed incubation system with aluminum foil and incubate it in a 31 °C water bath (Shanghai Jinghong Experimental Equipment Co., Ltd., DK-S28) for 3 h in the dark. After incubation, concentrate and buffer-exchange with PBS equilibration buffer, and store it in the dark at room temperature for 16 h - 24 h.

[0499] Table 8: In vitro recombination combination table of bispecific antibody mutants

[0500]

[0501] 3.3 Purification of bispecific antibody molecules

[0502] Select CaptoTM MMC Impres (Cytiva, 17371602) packing material for purification. Pre-equilibrate with a washing buffer (pH 7.5, 20 mM Tris-HCl + 1 M NaCl buffer system), and equilibrate the system with an equilibration buffer (pH 7.5, 20 mM Tris-HCl system). After sample loading, equilibrate with the equilibration buffer for 3 - 6 column volumes, and finally perform linear elution with an elution buffer (pH 7.5, 20 mM Tris-HCl + 1 M NaCl buffer system) to collect the target protein.

[0503] Example 10: Biacore Detection of the Binding and Dissociation Kinetics of Bispecific Antibody Mutants

[0504] The binding affinities of the antibody to recombinant human EGFR and human c-Met were detected using a Biacore T200 (GE Healthcare Life Sciences) molecular interaction analyzer.

[0505] The method for determining the binding affinity of the antibody to human EGFR was as follows: 40 μg / mL of goat anti-human IgG-Fc fragment antibody (Jackson ImmunoResearch) was conjugated to the surface of a CM5 chip (Cytiva, catalog number BR-1005-30) for capturing the antibody. 1 μg / mL of the ALK101 antibody was captured on the surface of the CM5 chip, and 40 nM and 10 nM of human EGFR were injected for binding to the antibody. The Biacore T200 system (GE Healthcare) was used to detect the binding and dissociation kinetic signals. The Biacore T200 Evaluation Software 3.0 was used to fit the binding and dissociation curves to calculate the affinity K D value.

[0506] The method for determining the binding affinity of the antibody to human c-Met was as follows: 40 μg / mL of goat anti-human IgG-Fc fragment antibody (Jackson ImmunoResearch) was conjugated to the surface of a CM5 chip (Cytiva, catalog number BR-1005-30) for capturing the antibody. 1 μg / mL of the ALK101 antibody was captured on the surface of the CM5 chip, and 40 nM and 10 nM of human c-Met were injected for binding to the antibody. The Biacore T200 system (GE Healthcare) was used to detect the binding and dissociation kinetic signals. The Biacore T200 Evaluation Software 3.0 was used to fit the binding and dissociation curves to calculate the affinity K D value.

[0507] The results are shown in Table 9. E2mut1-91Q-F405L-LF, E2mut2-91Q-F405L-LF, E2mut5-91Q-F405L-LF, E2mut12-91Q-F405L-LF, E2mut13-91Q-F405L-LF, E2mut16-91Q-F405L-LF, E2mut17-91Q-F405L-LF, E2mut25-91Q-F405L-LF, E2mut34-91Q-F405L-LF, and E2mut38-91Q-F405L-LF all have binding activity to human EGFR, and their affinity is comparable to or slightly weaker than that of JNJ-372. M5-91Q-K409R-LF has binding activity to human c-Met, and its affinity is comparable to that of JNJ-372. At the same time, the affinities of four bispecific antibody molecules, E2mut1-91Q×M5-91Q-FAE-LF, E2mut12-91Q×M5-91Q-FAE-LF, E2mut13-91Q×M5-91Q-FAE-LF, and E2mut25-91Q×M5-91Q-FAE-LF, were tested, and the results showed that the affinity of the monovalent form was consistent with that of the bivalent monoclonal antibody.

[0508] Table 9: Statistical Table of Antibody Mutant Affinity Determined by Biacore

[0509]

[0510]

[0511] Example 11: Cell Binding Experiment of Bispecific Antibody Mutants

[0512] Incubate 293F-EGFR and 293-cMet cells separately with different concentrations of JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, E2mut25-91Q×M5-91Q-LF and anti-KLH IgG1 antibody (starting concentration 100 μg / ml, 5-fold dilution, a total of 12 concentration gradients) at 4 °C for 30 min, then wash away the unbound antibody with staining buffer (PBS + 1 v / v% FBS), and incubate with a fluorescent secondary antibody goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) containing 5‰ (v / v) prepared with staining buffer in the dark at 4 °C for 30 minutes. Finally, collect the cells with a flow cytometer (BD, model C6 PLUS) to detect the fluorescent antibody bound to the cell surface. Analyze the raw data with FlowJo to obtain the MFI value, and fit the antibody dose-dependent binding curve with GraphPad and calculate the EC50.

[0513] Incubate MKN45 cells (endogenously co-expressing human EGFR and human cMet, used to detect the cooperative binding affinity of bispecific antibodies) with different concentrations of JNJ372, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF and anti-KLH IgG1 antibody (starting concentration 100 μg / ml, 4-fold dilution, a total of 11 concentration gradients) at 4 °C for 30 min, then wash away the unbound antibody with staining buffer (PBS + 1 v / v% FBS), and incubate with a fluorescent secondary antibody goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) containing 5‰ (v / v) prepared with staining buffer in the dark at 4 °C for 30 minutes. Finally, collect the cells with a flow cytometer (BD, model C6 PLUS) to detect the fluorescent antibody bound to the cell surface. Analyze the raw data with FlowJo to obtain the MFI value, and fit the antibody dose-dependent binding curve with GraphPad and calculate the EC50.

[0514] PC-9 cells (endogenously co-expressing human EGFR and human cMet, used to detect the cooperative binding affinity of bispecific antibodies) were incubated with different concentrations of JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and anti-KLH IgG1 antibody (starting concentration 25 μg / ml, 4-fold dilution, a total of 8 concentration gradients) at 4 °C for 30 min, then unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS), and incubated with a fluorescent secondary antibody goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) containing 5‰ (v / v) prepared with staining buffer in the dark at 4 °C for 30 minutes. Finally, cells were collected using a flow cytometer (BD, model C6 PLUS), and the fluorescent antibodies bound to the cell surface were detected. The original data was analyzed with FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted with GraphPad and the EC50 was calculated.

[0515] As Figures 10D to 10GAs shown, the binding EC50 values of JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and E2mut25-91Q×M5-91Q-LF to 293F-EGFR cells were 203.6 ng / ml, 70.01 ng / ml, 67.48 ng / ml, 55.53 ng / ml, and 337.4 ng / ml, respectively; the TOP values were 35956, 17708, 23177, 19445, and 23508, respectively; the binding EC50 values of JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and E2mut25-91Q×M5-91Q-LF to 293F-CMet cells were 24.13 ng / ml, 25.80 ng / ml, 26.95 ng / ml, 25.99 ng / ml, and 28.22 ng / ml, respectively; the binding EC50 values of JNJ372, E2mut12-91Q×M5-91Q-LF, and E2mut13-91Q×M5-91Q-LF to MKN45 cells were 65.77 ng / ml, 78.99 ng / ml, and 79.78 ng / ml, respectively; the binding EC50 values of E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and JNJ372 to PC-9 cells were 324.4 ng / ml, 142.0 ng / ml, 149.2 ng / ml, and 194.0 ng / ml, respectively; the results showed that the binding affinity of E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and E2mut25-91Q×M5-91Q-LF to the single antigen EGFR was weaker than that of the positive control JNJ-372; the binding affinity to the single antigen cMet was comparable to that of the positive control JNJ-372; and the binding affinity to the dual antigen EGFR&cMet was comparable to that of the positive control JNJ-372.

[0516] Example 12: Phosphorylation Inhibition Experiment of Bispecific Antibody Mutants

[0517] After digesting and centrifuging NCI-H1975 cells, they were resuspended in complete medium (RPMI-1640 medium + 10 v / v% FBS), and according to 2×10 4Cells were seeded at a density of [number] cells per well in a 96-well flat-bottom plate and incubated overnight in a CO₂ incubator. The next day, the medium in the 96-well plate was replaced with serum-free RPMI-1640 medium and incubated overnight in the CO₂ incubator. On the third day, antibodies JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and E2mut25-91Q×M5-91Q-LF at 2× the analytical concentration were prepared in serum-free RPMI-1640 medium (starting concentration 500 μg / ml, 3-fold dilution, a total of 12 concentration gradients). After discarding the medium in the 96-well plate, 50 μl of the diluted antibody was added and pre-incubated at 37°C for 30 min. EGF agonist at 2× the analytical concentration (concentration 8 ng / ml) was prepared in serum-free RPMI-1640 medium and placed in the incubator for later use. After the pre-incubation was completed, the 96-well flat-bottom plate was taken out of the incubator, and 50 μl of the diluted EGF agonist solution was added to each well and incubated at 37°C for another 30 min. After the incubation was completed, the 96-well flat-bottom plate was taken out, the supernatant was discarded, and 50 μl of 1× lysis buffer was added according to the KITs instructions (phospho-AKT(Ser473)kit (Cisbio, catalog number: 64AKSPEH) & Advanced phospho-ERK1 / 2(Thr202 / Tyr204)(Cisbio, catalog number: 64AERPEH)). After shaking at 350 rpm at room temperature for 30 min, 16 μl of the cell lysate and 4 μl of the mix antibody in the KITs were added to a HTRF 96well low volume plate (Cisbio, catalog number: 66PL96025), gently mixed with a pipette tip, the plate was sealed with a film, and centrifuged at 1000 rpm for 30 s in a centrifuge. Finally, the plate was incubated in the dark at room temperature (20°C - 25°C) for 4 - 24 h, and the absorbance values (A) at wavelengths 655 nm and 620 nm were measured with a microplate reader (BioTek, Synergy H1 model) to calculate the ratio (Ratio 665 / 620). The calculation formula was Ratio 665 / 620 = A655 / A620 × 10000, and the data of Ratio 665 / 620 was analyzed and processed with GraphPad Prism software.

[0518] After digesting and centrifuging BxPC-3 cells, they were resuspended in complete medium (RPMI-1640 medium + 10 v / v% FBS), and seeded at a density of 2×10 4Cells were seeded at a density of [number] cells per well in a 96-well flat-bottom plate and incubated overnight in a CO2 incubator. The next day, the medium in the 96-well plate was replaced with serum-free RPMI-1640 medium and incubated overnight in the CO2 incubator. On the third day, antibodies JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, E2mut25-91Q×M5-91Q-LF, and anti-KLH IgG1 (starting concentration 20 μg / ml, 3-fold dilution, a total of 10 concentration gradients) were prepared at 2× analytical concentration using serum-free RPMI-1640 medium. After discarding the medium in the 96-well plate, 50 μl of the above-diluted antibody was added and pre-incubated at 37°C for 30 min. Agonist HGF (concentration 300 ng / ml) was prepared at 2× analytical concentration using serum-free RPMI-1640 medium and placed in the incubator for later use. After the pre-incubation was completed, the 96-well flat-bottom plate was taken out of the incubator, and 50 μl of the diluted HGF agonist solution was added to each well and incubated at 37°C for another 30 min. After the incubation was completed, the 96-well flat-bottom plate was taken out, the supernatant was discarded, and 50 μl of 1× lysis buffer was added according to the instructions of the KITs (phospho-AKT(Ser473)kit (Cisbio, catalog number: 64AKSPEH) & Advanced phospho-ERK1 / 2(Thr202 / Tyr204) (Cisbio, catalog number: 64AERPEH)). After shaking at 350 rpm at room temperature for 30 min, 16 μl of the cell lysate and 4 μl of the mix antibody in the KITs were added to the HTRF 96well low volume plates (Cisbio, catalog number: 66PL96025). After gently mixing with a pipette tip, the plate was sealed with a film and centrifuged at 1000 rpm for 30 s in a centrifuge. Finally, the plate was incubated in the dark at room temperature (20°C - 25°C) for 4 - 24 h, and the absorbance values (A) at wavelengths 655 nm and 620 nm were measured using a microplate reader (BioTek, Synergy H1 model), and the ratio (Ratio 665 / 620) was calculated. The calculation formula was Ratio 665 / 620 = A655 / A620 × 10000, and the data of Ratio 665 / 620 was analyzed and processed using GraphPad Prism software.

[0519] Such as Figures 11F to 11MAs shown, under the condition of EGF activation, the IC 50 values of JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF and E2mut25-91Q×M5-91Q-LF for inhibiting AKT phosphorylation are 1377 ng / ml, 464713 ng / ml, 2988 ng / ml, 8899 ng / ml, 111137 ng / ml respectively; the IC 50 values for inhibiting ERK phosphorylation are 3383 ng / ml, 4711189 ng / ml, 8496 ng / ml, 42459 ng / ml, 575652 ng / ml respectively. It can be seen from the results that under the condition of EGF activation, the activity of E2mut12-91Q×M5-91Q-LF in inhibiting AKT and ERK phosphorylation is slightly weaker than that of the positive control JNJ-372, and the activities of E2mut1-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, E2mut25-91Q×M5-91Q-LF in inhibiting AKT and ERK phosphorylation are significantly weaker than that of the positive control JNJ-372. Under the condition of HGF activation, the IC 50 values of JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF and E2mut25-91Q×M5-91Q-LF for inhibiting AKT phosphorylation are 296.9 ng / ml, 410.4 ng / ml, 268.3 ng / ml, 404.5 ng / ml, 766.3 ng / ml respectively; the IC 50 values for inhibiting ERK phosphorylation are 457.8 ng / ml, 547.6 ng / ml, 517.9 ng / ml, 535.0 ng / ml, 1312 ng / ml respectively. It can be seen from the results that under the condition of HGF activation, the activities of E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q in inhibiting AKT and ERK phosphorylation are comparable to that of the positive control JNJ-372, and the activity of E2mut25-91Q×M5-91Q-LF in inhibiting AKT and ERK phosphorylation is slightly weaker than that of the positive control JNJ-372.

[0520] Example 13: ADCC Activity Experiment of Bispecific Antibody Mutants

[0521] Reporter gene method: Seed NCI-H1975 cells at 5×10 4Cells were seeded at a density of [number] cells per well in a 96-well white plate (Costar, catalog number: 3917) and incubated overnight in a CO₂ incubator. The next day, the 96-well white plate was removed from the CO₂ incubator, the culture supernatant was removed, and 40 μl of antibody diluted with assay buffer (RPMI-1640 medium + 2% v / v FBS) was added to each well. E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, JNJ-372, and anti-KLH IgG1 (starting concentration 10 μg / ml, 4-fold dilution, 10 concentration gradients) were pre-incubated with the target cells for 30 min. After the pre-incubation was completed, 40 μl of effector cells Jurkat ADCC expressing NFAT-Luc and FcγRIIIa resuspended with assay buffer (RPMI-1640 medium + 2% v / v FBS) ([number] cells per well) was added to each well and incubation was continued at 37 °C for 6 h. Finally, substrate One-Lite (Vazyme, catalog number: DD1203-03) was added and the luciferase signal was detected using a microplate reader (TECAN, model M1000pro). The higher the fluorescence reading, the stronger the ADCC effect. The ADCC effect curve as a function of antibody dose was fitted using GraphPad. 5 cells) and incubation was continued at 37 °C for 6 h. Finally, substrate One-Lite (Vazyme, catalog number: DD1203-03) was added and the luciferase signal was detected using a microplate reader (TECAN, model M1000pro). The higher the fluorescence reading, the stronger the ADCC effect. The ADCC effect curve as a function of antibody dose was fitted using GraphPad.

[0522] LDH method: First, PBMC effector cells were resuscitated and set aside, and allowed to recover overnight in a CO₂ incubator. After the target cells NCI-H1975 were digested and centrifuged, the supernatant was discarded, and the cell density was adjusted to 4×10 5 cells / ml using RPMI Medium 1640 without phenol red + 1% v / v FBS medium. 50 μl of target cells was added to each well in a 96-well low-attachment plate (Costar, catalog number: 7007). Then, the antibody was diluted with RPMI Medium 1640 without phenol red + 1% FBS medium. E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, JNJ-372, and anti-KLH IgG1 were prepared at 4× the analytical concentration (starting antibody concentration 66.67 μg / ml, 2 concentration gradients before 3-fold dilution; 7 concentration gradients after 5-fold dilution). 50 μl of the diluted antibody solution was added to each well, gently pipetted and mixed several times, and then incubated in the incubator for 30 min. The PBMC that had been resuscitated overnight was taken out, and the density of the PBMC was adjusted to 5×10 6cells / ml (effector-to-target ratio is 25:1). Add 100 μl of PBMC to each well of a 96-well low-attachment plate and co-culture in a CO2 incubator for 4 h. Half an hour before the end of the co-culture time, in the Tmax group, add 20 μl of cell lysis buffer from the LDH-cytotoxicity kit (BioVision, catalog number: K311-400) to each well, and then put the 96-well low-attachment plate back into the incubator for continued incubation. Take out the 96-well low-attachment plate and centrifuge (1500 rpm × 5 min). Aspirate 50 μl of the culture supernatant from each well into a new 96-well flat-bottom plate (Corning, catalog number: 3599), then add 50 μl of the diluted Dye Solution from the LDH-cytotoxicity kit, incubate in the dark on a shaker for 10 - 30 min, and finally read the plate with a multifunctional microplate reader (TECAN, model M1000 pro) and perform data analysis and processing with GraphPad Prism software. The killing calculation formula is: ADCC% = [(Sample - Buffer) / (Tmax - Ts)] × 100.

[0523] As Figure 12D shown, in the reporter gene assay, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, and E2mut13-91Q×M5-91Q-LF all had ADCC activity, and their EC 50 values were 50.92 ng / ml, 53.05 ng / ml, and 42.70 ng / ml respectively, and their ADCC activity was slightly weaker than that of the positive control JNJ-372.

[0524] As Figure 12E shown, when using PBMC as effector cells and NCI-H1975 as target cells, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, and E2mut13-91Q×M5-91Q-LF all had ADCC activity, and their EC 50 values were 33.66 ng / ml, 11.95 ng / ml, and 28.39 ng / ml respectively, and their ADCC activity was comparable to that of the positive control JNJ-372. While the negative control anti-KLH IgG1 could not induce an ADCC effect.

[0525] Example 14: Endocytosis Experiment of Bispecific Antibody Mutants

[0526] After digesting and centrifuging MKN45 cells, resuspend them with staining buffer (PBS + 1 v / v% FBS), and add 2×10 5Cells were seeded in a 96-well round bottom plate (Corning, catalog number: 3799), and antibodies E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and JNJ-372 (25 μg / ml) diluted with staining buffer (PBS + 1 v / v% FBS) were added and incubated at 4°C for 30 min. Then, unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS). After resuspending the cells with 200 μl of complete medium (RPMI-1640 medium + 10 v / v% FBS), the cells were divided into two parts. One part was continued to be incubated at 4°C for 24 h, and the other part was incubated at 37°C for 24 h. After the incubation time ended, the supernatant was discarded by centrifugation, and a fluorescent secondary antibody goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) containing 5‰ (v / v) prepared with staining buffer was added and incubated in the dark at 4°C for 30 minutes. Then, unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS). After fixation with PFA (Absin, catalog number: abs9179), the cells were collected on a flow cytometer (BD, C6 PLUS model) to detect the fluorescent antibodies bound to the cell surface. The original data was analyzed with FlowJo to obtain the MFI value, and the Top value was obtained by fitting the antibody dose-dependent binding curve with GraphPad. The formula for calculating the endocytosis rate is: internalization rate (%) = (1 - Top_37°C / Top_4°C) × 100.

[0527] The results are shown in Table 10.

[0528] Table 10: Detection results of endocytic activity of MKN45 cells 2

[0529] Group Top_4℃ Top_37℃ Internalization Rate, % JNJ-372 1273 324 75 E2mut12-91Q×M5-91Q-FAE-LF 1228 200 84 E2mut13-91Q×M5-91Q-FAE-LF 1171 235 80

[0530] The results showed that E2mut12-91Q×M5-91Q-FAE-LF, E2mut13-91Q×M5-91Q-FAE-LF, and JNJ-372 could all be endocytosed by MKN45 cells, and their endocytic activities were comparable. The endocytosis rates at 24 h were 75%, 84%, and 80% respectively.

[0531] After digesting and centrifuging PC-9 cells, they were resuspended with staining buffer (PBS + 1 v / v% FBS), and 2×10 5Cells were seeded in a 96-well round-bottom plate (Corning, catalog number: 3799), and antibodies E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and JNJ-372 (25 μg / ml) diluted with staining buffer (PBS + 1 v / v% FBS) were added and incubated at 4°C for 30 min. Then, unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS). After resuspending the cells with 200 μl of complete medium (RPMI-1640 medium + 10 v / v% FBS), the cells were divided into two parts. One part was continued to be incubated at 4°C for 24 h, and the other part was incubated at 37°C for 24 h. After the incubation time ended, the supernatant was discarded by centrifugation, and a fluorescent secondary antibody goat anti-human IgGPE (SouthernBiotech, Cat#2040-09) containing 5‰ (v / v) prepared with staining buffer was added and incubated in the dark at 4°C for 30 minutes. Then, unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS). After fixation with PFA (Absin, catalog number: abs9179), cells were collected on a flow cytometer (BD, C6 PLUS model) to detect the fluorescent antibodies bound to the cell surface. The original data was analyzed with FlowJo to obtain the MFI value, and the Top value was obtained by fitting the antibody dose-dependent binding curve with GraphPad. The formula for the endocytosis rate is: internalization rate (%) = (1 - Top_37°C / Top_4°C) × 100.

[0532] The results are shown in Table 11.

[0533] Table 11: Detection results of endocytic activity of PC-9 cells

[0534] Group Top_4℃ Top_37℃ Internalization Rate, % JNJ-372 2284 425 81 E2mut1-91Q×M5-91Q-FAE-LF 2749 335 88 E2mut12-91Q×M5-91Q-FAE-LF 2992 403 87 E2mut13-91Q×M5-91Q-FAE-LF 3159 459 85

[0535] The results showed that E2mut1-91Q×M5-91Q-FAE-LF, E2mut12-91Q×M5-91Q-FAE-LF, E2mut13-91Q×M5-91Q-FAE-LF, and JNJ-372 could all be endocytosed by MKN45 cells, and their endocytic activities were comparable. The endocytosis rates at 24 h were 88%, 87%, 85%, and 81% respectively.

[0536] After digesting and centrifuging NUGC4 cells (endogenously expressing human EGFR and human cMet) and BaF3-EGFR-cMet (overexpressing human EGFR and human cMet), they were resuspended with staining buffer (PBS + 1 v / v% FBS), and 2×10 5Cells were seeded in 96-well round-bottom plates (Corning, catalog number: 3799), and antibodies E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, E2mut25-91Q×M5-91Q-LF, and JNJ-372 (100 μg / ml) diluted with staining buffer (PBS + 1 v / v% FBS) were added and incubated at 4°C for 30 min. Then, unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS). After resuspending the cells with 200 μl of complete medium (RPMI-1640 medium + 10 v / v% FBS), the cells were divided into two parts. One part was continued to be incubated at 4°C for 24 h, and the other part was incubated at 37°C for 24 h. After the incubation time ended, the supernatant was discarded by centrifugation, and a fluorescent secondary antibody goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) containing 5‰ (v / v) prepared with staining buffer was added and incubated in the dark at 4°C for 30 minutes. Then, unbound antibodies were washed away with staining buffer (PBS + 1 v / v% FBS). After fixation with PFA (Absin, catalog number: abs9179), cells were collected on a flow cytometer (BD, model C6 PLUS) to detect the fluorescent antibodies bound to the cell surface. The original data were analyzed with FlowJo to obtain the MFI value, and the Top value was obtained by fitting the antibody dose-dependent binding curve with GraphPad. The formula for the endocytosis rate is: internalization rate (%) = (1 - Top_37°C / Top_4°C) × 100.

[0537] The results are shown in Tables 12 and 13.

[0538] Table 12: Detection results of endocytic activity of NUGC4 cells

[0539] Group Top_4℃ Top_37℃ Internalization Rate, % JNJ-372 7348 2949 60 E2mut1-91Q×M5-91Q-FAE-LF 5059 2356 53 E2mut12-91Q×M5-91Q-FAE-LF 5940 1958 67 E2mut13-91Q×M5-91Q-FAE-LF 5485 2377 57 E2mut25-91Q×M5-91Q-FAE-LF 6440 2050 68

[0540] Table 13: Detection results of endocytic activity of BaF3-EGFR-cMet cells

[0541] Group Top_4℃ Top_37℃ Internalization Rate, % JNJ-372 13553 2608 81 E2mut1-91Q×M5-91Q-FAE-LF 24752 3907 84 E2mut12-91Q×M5-91Q-FAE-LF 18826 4428 76 E2mut13-91Q×M5-91Q-FAE-LF 18101 4804 73 E2mut25-91Q×M5-91Q-FAE-LF 14777 3578 76

[0542] The results showed that E2mut1-91Q×M5-91Q-FAE-LF, E2mut12-91Q×M5-91Q-FAE-LF, E2mut13-91Q×M5-91Q-FAE-LF, E2mut25-91Q×M5-91Q-FAE-LF, and JNJ-372 could all be endocytosed by NUGC4 cells and BaF3-EGFR-cMet cells.

[0543] Example 15: H1975 Xenograft Model of Bispecific Antibody Mutants

[0544] This example evaluates the inhibitory effects of the present invention's E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-LF, and ALK101-31Q-LF on the NDG mouse xenograft human lung adenocarcinoma cell NCI-H1975 model. ALK101-15Q-LF, ALK101-17Q-LF, and ALK101-31Q-LF are selected from the bispecific antibodies in Table 8.

[0545] 1. Test procedure

[0546] Female NDG mice at 6-8 weeks old (purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd., animal certificate number: 20170012022482) were subcutaneously inoculated with 5×10 6 NCI-H1975 cells on the right side. When the average tumor volume was approximately 125 mm 3 , appropriate animals were selected and randomly divided into 6 groups with 6 animals in each group according to the tumor volume. They were respectively:

[0547] G1 Normal saline control group (solvent control group);

[0548] G2 JNJ-372 (3 mg / kg) group (positive control group);

[0549] G3 E2mut34-91A×M5-91A-FAE-LF (3 mg / kg) group (treatment group);

[0550] G4 ALK101-15Q-LF (3 mg / kg) group (treatment group);

[0551] G5 ALK101-31Q-LF (3 mg / kg) group (treatment group);

[0552] G6 ALK101-17Q-LF (3 mg / kg) group (treatment group).

[0553] Intraperitoneal injection was performed, administered 2 times a week for 2 consecutive weeks, and the experiment ended 28 days after the initial administration. The tumor volume and body weight were measured 2 times a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the relative tumor proliferation rate was calculated, TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%. (Ti: the average tumor volume of the treatment group on the i-th day of administration, T0: the average tumor volume of the treatment group on the 0-th day of administration; Vi: the average tumor volume of the solvent control group on the i-th day of administration, V0: the average tumor volume of the solvent control group on the 0-th day of administration).

[0554] The results are shown in Table 14 andFigure 14 as shown

[0555] Table 14: Pharmacodynamic analysis table of each group in the NCI-H1975 human lung cancer subcutaneous tumor model

[0556]

[0557]

[0558] Note: 1. Data are expressed as "mean ± standard error";

[0559] 2. TGI% = [1 - (Ti - T0) / (Vi - V0)] × 100%;

[0560] 3. P values were obtained by comparing the tumor volumes of each group using the T-test method.

[0561] The results showed that on the 28th day after drug administration, the average tumor volume of the normal saline control group was 2070 mm 3 . The average tumor volume of the JNJ-372 (3 mg / kg) group was 272 mm 3 , and compared with the normal saline control group, the tumor inhibition rate was 92.5%, significantly inhibiting tumor growth; the average tumor volume of the E2mut34-91A×M5-91A-FAE-LF (3 mg / kg) group was 223 mm 3 , and compared with the normal saline control group, the tumor inhibition rate was 95.0%, significantly inhibiting tumor growth. The average tumor volume of the ALK101-15Q-LF (3 mg / kg) group was 600 mm 3 , and compared with the normal saline control group, the tumor inhibition rate was 75.6%, significantly inhibiting tumor growth. The average tumor volume of the ALK101-31Q-LF (3 mg / kg) group was 348 mm 3 , and compared with the normal saline control group, the tumor inhibition rate was 88.5%, significantly inhibiting tumor growth. The average tumor volume of the ALK101-17Q-LF (3 mg / kg) group was 394 mm 3 , and compared with the normal saline control group, the tumor inhibition rate was 86.1%, significantly inhibiting tumor growth. The results indicated that in the NDG mouse transplanted NCI-H1975 model, at the dose level of 3 mg / kg, E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-L and ALK101-31Q-LF had significant tumor inhibitory effects.

[0562] Example 16: H1975 (L858R / T790M / C797S) Xenograft Model of Bispecific Antibody Mutants

[0563] This example evaluates the inhibitory effects of the present invention's E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-LF, and ALK101-31Q-LF on a model of NDG mice transplanted with human lung adenocarcinoma cells NCI-H1975 (L858R / T790M / C797S).

[0564] 1. Test procedure

[0565] Female NDG mice at 6 - 8 weeks old (purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd., animal certificate number: 20170012022482) were subcutaneously inoculated with 5×10 6 NCI-H1975 cells on the right side. When the average tumor volume was approximately 186 mm 3 , appropriate animals were selected and randomly divided into 6 groups with 5 animals in each group according to the tumor volume. They were respectively:

[0566] G1 Normal saline control group (solvent control group);

[0567] G2 JNJ-372 (2 mg / kg) group (positive control group);

[0568] G3 E2mut34-91A×M5-91A-FAE-LF (2 mg / kg) group (treatment group);

[0569] G4 ALK101-15Q-LF (2 mg / kg) group (treatment group);

[0570] G5 ALK101-31Q-LF (2 mg / kg) group (treatment group);

[0571] G6 ALK101-17Q-LF (2 mg / kg) group (treatment group).

[0572] Intraperitoneal injection was performed 2 times a week for a total of 5 times, and the experiment ended 28 days after the initial administration. The tumor volume and body weight were measured 2 times a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the relative tumor proliferation rate was calculated, TGI(%) = [1 - (Ti - T0) / (Vi - V0)]×100%. (Ti: the average tumor volume of the treatment group on the i-th day of administration, T0: the average tumor volume of the treatment group on the 0-th day of administration; Vi: the average tumor volume of the solvent control group on the i-th day of administration, V0: the average tumor volume of the solvent control group on the 0-th day of administration).

[0573] The results are shown in Table 15 and Figure 15 as follows.

[0574] Table 15: Table of Pharmacodynamic Analysis of Each Group in the H1975 (L858R / T790M / C797S) Human Lung Cancer Subcutaneous Tumor Model

[0575]

[0576] Note: 1. Data are expressed as "mean ± standard error".

[0577] 2. TGI% = [1 - (Ti - T0) / (Vi - V0)] × 100%;

[0578] 3. The P value was obtained by comparing the tumor volumes of each group using the T-test method.

[0579] The results showed that on the 28th day after drug administration, the average tumor volume of the normal saline control group was 1678 mm 3 . The average tumor volume of the JNJ-372 (2 mg / kg) group was 4 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 112.2%, significantly inhibiting tumor growth; the average tumor volume of the E2mut34-91A×M5-91A-FAE-LF (2 mg / kg) group was 7 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 112.0%, significantly inhibiting tumor growth. The average tumor volume of the ALK101-15Q-LF (2 mg / kg) group was 9 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 111.9%, significantly inhibiting tumor growth. The average tumor volume of the ALK101-31Q-LF (2 mg / kg) group was 6 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 112.1%, significantly inhibiting tumor growth. The average tumor volume of the ALK101-17Q-LF (2 mg / kg) group was 0 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 112.5%, significantly inhibiting tumor growth. The results indicated that in the NDG mouse xenograft NCI-H1975 (L858R / T790M / C797S) model, at the dose level of 2 mg / kg, E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-L and ALK101-31Q-LF had significant tumor inhibitory effects.

[0580] Example 17: H292 Xenograft Model of Bispecific Antibody Mutants

[0581] This example evaluates the inhibitory effects of the present invention's E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-LF, and ALK101-31Q-LF on the NDG mouse xenograft human lung adenocarcinoma cell H292 model.

[0582] 1. Test process

[0583] Female NDG mice at 6-8 weeks of age (purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd., animal certificate number: 20170012022482) were subcutaneously inoculated with 5×10 6 H292 cells on the right side. When the average tumor volume was approximately 223 mm 3 , appropriate animals were selected and randomly divided into 6 groups with 5 animals in each group according to the tumor volume. They were respectively:

[0584] G1 Normal saline control group (solvent control group);

[0585] G2 JNJ-372 (1 mg / kg) group (positive control group);

[0586] G3 E2mut34-91A×M5-91A-FAE-LF (1 mg / kg) group (treatment group);

[0587] G4 ALK101-15Q-LF (1 mg / kg) group (treatment group);

[0588] G5 ALK101-31Q-LF (1 mg / kg) group (treatment group);

[0589] G6 ALK101-17Q-LF (1 mg / kg) group (treatment group).

[0590] Intraperitoneal injection was performed. It was administered once in the first week, and starting from the second week, it was administered twice a week for a total of 6 times. The experiment ended 28 days after the initial administration. The tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized, and the relative tumor proliferation rate was calculated, TGI(%) = [1 - (Ti - T0) / (Vi - V0)]×100%. (Ti: the average tumor volume of the treatment group on the i-th day of administration, T0: the average tumor volume of the treatment group on the 0-th day of administration; Vi: the average tumor volume of the solvent control group on the i-th day of administration, V0: the average tumor volume of the solvent control group on the 0-th day of administration).

[0591] The results are shown in Table 16 and Figure 16 as follows.

[0592] Table 16: Analysis table of the efficacy of each group in the H292 human lung cancer subcutaneous tumor model

[0593]

[0594] Note: 1. Data are expressed as "mean ± standard error".

[0595] 2. TGI% = [1 - (Ti - T0) / (Vi - V0)] × 100%;

[0596] 3. P values were obtained by comparing the tumor volumes of each group using the T - test method.

[0597] The results showed that on the 28th day after administration, the average tumor volume of the normal saline control group was 1713 mm 3 . The average tumor volume of the JNJ - 372 (1 mg / kg) group was 625 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 73%, significantly inhibiting tumor growth; the average tumor volume of the E2mut34 - 91A×M5 - 91A - FAE - LF (1 mg / kg) group was 659 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 70.8%, significantly inhibiting tumor growth. The average tumor volume of the ALK101 - 15Q - LF (1 mg / kg) group was 1049 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 44.6%. The average tumor volume of the ALK101 - 31Q - LF (1 mg / kg) group was 106 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 107.9%, significantly inhibiting tumor growth. The average tumor volume of the ALK101 - 17Q - LF (1 mg / kg) group was 152 mm 3 . Compared with the normal saline control group, the tumor inhibition rate was 104.7%, significantly inhibiting tumor growth. The results indicated that in the NDG mouse xenograft H292 model, at the dose level of 1 mg / kg, E2mut34 - 91A×M5 - 91A - FAE - LF, ALK101 - 17Q - L and ALK101 - 31Q - LF had significant tumor - inhibiting effects.

[0598] Example 18: Thermal Stability of Bispecific Antibodies

[0599] DSF (Differential Scanning Fluorimetry) technology is a method for detecting protein denaturation based on the change in the endogenous fluorescence of proteins. When a protein is in its folded state, the emission light of its internal hydrophobic amino acids such as tryptophan at 330 nm is greater than that at 350 nm. When the protein is heated or treated with chemical denaturants, the protein unfolds and tryptophan is exposed to the liquid phase environment. At this time, the maximum emission light will shift, and finally the emission light at 350 nm will be greater than that at 330 nm. Therefore, we can use the change in fluorescence value to detect the process of protein unfolding caused by temperature or chemical denaturants, and then calculate the melting temperature, that is, the Tm value. Set 20 °C as the starting temperature and 95 °C as the ending temperature, with a heating rate of 1 °C / min, and investigate the stability of the bispecific antibody in the buffer system (20 mM histidine - histidine hydrochloride buffer, 150 mM arginine hydrochloride, pH 6.0).

[0600] Replace the bispecific antibody sample into the above buffer system, control the sample concentration at about 130 mg / mL, and use nanoDSF for detection. The results are shown in Table 17. The thermal transition temperatures (Tm) of the bispecific antibodies E2mut34 - 91A×M5 - 91A - FAE - LF, ALK101 - 15Q, ALK101 - 17Q, and ALK101 - 31Q provided in this application are all above 62 °C, showing good thermal stability.

[0601] Table 17: Thermal stability of bispecific antibodies

[0602] Sample Tm1 (℃) Tm2 (℃) Tm3 (℃) E2mut34-91A×M5-91A-FAE-LF 67.2 78.6 / ALK101-15Q 62.5 70.6 76.7 ALK101-17Q 62.4 67.8 75.6 ALK101-31Q 65.8 74.3 /

[0603] Note: " / " represents non - existence

[0604] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-EGFR antibody or an antigen-binding fragment thereof, wherein the anti-EGFR antibody comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprises HCDR1 to HCDR3, and the light-chain variable region comprises LCDR1 to LCDR3, wherein: The amino acid sequence of HCDR1 is as shown in SEQ ID NO:25 or SEQ ID NO:26, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:27, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:37; and The amino acid sequence of LCDR1 is as shown in SEQ ID NO:38, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:39, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:

76.

2. The anti-EGFR antibody or an antigen-binding fragment thereof according to claim 1, wherein, The amino acid sequence of HCDR3 is as shown in any one of SEQ ID NO:28 to SEQ ID NO:

36.

3. The anti-EGFR antibody or an antigen-binding fragment thereof according to claim 1, wherein, The amino acid sequence of the heavy-chain variable region of the anti-EGFR antibody is selected from any one of SEQ ID NO:1 to SEQ ID NO:10; and The amino acid sequence of the light-chain variable region of the anti-EGFR antibody is selected from SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:

77.

4. The anti-EGFR or an antigen-binding fragment thereof according to any one of claims 1 to 3, wherein, The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:1, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:2, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:3, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:4, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:5, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:6, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:8, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:42; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:44; Or The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

77.

5. The anti-EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein, The anti-EGFR antibody comprises non-CDR regions, and the non-CDR regions are from a species other than murine, such as from a human antibody.

6. The anti-EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein, The constant region of the anti-EGFR antibody is selected from the constant regions of human IgG1, IgG2, IgG3 or IgG4.

7. The anti-EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein, The heavy chain constant region of the anti-EGFR antibody is Ig gamma-1chain C region or Ig gamma-4chain C region; the light chain constant region is Ig kappa chain C region or Ig lambda chain C region.

8. The anti-EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein, The heavy chain constant region sequence of the anti-EGFR antibody is selected from SEQ ID NO:79, 80 and 81.

9. The anti-EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein, The heavy chain of the anti-EGFR antibody is selected from SEQ ID NO:17, 18, 19, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 and 75; and The light chain of the anti-EGFR antibody is selected from SEQ ID NO:11, 12, 13 and 57.

10. The anti-EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein: The amino acid sequence of the heavy chain is as shown in SEQ ID NO:17, and the amino acid sequence of the light chain is as shown in SEQ ID NO:11; The amino acid sequence of the heavy chain is as shown in SEQ ID NO:17, and the amino acid sequence of the light chain is as shown in SEQ ID NO:12; The amino acid sequence of the heavy chain is as shown in SEQ ID NO:17, and the amino acid sequence of the light chain is as shown in SEQ ID NO:13; The amino acid sequence of the heavy chain is as shown in SEQ ID NO:18, and the amino acid sequence of the light chain is as shown in SEQ ID NO:11; The amino acid sequence of the heavy chain is as shown in SEQ ID NO:18, and the amino acid sequence of the light chain is as shown in SEQ ID NO:12; The amino acid sequence of the heavy chain is as shown in SEQ ID NO:18, and the amino acid sequence of the light chain is as shown in SEQ ID NO:13; The amino acid sequence of the heavy chain is as shown in SEQ ID NO:19, and the amino acid sequence of the light chain is as shown in SEQ ID NO:11; The amino acid sequence of the heavy chain is as shown in SEQ ID NO:19, and the amino acid sequence of the light chain is as shown in SEQ ID NO:12; The amino acid sequence of the heavy chain is as shown in SEQ ID NO:19, and the amino acid sequence of the light chain is as shown in SEQ ID NO:13; Or The amino acid sequence of the heavy chain is as shown in SEQ ID NO:17, 18, 19, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75, and the amino acid sequence of the light chain is as shown in SEQ ID NO:

57.

11. The anti-EGFR antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein, The anti-EGFR antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.

12. An isolated nucleic acid molecule encoding the anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1 to 11.

13. A recombinant vector comprising the isolated nucleic acid molecule according to claim 12.

14. A host cell comprising the isolated nucleic acid molecule according to claim 12, or the recombinant vector according to claim 12.

15. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein, The antibody or its antigen-binding fragment is the anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1 to 11; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug; Preferably, the antibody or its antigen-binding fragment is linked to the small molecule drug through a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond; Preferably, the molar ratio of the antibody or its antigen-binding fragment to the small molecule drug is 1:(2 - 8); Preferably, the molar ratio of the antibody or its antigen-binding fragment to the small molecule drug is 1:(2 - 4).

16. A pharmaceutical composition comprising an effective amount of the anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1 to 11, and one or more pharmaceutically acceptable excipients.

17. The pharmaceutical composition according to claim 16, which further comprises an effective amount of an anti-MET antibody or its antigen-binding fragment; Preferably, the anti-MET antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein: The amino acid sequence of HCDR1 is as shown in SEQ ID NO:45, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:46, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:47; or the amino acid sequence of HCDR1 is as shown in SEQ ID NO:48, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:49, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:50; And The amino acid sequence of LCDR1 is as shown in SEQ ID NO:51, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:52, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

53.

18. The pharmaceutical composition according to any one of claims 16 to 17, wherein the amino acid sequence of the heavy chain variable region of the anti-MET antibody is selected from SEQ ID NO:54 and SEQ ID NO:55; and the amino acid sequence of the light chain variable region of the anti-MET antibody is as shown in SEQ ID NO:56; Preferably, the anti-MET antibody: has an amino acid sequence of the heavy chain as shown in SEQ ID NO:16 or SEQ ID NO:21, and an amino acid sequence of the light chain variable region as shown in SEQ ID NO:

14.

19. A combined pharmaceutical product, comprising a first pharmaceutical product and a second pharmaceutical product in separate packages, wherein: the first pharmaceutical product contains an effective amount of the anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1 to 11, and one or more pharmaceutically acceptable excipients; the second pharmaceutical product contains an effective amount of an anti-MET antibody or its antigen-binding fragment, and one or more pharmaceutically acceptable excipients; Optionally, the combined pharmaceutical product further contains a product instruction manual.

20. The combined pharmaceutical product according to claim 19, wherein the anti-MET antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein: the amino acid sequence of HCDR1 is as shown in SEQ ID NO:45, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:46, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:47; or the amino acid sequence of HCDR1 is as shown in SEQ ID NO:48, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:49, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:50; And the amino acid sequence of LCDR1 is as shown in SEQ ID NO:51, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:52, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

53.

21. The combined pharmaceutical product according to any one of claims 19 to 20, wherein The amino acid sequence of the heavy chain variable region of the anti-MET antibody is selected from SEQ ID NO:54 and SEQ ID NO:55; and the amino acid sequence of the light chain variable region of the anti-MET antibody is as shown in SEQ ID NO:56; Preferably, the anti-MET antibody: The amino acid sequence of the heavy chain is as shown in SEQ ID NO:16 or SEQ ID NO:21, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

14.

22. Bispecific antibody, comprising: A first protein functional region targeting EGFR, and A second protein functional region targeting a target different from EGFR; Wherein, the first protein functional region comprises the heavy chain variable region of the anti-EGFR antibody or its antigen-binding fragment as claimed in any one of claims 1 to 11; preferably, it comprises the heavy chain variable region and the light chain variable region of the anti-EGFR antibody or its antigen-binding fragment as claimed in any one of claims 1 to 11.

23. The bispecific antibody according to claim 22, wherein The second protein functional region comprises the heavy chain variable region of the anti-MET antibody or its antigen-binding fragment; preferably, it comprises the heavy chain variable region and the light chain variable region of the anti-MET antibody or its antigen-binding fragment; Wherein, the anti-MET antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein: The amino acid sequence of HCDR1 is as shown in SEQ ID NO:45, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:46, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:47; or the amino acid sequence of HCDR1 is as shown in SEQ ID NO:48, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:49, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:50; And The amino acid sequence of LCDR1 is as shown in SEQ ID NO:51, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:52, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:

53.

24. The bispecific antibody according to any one of claims 22 to 23, wherein The amino acid sequence of the heavy chain variable region of the anti-MET antibody is selected from SEQ ID NO:54 and SEQ ID NO:55; and The amino acid sequence of the light chain variable region of the anti-MET antibody is as shown in SEQ ID NO:56; Preferably, the anti-MET antibody: The amino acid sequence of the heavy chain is as shown in SEQ ID NO:16 or SEQ ID NO:21, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

14.

25. The bispecific antibody according to any one of claims 22 to 24, wherein Among them, The first protein functional region and the second protein functional region are independently a fusion protein of a single-chain antibody or a half-molecule type monovalent antibody (IgG half molecule, IgG-HM).

26. The bispecific antibody according to any one of claims 22 to 25, wherein the first protein functional region is a half-molecule type monovalent antibody, and the second protein functional region is a half-molecule type monovalent antibody.

27. The bispecific antibody according to any one of claims 22 to 26, wherein the first protein functional region is a half-molecule type monovalent antibody targeting EGFR, and the second protein functional region is a half-molecule type monovalent antibody targeting MET.

28. The bispecific antibody according to any one of claims 25 to 27, wherein the heavy chain constant regions of the two half-molecule type monovalent antibodies respectively contain a first CH3 region and a second CH3 region, the sequences of the first CH3 region and the second CH3 region are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than the homodimer interaction of each of the first CH3 region and the second CH3 region.

29. The bispecific antibody according to any one of claims 25 to 28, wherein the heavy chain constant region of the half-molecule type monovalent antibody is the heavy chain constant region of human IgG1 and has a Knob mutation (such as S354C and T366W mutations); and the heavy chain constant region of the half-molecule type monovalent antibody is the heavy chain constant region of human IgG1 and has a Hole mutation (such as Y349C, T366S, L368A and Y407V mutations).

30. The bispecific antibody according to any one of claims 25 to 29, wherein the heavy chain constant region of the half-molecule type monovalent antibody is the heavy chain constant region of human IgG1, and according to the EU Numbering System, the 435th position of the heavy chain constant region of one half-molecule type monovalent antibody is mutated to amino acid R, and the 436th position is mutated to amino acid F.

31. The bispecific antibody according to any one of claims 25 to 30, wherein the heavy chain constant region of the half-molecule type monovalent antibody is the heavy chain constant region of human IgG1, and according to the EU Numbering System, the 405th position of the heavy chain constant region of one half-molecule type monovalent antibody is mutated to amino acid L, and the 409th position of the heavy chain constant region of the other half-molecule type monovalent antibody is mutated to amino acid R.

32. The bispecific antibody according to any one of claims 22 to 31, which is in the IgG form, preferably in the IgG1 form; Preferably, the sequences of the light chains in the bispecific antibody are the same; Preferably, the bispecific antibody has two light chains with the same sequence; Preferably, the bispecific antibody is composed of the following peptide chains: (1) Peptide chains selected from SEQ ID NO: 17 to SEQ ID NO: 19, and SEQ ID NO: 58 to SEQ ID NO: 75 (2) A peptide chain selected from SEQ ID NO:16 and SEQ ID NO:20, and (3) A peptide chain selected from SEQ ID NO:11 to SEQ ID NO:13, and the peptide chain in SEQ ID NO:57, Among them, The peptide chains in (3) are two identical copies; Preferably, the peptide chains in (1) and (2), the peptide chains in (2) and (3), and the peptide chains in (1) and (3) are linked by one or more disulfide bonds (such as 2 or 3 disulfide bonds).

33. The bispecific antibody according to any one of claims 22 to 32, which is composed of the following peptide chains: The peptide chain shown in SEQ ID NO:17, the peptide chain shown in SEQ ID NO:16, and the peptide chain shown in SEQ ID NO:12, wherein the peptide chain shown in SEQ ID NO:12 is two identical copies; The peptide chain shown in SEQ ID NO:17, the peptide chain shown in SEQ ID NO:16, and the peptide chain shown in SEQ ID NO:13, wherein the peptide chain shown in SEQ ID NO:13 is two identical copies; The peptide chain shown in SEQ ID NO:18, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:12, wherein the peptide chain shown in SEQ ID NO:12 is two identical copies; The peptide chain shown in SEQ ID NO:18, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:13, wherein the peptide chain shown in SEQ ID NO:13 is two identical copies; The peptide chain shown in SEQ ID NO:19, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:12, wherein the peptide chain shown in SEQ ID NO:12 is two identical copies; The peptide chain shown in SEQ ID NO:19, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:13, wherein the peptide chain shown in SEQ ID NO:13 is two identical copies; The peptide chain shown in any one of SEQ ID NO:17, 18, 19, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75, the peptide chain shown in SEQ ID NO:16, and the peptide chain shown in SEQ ID NO:57, wherein the peptide chain shown in SEQ ID NO:57 is two identical copies; Or The peptide chain shown in any one of SEQ ID NO:17, 18, 19, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75, the peptide chain shown in SEQ ID NO:20, and the peptide chain shown in SEQ ID NO:57, wherein the peptide chain shown in SEQ ID NO:57 is two identical copies.

34. An isolated nucleic acid molecule encoding the bispecific antibody according to any one of claims 22 to 33.

35. A recombinant expression vector comprising the isolated nucleic acid molecule according to claim 34.

36. A recombinant host cell comprising the recombinant expression vector according to claim 35, preferably, the recombinant host cell is a recombinant CHO-K1 cell.

37. A conjugate comprising a bispecific antibody and a small molecule drug, wherein, The bispecific antibody is the bispecific antibody according to any one of claims 22 to 33; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug; Preferably, the bispecific antibody is linked to the small molecule drug through a linker; for example, the linker is a hydrazone bond, a disulfide bond or a peptide bond; Preferably, the molar ratio of the bispecific antibody to the small molecule drug is 1:(2-8); Preferably, the molar ratio of the bispecific antibody to the small molecule drug is 1:(2-4).

38. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 22 to 33 and one or more pharmaceutically acceptable excipients.

39. Use of the anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1 to 11 or the bispecific antibody according to any one of claims 22 to 33 in the preparation of a drug for treating or preventing tumors; Preferably, the tumor is a tumor with high expression of EGFR and / or MET; Preferably, the tumor is one or more selected from glioma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological tumor, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, brain cancer, thyroid cancer and head and neck cancer; Preferably, the lung cancer is small cell lung cancer or non-small cell lung cancer.

40. The anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1 to 11 or the bispecific antibody according to any one of claims 22 to 33 for treating or preventing tumors; Preferably, the tumor is a tumor with high expression of EGFR and / or MET; Preferably, the tumor is one or more selected from glioma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological tumor, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, brain cancer, thyroid cancer and head and neck cancer; Preferably, the lung cancer is small cell lung cancer or non-small cell lung cancer.

41. A method for treating or preventing tumors, comprising the step of administering to a subject in need thereof an effective amount of the anti-EGFR antibody or its antigen-binding fragment according to any one of claims 1 to 11 or the bispecific antibody according to any one of claims 22 to 33; Preferably, the tumor is a tumor with high expression of EGFR and / or MET; Preferably, the tumor is one or more selected from glioma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological tumor, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, brain cancer, thyroid cancer, and head and neck cancer; Preferably, the lung cancer is small cell lung cancer or non-small cell lung cancer.

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