Anti-egfr mabs, bispecific antibodies thereof, pharmaceutical compositions and uses

Abstract

This invention belongs to the field of biomedicine and relates to an anti-EGFR monoclonal antibody, its bispecific antibody, pharmaceutical composition, and uses. Specifically, this invention relates to an anti-EGFR antibody or its antigen-binding fragment. This invention also relates to an anti-EGFR-anti-Met bispecific antibody. Both the anti-EGFR monoclonal antibody and the bispecific antibody of this invention exhibit good antitumor activity.

Description

Anti-EGFR monoclonal antibody, bispecific antibody, pharmaceutical composition and use thereof Technical Field

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

[0002] EGFR (Epidermal Growth Factor Receptor) is a receptor for epidermal growth factor (EGF) and belongs to the ErbB receptor family. EGFR is a 170 kDa transmembrane glycoprotein and a receptor-type tyrosine kinase. In response to ligands such as epidermal growth factor (EGF) and transforming growth factor α (TGFα), EGFR converts from a monomer to a dimer, becoming activated. This further activates downstream signaling pathways, such as phosphorylation of kinases such as Akt and ERK, regulating 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). Numerous studies have shown that EGFR is overexpressed or abnormally expressed in most tumors, such as glial cell carcinoma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer (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.). Abnormal EGFR function is associated with the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis (Castillo L, Etienne Grimaldi MC, Fischel JL, et al. Pharmacological background of EGFR targeting. Ann Oncol 2004; 15: 1007-12.). Its abnormal function is mainly manifested in two aspects: one is excessive abnormal expression in tumor tissues, and the other is the persistent activation of EGFR mutants in tumor cells (without the need for ligand stimulation or the formation of a self-circulating stimulation pathway).

[0003] c-mesenchymal-epithelial transition factor (c-Met, cMet, or Met) is a member of the receptor tyrosine kinase family. Binding of the Met receptor to its ligand, hepatocyte growth factor (HGF), induces Met dimerization and activates it, thereby activating downstream signaling pathways, such as the phosphorylation of kinases such as 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 due to Met mutations promotes angiogenesis and cancer metastasis.

[0004] Lung cancer is the leading cancer with the highest morbidity and mortality, with non-small cell lung cancer (NSCLC) accounting for 90% of lung cancer patients. EGFR mutation is the primary driver of NSCLC, accounting for over 40% of Asian NSCLC patients. EGFR kinase inhibitors (EGFR-TKIs) are the primary treatment, but the rate of drug resistance in patients is high and the mechanisms are complex, with Met amplification occurring in 7-15% of patients. In addition, Met exon 14 skipping mutations, Met fusions, Met amplification, and overexpression are also primary drivers of NSCLC. Dual antibodies targeting EGFR and Met can simultaneously inhibit both signaling pathways and are expected to be used in patients for whom EGFR-TKI is ineffective or resistant.

[0005] Currently, there are several publicly reported EGFR×Met bispecific antibodies. US9328173B2 (Eli Lilly), WO2018221969A1 (Jonggondang Biopharmaceuticals Co., Ltd.), and WO2022104236A2 (Ab Theraputics) disclose methods for constructing "2+2" EGFR×Met bispecific antibodies, where both the anti-EGFR and anti-Met ends are bivalent. Because EGFR antibodies can be dermatologically toxic, "2+2" EGFR×Met bispecific antibodies pose potentially higher safety risks. Johnson & Johnson's Amivantamab (US2017275367A1) discloses methods for constructing "1+1" EGFR×Met bispecific antibodies, where both the anti-EGFR and anti-Met ends are monovalent. This antibody has been approved for marketing and has a good safety profile.

[0006] The construction of a "1+1" type EGFR×Met bispecific antibody presents huge technical challenges, and it is necessary to solve the problems of correct pairing of light and heavy chains and heterodimerization of heavy chains. As reported in US2017275367A1, Amivantamab uses Fab arm exchange technology. However, this method requires the preparation of anti-EGFR antibodies and anti-Met antibodies separately, and then obtains bispecific antibodies through in vitro recombination, which increases production costs. Common light chain technology is one of the methods for constructing bispecific antibodies. Since there is no light and heavy chain mispairing problem, it greatly facilitates the preparation of bispecific antibodies.

[0007] 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 present inventors have developed bispecific antibodies, specifically anti-EGFR-anti-Met bispecific antibodies. This invention utilizes common light chain technology to construct an EGFR×Met bispecific antibody that inhibits EGF- and HGF-induced Akt and ERK phosphorylation; promotes EGFR and Met internalization; and kills tumor cells through ADCC. The bispecific antibody of the present invention exhibits excellent anti-tumor activity. This invention provides the following:

[0009] One aspect of the present invention relates to 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, wherein the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

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

[0011] The amino acid sequence of LCDR1 is shown in SEQ ID NO: 38, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 39, and the amino acid sequence of LCDR3 is 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 antigen-binding fragment thereof, wherein,

[0013] The amino acid sequence of HCDR3 is 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 antigen-binding fragment thereof, 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 antigen-binding fragment thereof, wherein,

[0018] 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: 42;

[0019] 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: 42;

[0020] 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: 42;

[0021] 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: 42;

[0022] 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: 42;

[0023] 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: 42;

[0024] 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: 42;

[0025] 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: 42;

[0026] 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: 42;

[0027] 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: 42;

[0028] 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: 43;

[0029] 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: 43;

[0030] 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: 43;

[0031] 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: 43;

[0032] 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: 43;

[0033] 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: 43;

[0034] 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: 43;

[0035] 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: 43;

[0036] 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: 43;

[0037] 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: 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 antigen-binding fragment thereof, wherein,

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

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

[0053] In some embodiments of the present invention, the anti-EGFR antibody or antigen-binding fragment thereof, 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; and 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 antigen-binding fragment thereof, 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 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 the group consisting of 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 antigen-binding fragment thereof, wherein:

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

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

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

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

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

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

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

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

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

[0067] or

[0068] The amino acid sequence of the heavy chain is 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 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 antigen-binding fragment thereof according to any one of the present invention.

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

[0073] Another aspect of the present invention relates to a host cell comprising 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 a pharmaceutical composition comprising an effective amount of the anti-EGFR antibody or antigen-binding fragment thereof according to any one of the present invention, and one or more pharmaceutically acceptable excipients.

[0075] In some embodiments of the present invention, the pharmaceutical composition further comprises an effective amount of an anti-Met antibody or an antigen-binding fragment thereof;

[0076] 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:

[0077] the amino acid sequence of HCDR1 is set forth in SEQ ID NO:45, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:46, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:47; or the amino acid sequence of HCDR1 is set forth in SEQ ID NO:48, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:49, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:50;

[0078] and

[0079] 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.

[0080] In some embodiments of the present invention, in the pharmaceutical composition,

[0081] 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

[0082] The amino acid sequence of the light chain variable region of the anti-Met antibody is shown in SEQ ID NO: 56;

[0083] Preferably, the anti-Met antibody:

[0084] 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.

[0085] Yet another aspect of the present invention relates to a combination pharmaceutical product comprising a first pharmaceutical product and a second pharmaceutical product in separate packages, wherein:

[0086] The first pharmaceutical product comprises an effective amount of the anti-EGFR antibody or antigen-binding fragment thereof according to any one of the present invention, and one or more pharmaceutically acceptable excipients;

[0087] The second drug product comprises an effective amount of an anti-Met antibody or an antigen-binding fragment thereof, and one or more pharmaceutically acceptable excipients;

[0088] Optionally, the combination drug product further comprises a product instruction sheet.

[0089] In some embodiments of the present invention, the combination drug product, wherein

[0090] The anti-Met antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

[0091] the amino acid sequence of HCDR1 is set forth in SEQ ID NO:45, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:46, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:47; or the amino acid sequence of HCDR1 is set forth in SEQ ID NO:48, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:49, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:50;

[0092] and

[0093] 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.

[0094] In some embodiments of the present invention, the combination drug product, wherein

[0095] 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

[0096] The amino acid sequence of the light chain variable region of the anti-Met antibody is shown in SEQ ID NO: 56;

[0097] Preferably, the anti-Met antibody:

[0098] 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.

[0099] Yet another aspect of the present invention relates to a bispecific antibody comprising:

[0100] Targeting the first protein domain of EGFR, and

[0101] Targeting a second protein domain that is different from the EGFR target (e.g., Met);

[0102] Wherein, the first protein functional region comprises the heavy chain variable region of the anti-EGFR antibody or antigen-binding fragment thereof described in any one of the present invention, or comprises the heavy chain variable region and light chain variable region of the anti-EGFR antibody or antigen-binding fragment thereof described in any one of the present invention.

[0103] 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.

[0104] In some embodiments of the present invention, the bispecific antibody is a "1+1" type EGFR×Met bispecific antibody.

[0105] In some embodiments of the present invention, the bispecific antibody, wherein

[0106] The second protein functional region comprises a heavy chain variable region of an anti-Met antibody or an antigen-binding fragment thereof; or comprises a heavy chain variable region and a light chain variable region of an anti-Met antibody or an antigen-binding fragment thereof;

[0107] in,

[0108] The anti-Met antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein:

[0109] the amino acid sequence of HCDR1 is set forth in SEQ ID NO:45, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:46, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:47; or the amino acid sequence of HCDR1 is set forth in SEQ ID NO:48, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:49, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:50;

[0110] and

[0111] 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.

[0112] In some embodiments of the present invention, the bispecific antibody, wherein

[0113] 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

[0114] The amino acid sequence of the light chain variable region of the anti-Met antibody is shown in SEQ ID NO: 56;

[0115] Preferably, the anti-Met antibody:

[0116] 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.

[0117] In some embodiments of the present invention, the bispecific antibody, wherein

[0118] Wherein, 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 monovalent antibody (IgG half molecule, IgG-HM).

[0119] In some embodiments of the present invention, the bispecific antibody, wherein

[0120] The first protein functional region is a half-molecule monovalent antibody, and

[0121] The second protein functional region is a half-molecule monovalent antibody.

[0122] In some embodiments of the present invention, the bispecific antibody, wherein

[0123] The first protein functional region is a half-molecule monovalent antibody targeting EGFR, and

[0124] The second protein functional region is a half-molecule monovalent antibody targeting Met.

[0125] In some embodiments of the present invention, the bispecific antibody, wherein

[0126] 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 heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region.

[0127] In some embodiments of the present invention, the bispecific antibody, wherein

[0128] The heavy chain constant regions of the two half-molecule monovalent antibodies respectively comprise a first CH3 region and a second CH3 region, and according to the EU numbering system, position 405 of the first CH3 region is mutated to Ala, Asp, Glu, His, Ile, Met, Asn, Gln, Thr, Val, Tyr, Leu, Lys, Ser or Trp; and position 409 of the second CH3 region is mutated to amino acid Ala, Asp, Glu, Phe, Gly, His, Ile, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr.

[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 contained therein respectively include a first CH3 region and a second CH3 region, and according to the EU Numbering System, position 405 of the first CH3 region is mutated to Leu; position 409 of the second CH3 region is mutated to amino acid Arg.

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

[0132] The heavy chain constant region of the half-molecule monovalent antibody is a heavy chain constant region of human IgG1.

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

[0134] The heavy chain constant region of the half-molecule monovalent antibody is a heavy chain constant region of human IgG1 and has a Knob mutation (eg, S354C and T366W mutations); and

[0135] The heavy chain constant region of the half-molecule monovalent antibody is a heavy chain constant region of human IgG1 and has Hole mutations (eg, Y349C, T366S, L368A, and Y407V mutations).

[0136] In the present invention, the mutation positions of the knob and hole are numbered according to the EU numbering system. In some embodiments of the present invention, the knob mutation refers to the S354C and T366W mutations. In some embodiments of the present invention, the hole mutation refers to the Y349C, T366S, L368A, and Y407V mutations.

[0137] 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, position 435 of the heavy chain constant region of one half-molecule monovalent antibody is mutated to the amino acid Arg (R), and position 436 is mutated to the amino acid Phe (F).

[0138] In some embodiments of the present invention, the bispecific antibody is in IgG form, preferably in IgG1 form;

[0139] Preferably, the sequences of the light chains in the bispecific antibody are identical;

[0140] Preferably, the bispecific antibody has two light chains with identical sequences;

[0141] Preferably, the bispecific antibody consists of the following peptide chains:

[0142] (1) a peptide chain selected from SEQ ID NO: 17 to SEQ ID NO: 19, and SEQ ID NO: 58 to SEQ ID NO: 75,

[0143] (2) a peptide chain selected from SEQ ID NO: 16 and SEQ ID NO: 20, and

[0144] (3) a peptide chain selected from SEQ ID NO: 11 to SEQ ID NO: 13, and a peptide chain in SEQ ID NO: 57,

[0145] Among them, the peptide chain in (3) is two identical copies;

[0146] 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 (eg, 2 or 3 disulfide bonds).

[0147] In some embodiments of the present invention, the bispecific antibody is composed of the following peptide chains:

[0148] The peptide chain represented by SEQ ID NO: 17, the peptide chain represented by SEQ ID NO: 16, and the peptide chain represented by SEQ ID NO: 12, wherein the peptide chain represented by SEQ ID NO: 12 is two identical copies;

[0149] The peptide chain represented by SEQ ID NO: 17, the peptide chain represented by SEQ ID NO: 16, and the peptide chain represented by SEQ ID NO: 13, wherein the peptide chain represented by SEQ ID NO: 13 is two identical copies;

[0150] The peptide chain represented by SEQ ID NO: 18, the peptide chain represented by SEQ ID NO: 20, and the peptide chain represented by SEQ ID NO: 12, wherein the peptide chain represented by SEQ ID NO: 12 is two identical copies;

[0151] The peptide chain represented by SEQ ID NO: 18, the peptide chain represented by SEQ ID NO: 20, and the peptide chain represented by SEQ ID NO: 13, wherein the peptide chain represented by SEQ ID NO: 13 is two identical copies;

[0152] The peptide chain represented by SEQ ID NO: 19, the peptide chain represented by SEQ ID NO: 20, and the peptide chain represented by SEQ ID NO: 12, wherein the peptide chain represented by SEQ ID NO: 12 is two identical copies;

[0153] The peptide chain represented by SEQ ID NO: 19, the peptide chain represented by SEQ ID NO: 20, and the peptide chain represented by SEQ ID NO: 13, wherein the peptide chain represented by SEQ ID NO: 13 is two identical copies;

[0154] The peptide chain represented by any one of SEQ ID NOs: 17, 18, 19, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75, and the peptide chain represented by SEQ ID NO: 16 and the peptide chain represented by SEQ ID NO: 57, wherein the peptide chain represented by SEQ ID NO: 57 is two identical copies;

[0155] or

[0156] The peptide chain represented 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 represented by SEQ ID NO: 20, and the peptide chain represented by SEQ ID NO: 57, wherein the peptide chain represented by SEQ ID NO: 57 is two identical copies.

[0157] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the bispecific antibody according to any one of the present invention.

[0158] Yet another aspect of the present invention relates to a recombinant expression vector comprising the isolated nucleic acid molecule of the present invention.

[0159] 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.

[0160] Another aspect of the present invention relates to a pharmaceutical composition comprising the bispecific antibody according to any one of the present invention, and one or more pharmaceutically acceptable excipients.

[0161] Another aspect of the present invention relates to use of the anti-EGFR antibody or antigen-binding fragment thereof according to any one of the present invention or the bispecific antibody according to any one of the present invention in the preparation of a medicament for treating or preventing tumors;

[0162] Preferably, the tumor is a tumor with high expression of EGFR and / or Met;

[0163] Preferably, the tumor is one or more selected from glial cell carcinoma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic tumor, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, brain cancer, thyroid cancer and head and neck cancer;

[0164] Preferably, the lung cancer is non-small cell lung cancer.

[0165] The anti-EGFR antibody or antigen-binding fragment thereof according to any one of the present invention or the bispecific antibody according to any one of the present invention is used for treating or preventing tumors;

[0166] Preferably, the tumor is a tumor with high expression of EGFR and / or Met;

[0167] Preferably, the tumor is one or more selected from glial cell carcinoma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic tumor, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, brain cancer, thyroid cancer and head and neck cancer;

[0168] Preferably, the lung cancer is non-small cell lung cancer.

[0169] 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 thereof an effective amount of the anti-EGFR antibody or antigen-binding fragment thereof described in any one of the present invention, or the bispecific antibody described in any one of the present invention;

[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 glial cell carcinoma, renal cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic tumor, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, brain cancer, thyroid cancer and head and neck cancer;

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

[0173] In some embodiments of the present invention, the method for treating or preventing tumors is administered before or after surgery, and / or before or after radiotherapy.

[0174] In some embodiments of the present invention, the method for treating or preventing tumors, wherein:

[0175] The single dosage of the anti-EGFR antibody or antigen-binding fragment thereof 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;

[0176] 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;

[0177] Preferably, the administration is by intravenous drip or intravenous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0178] Figure 1: Schematic diagram of the structure of the bispecific antibody E2mut34-91A×M5-91A-FAE-LF.

[0179] Figure 2: Schematic diagram of the structure of the bispecific antibody E2mut34-69×M5-69-FAE-LF.

[0180] Figure 3: Schematic diagram of the structure of the bispecific antibody JNJ-372.

[0181] Figure 4: EGFR ELISA binding assay results.

[0182] Figure 5: Met ELISA binding assay results.

[0183] Figure 6: EGFR ELISA blocking assay results.

[0184] Figure 7: Met ELISA blocking assay results.

[0185] Figures 8A to 8C show that the antibodies E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 all bind to recombinant human EGFR. Figures 8A and 8B show binding and dissociation curves of the antibody E2-mut34-91A×M5-91A-FAE-LF and human EGFR, respectively; Figure 8C shows binding and dissociation curves of the antibody JNJ-372 and human EGFR.

[0186] Figures 9A to 9C show that antibodies E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 all bind to recombinant human Met. Figure 9A shows the binding and dissociation curves of antibody E2-mut34-91A×M5-91A-FAE-LF and human Met; Figure 9B shows the binding and dissociation curves of antibody E2-mut34-69×M5-69-FAE-LF and human Met; Figure 9C shows the binding and dissociation curves of antibody JNJ-372 and human Met.

[0187] Figures 10A to 10G show binding experiments of test substances at the cellular level.

[0188] Figure 11A to Figure 11M: Detection results of the test substances on AKT or ERK phosphorylation.

[0189] FIG12A shows ADCC assay results using a reporter gene assay.

[0190] FIG12B shows ADCC assay results using a reporter gene assay.

[0191] FIG12C shows ADCC assay results using the LDH assay.

[0192] FIG12D shows ADCC assay results using a reporter gene assay.

[0193] FIG12E : ADCC experimental results detected by LDH method.

[0194] Figure 13: Inhibitory effect of the test substance on the NCI-H1975 human lung cancer model transplanted into CB-17 SCID mice.

[0195] Figure 14: Inhibitory effect of the test substance on the NCI-H1975 human lung cancer subcutaneous tumor model.

[0196] Figure 15: Inhibitory effect of the test substance on the H1975 (L858R / T790M / C797S) human lung cancer subcutaneous tumor model.

[0197] Figure 16: Inhibitory effect of the test substance on the H292 human lung cancer subcutaneous tumor model. DETAILED DESCRIPTION

[0198] Scientific and technical terms

[0199] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0200] As used herein, the term EC 50 It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0201] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 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 (C1q) of the classical complement system. The VH and VL regions can also be further subdivided 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 from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site. The assignment of amino acids to regions or domains follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the IMGT numbering system definition, see Ehrenmann F, Kaas Q, Lefranc MP. 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.

[0202] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0203] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for possible spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology 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 it can also be obtained by using recombinant DNA technology (see, for example, US Patent 4,816,567).

[0204] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, 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 an antibody is a diabodies, in which V H and V LThe domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993;90:6444-6448 and Poljak RJ et al., Structure 1994;2:1121-1123).

[0205] As used herein, the term "single chain antibody (ScFv)" refers to a single chain antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ) molecule. Where V L and V H The domains are paired to form monovalent molecules via linkers that enable their production as a single polypeptide chain (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 may have the general structure: NH2-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L -COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having 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.

[0206] As used herein, the term "isolated" or "isolated" refers to something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and a highly pure identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0207] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to 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. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0208] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0209] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity 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 Affinity of M or less (K D ) binds to the antigen.

[0210] 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 the antibody and the 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, antibodies bind with a dissociation equilibrium 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 dissociation equilibrium constant (K D ) binding antigen (eg, EGFR protein). K can be determined using methods known to those skilled in the art. D , for example, using the Fortebio molecular interaction instrument.

[0211] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0212] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0213] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0214] As used herein, when referring to the amino acid sequence of EGFR protein, it includes the full length of EGFR protein, including fragments of EGFR ECD; it also includes a fusion protein of the full length of EGFR protein or a fusion protein of EGFR ECD, such as a fragment fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and / or additions) may be naturally produced or artificially introduced into the amino acid sequence of EGFR protein without affecting its biological function. Therefore, in the present invention, the term "addition), protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of EGFR protein, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0215] As used herein, when referring to the amino acid sequence of the Met protein, it includes the full-length Met protein, fragments including the Met ECD; it also includes fusion proteins of the full-length Met protein or fusion proteins of the Met ECD, such as fragments fused with the Fc protein fragment (mFc or hFc) of mouse 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 be naturally or artificially introduced into the amino acid sequence of the Met protein without affecting its biological function. Therefore, in the present invention, the term "addition) protein should include all such sequences, including natural or artificial variants thereof. Furthermore, when describing a sequence fragment of the Met protein, it also includes the corresponding sequence fragments in its natural or artificial variants.

[0216] As used herein, the term "ADCC" refers to antibody-dependent cell-mediated cytotoxicity. The Fab segment of an antibody binds to antigenic epitopes on virus-infected cells or tumor cells, while its Fc segment binds to Fc receptors (FcRs) on the surface of killer cells (NK cells, macrophages, etc.), mediating direct killing of target cells by these cells.

[0217] In the present invention, unless otherwise specified, the "first" (e.g., the first protein functional region or the first drug product) and "second" (e.g., the second protein functional region or the second drug product) are for the purpose of distinguishing references or clarifying expressions, and do not have a typical order meaning.

[0218] In the present invention, the term "half molecule monovalent antibody (IgG half molecule, IgG-HM)" refers to an antibody molecule composed of one heavy chain and one light chain of an IgG antibody (e.g., IgG1, IgG2, IgG3 or IgG4), which is a monovalent antibody (for example, see Feng Yifan et al., Construction and Activity Analysis of HIV-1 Specific Monoclonal Antibody 2G12 Monovalent Antibody, Chinese Journal of Virology, Vol. 5, No. 3, May 2015, p171-175).

[0219] The partial sequences involved in the present invention are as follows (the underlined parts are CDRs according to the Kabat numbering scheme): (1) E2-M1-VH

[0220] (2)E2-M2-VH

[0221] (3)E2-M5-VH

[0222] (4)E2-M12-VH

[0223] (5)E2-M13-VH

[0224] (6)E2-M16-VH

[0225] (7)E2-M17-VH

[0226] (8)E2-M25-VH

[0227] (9)E2-M34-VH

[0228] (10)E2-M38-VH

[0229] (11)E2-LC

[0230] The sequences of the three light chain CDRs are as follows:

[0231] LCDR1: RASQSVSSWLA (SEQ ID NO: 38)

[0232] LCDR2: GASNRAT (SEQ ID NO: 39)

[0233] LCDR3: QVGSTPLT (SEQ ID NO: 40)

[0234] The light chain variable region VL sequence is as follows:

[0235] (12)E2-10-LC-13-91A

[0236] The sequences of the three light chain CDRs are as follows:

[0237] LCDR1: RASQSVSSWLA (SEQ ID NO: 38)

[0238] LCDR2: GASNRAT (SEQ ID NO: 39)

[0239] LCDR3: LQAGSTPLT (SEQ ID NO: 41)

[0240] The light chain variable region VL sequence is as follows:

[0241] (13)E2-10-LC-69

[0242] The sequences of the three light chain CDRs are as follows:

[0243] LCDR1: RASQSVSSWLA (SEQ ID NO: 38)

[0244] LCDR2: GASNRAT (SEQ ID NO: 39)

[0245] LCDR3: LQAGSTPLT (SEQ ID NO: 41)

[0246] The light chain variable region VL sequence is as follows:

[0247] (14) JNJ-372-ML (the italicized part is the light chain variable region)

[0248] The sequences of the three light chain CDRs are as follows:

[0249] The light chain variable region VL sequence is as follows:

[0250] (15) JNJ-372-EL (the italicized part is the light chain variable region)

[0251] (16) M5-HC-K409R (the italicized part is the heavy chain variable region)

[0252] The sequences of the three heavy chain CDRs are as follows:

[0253] HCDR1: SSVYYWS (SEQ ID NO: 45)

[0254] HCDR2: VIYPSGNTYYSPSLKS (SEQ ID NO: 46)

[0255] HCDR3:TIYDLFDI(SEQ ID NO:47)

[0256] The heavy chain variable region VH sequence is as follows:

[0257] The heavy chain constant region sequence is as follows:

[0258] (17) E2mut34-HC-F405L (the italicized part is the heavy chain variable region)

[0259] The heavy chain constant region sequence is as follows:

[0260] (18) E2mut34-HC-hole (the italicized part is the heavy chain variable region)

[0261] The constant region sequence is as follows:

[0262] (19) E2mut34-HC-holeRF (the italicized part is the heavy chain variable region)

[0263] The constant region sequence is as follows:

[0264] (20) M5-HC-knob (the italicized part is the heavy chain variable region)

[0265] The constant region sequence is as follows:

[0266] (21) JNJ-372-MH (the italicized part is the heavy chain variable region)

[0267] The sequences of the three heavy chain CDRs are as follows:

[0268] The heavy chain variable region VH is as follows:

[0269] (22) JNJ-372-EH (the italicized part is the heavy chain variable region)

[0270] (23)EGFR-his-tag

[0271] (24)Met-his-tag

[0272] (25)E2-10-LC-13-91Q

[0273] The sequences of the three light chain CDRs are as follows:

[0274] LCDR1: RASQSVSSWLA (SEQ ID NO: 38)

[0275] LCDR2: GASNRAT (SEQ ID NO: 39)

[0276] LCDR3: LQQGSTPLT (SEQ ID NO: 76)

[0277] The light chain variable region VL sequence is as follows:

[0278] (26) E2mut1-HC-F405L (the italicized part is the heavy chain variable region)

[0279] (27) E2mut2-HC-F405L (the italicized part is the heavy chain variable region)

[0280] (28) E2mut5-HC-F405L (the italicized part is the heavy chain variable region)

[0281] (29) E2mut12-HC-F405L (the italicized part is the heavy chain variable region)

[0282] (30) E2mut13-HC-F405L (the italicized part is the heavy chain variable region)

[0283] (31) E2mut16-HC-F405L (the italicized part is the heavy chain variable region)

[0284] (32) E2mut17-HC-F405L (the italicized part is the heavy chain variable region)

[0285] (33) E2mut25-HC-F405L (the italicized part is the heavy chain variable region)

[0286] (34) E2mut38-HC-F405L (the italicized part is the heavy chain variable region)

[0287] (35) E2mut1-HC-hole (the italicized part is the heavy chain variable region)

[0288] (36) E2mut2-HC-hole (the italicized part is the heavy chain variable region)

[0289] (37) E2mut5-HC-hole (the italicized part is the heavy chain variable region)

[0290] (38) E2mut12-HC-hole (the italicized part is the heavy chain variable region)

[0291] (39) E2mut13-HC-hole (the italicized part is the heavy chain variable region)

[0292] (40) E2mut16-HC-hole (the italicized part is the heavy chain variable region)

[0293] (41) E2mut17-HC-hole (the italicized part is the heavy chain variable region)

[0294] (42) E2mut25-HC-hole (the italicized part is the heavy chain variable region)

[0295] (43) E2mut38-HC-hole (the italicized part is the heavy chain variable region)

[0296] Advantageous Effects of the Invention

[0297] The bispecific antibody of the present invention achieves one or more of the following technical effects (1)-(5):

[0298] (1) Having good affinity and / or specificity for the target EGFR and / or Met;

[0299] (2) High ADCC activity;

[0300] (3) has a high endocytosis rate;

[0301] (4) has good anti-tumor activity;

[0302] (5) The production process is simple and the cost is low.

[0303] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0304] Preparation Example 1: Sequence Design and Preparation of Anti-EGFR Monoclonal Antibodies

[0305] Ten anti-EGFR monoclonal antibodies were designed and screened, designated E2-M1, E2-M2, E2-M5, E2-M12, E2-M13, E2-M16, E2-M17, E2-M25, E2-M34, and E2-M38. The amino acid sequences of the heavy chain variable regions of these ten anti-EGFR monoclonal antibodies are shown in SEQ ID NOs: 1-10, respectively, and the heavy chain constant regions are selected from the amino acid sequences shown in SEQ ID NOs: 79, 80, and 81. The amino acid sequences of the light chains are all shown in SEQ ID NO: 11. The HCDRs and LCDRs are identified using the Kabat numbering system and are underlined, as shown in Table A below.

[0306] Table A

[0307] In the above antibody, 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.

[0308] Example 1: Determination of affinity of E2 mutants for EGFR using octet Red 96e

[0309] The kinetic parameters of binding of the 10 previously prepared anti-EGFR monoclonal antibodies to the EGFR-his-tag antigen were determined using a protein A capture assay. The monoclonal antibodies and the control antibody JNJ-372-E-LF (prepared in Preparation Example 2 below) were bound to a Protein A probe (Cat No: 18-5010; Lot: 2001131) at a concentration of 1 μg / ml. The EGFR-his-tag antigen was diluted two-fold in 1X Fortebio working solution (1X PBS + 0.05% Tween 20) from 50 nM to form four concentration gradients, allowing the antibodies to bind and dissociate in 1X Fortebio working solution.

[0310] The kinetic parameters of the binding of E2 mutants to EGFR-his-tag are shown in Table B .

[0311] Table B

[0312] The results showed that the affinities of the 10 E2 mutants all reached the nM level, among which E2-M1, E2-M2, E2-M25, E2-M34, and E2-M38 had better affinities than the control antibody JNJ-372-E-LF, and E2-M34 had the highest affinity.

[0313] Preparation Example 2: Design and Preparation of the First Batch of EGFR×Met Bispecific Antibodies

[0314] 1. Construction of bispecific antibody molecules

[0315] E2mut34 was selected from the 10 high-affinity EGFR antibody heavy chain variable regions prepared in Preparation Example 1 to construct the heavy chain of the EGFR-terminal parent antibody of the EGFR×Met bispecific antibody (see SEQ ID NOs: 17-19). In addition, the heavy chain of a previously disclosed Met antibody (SEQ ID NO: 410 in CN105705519A) was used as the heavy chain of the Met-terminal parent antibody of the EGFR×Met bispecific antibody (see SEQ ID NOs: 16 and 20) after making corresponding modifications in the constant region as needed for bispecific antibody preparation. E2-10-LC-13-91A or E2-10-LC-69 was used as the common light chain.

[0316] Qingke Biotechnology was commissioned to synthesize the DNA sequences encoding the four light chains E2-10-LC-13-91A, E2-10-LC-69, JNJ-372-ML, and JNJ-372-EL in Table 1. The DNA sequences were digested with SapI (purchased from NEB, catalog number: R0569L) and ligated into the HXT2 vector (Junshi Biosciences' own modified vector, derived from pTT5) to obtain four expression vectors, named HXT2-E2-10-LC-13-91A, HXT2-E2-10-LC-69, HXT2-JNJ-372-ML, and HXT2-JNJ-372-EL, respectively.

[0317] Among them, HXT2-JNJ-372-ML is the Met-terminal antibody light chain of Amivantamab, and HXT2-JNJ-372-EL is the EGFR-terminal antibody light chain of Amivantamab (sequence source: IMGT official website).

[0318] The DNA sequences encoding the seven heavy chains M5-HC-K409R, E2mut34-HC-F405L, E2mut34-HC-hole, E2mut34-HC-holeRF, M5-HC-knob, JNJ-372-MH, and JNJ-372-EH in Table 1 were synthesized by Qingke Biotechnology, digested with SapI (purchased from NEB, Cat. No. R0569L), and ligated into the HXT1S vector (Junshi Biosciences). The modified vector was derived from pTT5) to obtain seven expression vectors, which were 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.

[0319] Among them, HXT1S-JNJ-372-MH is the Met-terminal antibody heavy chain of Amivantamab, and HXT1S-JNJ-372-EH is the EGFR-terminal antibody heavy chain of Amivantamab (sequence source: IMGT official website).

[0320] Table 1: Heavy and light chain names

[0321] 2. Transient protein expression and purification

[0322] The required heavy chain, light chain and expression vector are shown in Table 2. Among them:

[0323] E2mut34-91A-F405L-LF and M5-91A-K409R-LF were used for subsequent preparation of bispecific antibodies (in vitro recombinant method);

[0324] E2mut34-69-F405L-LF and M5-69-K409R-LF were used for subsequent preparation of bispecific antibodies (in vitro recombinant method);

[0325] JNJ-372-M-LF and JNJ-372-E-LF were used for the subsequent preparation of bispecific antibodies (in vitro recombinant method).

[0326] ALK101-2 and ALK101-4 used a common light chain and "knob-into-hole" technology (see WO1996027011A1) to prepare bispecific antibodies.

[0327] Table 2: Combinations of heavy and light chains and corresponding expression vectors

[0328] 2.1 Expression of bispecific antibody molecules

[0329] Count the CHO-K1 cells (owned by Suzhou Junmeng) in culture. When the cell density is between 2 and 6*10 6 / ml, and then subculture and expand using CD CHO medium (purchased from Thermofisher, product number: 12490-001). The day before transfection, the cell density was diluted to 1.8-2.5*10 6 / ml, the next day when the cell density reached about 3.5-5.0*10 6 / ml for transfection. First add one tenth of the transfection volume of CD CHO culture medium, add 1-2μg / ml of plasmid (made by the company) according to the combination table shown in Table 2, and finally add 3-14μg / 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. The transfected mixture is placed on a shaker for culture. On the first day after transfection, 6% of glycoform regulator (purchased from Aupuma, catalog number: R170026) is added, and 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) are supplemented. Then, the feed is supplemented every two days, and the samples are collected 5-9 days after transfection.

[0330] 3. In vitro Reconstitution and Purification

[0331] 3.1 Affinity capture of bispecific antibody molecules

[0332] After the incubation period, the cell supernatant was collected by centrifugation at 1000 g for 5 min in a floor-standing centrifuge (ThermoFisher, R404A) and the precipitate was discarded. The supernatant was then collected by centrifugation at 8000 g for 30 min and sterile filtered using a 0.22 μm filter cup (Jet, FPE-214-000). Purification was performed using a protein purifier (GE, AKTA Avant). A Mabselect Sure LX column (Cytiva, 17547403) was equilibrated with PBS equilibration buffer (Wuxi Aorui Dongyuan Biotechnology Co., Ltd., ZLI-9061). After the sample loading is completed, the sample is first eluted with affinity chromatography elution buffer A (pH 5.5, 45mM acetic acid-sodium acetate + 1M sodium chloride system), then eluted with eluent B (pH 5.5, 45mM acetic acid-sodium acetate system), and finally eluted with affinity elution buffer (pH 3.6, 10mM acetic acid-sodium acetate buffer). The sample is neutralized with 1M Tris (purchased from Merck, product number: E300016981946) buffer and the pH is adjusted to 5.5-6 for the next in vitro reconstitution.

[0333] 3.2 In vitro recombination of bispecific antibody molecules

[0334] Referring to the technical solution disclosed in WO2011131746, a bispecific antibody was prepared using Fab arm exchange technology as follows:

[0335] The two parent antibodies after affinity were concentrated and exchanged into PBS equilibration buffer (purchased from Wuxi Aorui Dongyuan Biotechnology Co., Ltd., ZLI-9061), and the protein concentration was set at 1±0.05 mg / ml.

[0336] 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, add PBS to quantify to 30 ml, then filter with a 0.22 μm filter (purchased from Sartorius, 16541-K), and finally wrap with aluminum foil and store in the dark.

[0337] To construct the incubation system: Take the parent antibody treated in the above steps, as shown in Table 3, and add 2 ml of the M parent antibody, 2.4 ml of the E parent antibody, and 0.489 ml of 750 mM cysteamine hydrochloride to a 15 ml centrifuge tube (Genemore, G3210015) at a molar ratio of M:E = 1:1.2. Seal the constructed incubation system with aluminum foil and incubate in a 31°C water bath (Shanghai Jinghong Laboratory Equipment Co., Ltd., DK-S28) for 3 hours in the dark. After incubation, concentrate and replace the buffer with PBS equilibration buffer and incubate at room temperature in the dark for 16 to 24 hours. The molecular structures of the bispecific antibodies prepared by the in vitro recombinant method are shown in Figures 1 to 3.

[0338] Table 3: In vitro recombination combinations of bispecific antibodies

[0339] 3.3 Purification of bispecific antibody molecules

[0340] Capto™ MMC Impres (Cytiva, 17371602) was used for purification. Pre-equilibration was performed using wash buffer (pH 7.5, 20mM Tris-HCl + 1M NaCl buffer system) and equilibration was performed using equilibration buffer (pH 7.5, 20mM Tris-HCl system). After loading, 3-6 column volumes of equilibration buffer were used. Finally, linear elution was performed using elution buffer (pH 7.5, 20mM Tris-HCl + 1M NaCl system) to collect the target protein. The three bispecific antibodies listed in Table 3 were thus prepared.

[0341] Example 2: ELISA binding assay

[0342] 4.1 EGFR ELISA binding assay

[0343] Use PBS (purchased from Hyclone, product number: SH30256.01) to dilute EGFR-his-tag (purchased from Suzhou Junmeng, batch number: 20210803. Sequence source: nuiprot official website) to 5.0μg / ml, add 100μl / well to the enzyme-labeled plate, and let it sit in a 37℃ constant temperature incubator for 60 minutes; wash the plate; add 200μl / well 2% BSA (purchased from Sigma, product number: B2064) to the plate, incubate in a 37℃ constant temperature incubator for 60 minutes, wash the plate; dilute the sample with 2% BSA to 10μg / ml, dilute it 3 times to 0.056ng / ml, add 100μl / well to the enzyme-labeled plate, and incubate in a 37℃ constant temperature incubator for 60 minutes. The plate was washed for 10 minutes; horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Fc-specific) antibody (purchased from Sigma, catalog number: A0170) was diluted 5000-fold with 2% BSA and added to the ELISA plate at 100 μl / well. The plate was incubated in a 37°C constant temperature incubator for 60 minutes and washed; the color developing solution TMB (purchased from Sigma, catalog number: T2885) 0.1 mg / ml was added at 100 μl / well. Avoid bubbles and color development at 37°C in the dark for 10 minutes; finally, 2 M hydrochloric acid solution was added to terminate the reaction at 100 μl / well. Avoid bubbles and complete the microplate reader reading within 10 minutes (detection wavelength 450 nm; reference wavelength 620 nm). The EC was fitted using a four-parameter logarithmic regression (4PL) model. 50 As shown in Figure 4, E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 bind to EGFR EC 50 The values ​​were 13.57, 11.71 and 33.90 ng / ml respectively. The binding ability of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF to EGFR was better than that of JNJ-372.

[0344] 4.2 Met ELISA binding assay

[0345] Use PBS (purchased from Hyclone, product number: SH30256.01) to dilute Met-his-tag (purchased from Suzhou Junmeng, batch number: 20220404. Sequence source: nuiprot official website) to 2.0 μg / ml, add 100 μl / well to the enzyme-labeled plate, and let it sit in a 37°C constant temperature incubator for coating for 60 minutes; wash the plate; add 200 μl / well 2% BSA (purchased from Sigma, product number: B2064) to the plate, incubate in a 37°C constant temperature incubator for 60 minutes, and wash the plate; dilute the sample to 10 μg / ml with 2% BSA, dilute it 3 times in a gradient to 0.056 ng / ml, add 100 μl / well to the enzyme-labeled plate, and incubate in a 37°C constant temperature incubator for 60 minutes. Wash the plate; dilute horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Fc-specific) antibody (purchased from Sigma, catalog number: A0170) 5000-fold with 2% BSA, add 100 μl / well to the ELISA plate, incubate in a 37°C constant temperature incubator for 60 minutes, and wash the plate; add 0.1 mg / ml TMB (purchased from Sigma, catalog number: T2885) at 100 μl / well, avoid bubbles, and color at 37°C in the dark for 10 minutes; finally, add 2M hydrochloric acid solution to terminate the reaction, 100 μl / well, avoid bubbles, and complete the microplate reader reading within 10 minutes (detection wavelength 450 nm; reference wavelength 620 nm), and use the four-parameter logarithmic regression (4PL) model to fit the EC 50 As shown in Figure 5, the EC values ​​of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 for binding to Met 50 The values ​​were 7.461, 6.558 and 8.416 ng / ml respectively. The binding ability of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF to Met was comparable to that of JNJ-372.

[0346] Example 3: ELISA blocking experiment

[0347] 5.1 EGFR ELISA blocking assay

[0348] EGFR-his-tag (purchased from Suzhou Junmeng, batch number: 20210803) was diluted to 2.0 μg / ml with PBS (purchased from Hyclone, product number: SH30256.01), and 100 μl / well was added to the ELISA plate, and the plate was incubated in a 37°C constant temperature incubator for 60 minutes; the plate was washed; 200 μl / well 2% BSA (purchased from Sigma, product number: B2064) was added to the plate, and the plate was incubated in a 37°C constant temperature incubator for 60 minutes; the plate was washed; EGF mFc (purchased from Acro, product number: EGF-H525b) was diluted to 3.0 μg / ml with 2% BSA, and then 3.0 μg / ml of EGF was added. The mFc sample was diluted to 400 μg / ml, serially diluted 3-fold to 0.0023 μg / ml, and added to the ELISA plate at 100 μl / well. The plate was incubated in a 37°C incubator for 60 minutes and washed. Horseradish peroxidase (HRP)-conjugated anti-mouse Fc antibody (purchased from Sigma, Catalog No. A2554) was diluted 5000-fold with 2% BSA and added to the ELISA plate at 100 μl / well. The plate was incubated in a 37°C incubator for 60 minutes and washed. The color development solution TMB (purchased from Sigma, Catalog No. T2885) at 0.1 mg / ml was added at 100 μl / well. The color was developed at 37°C in the dark for 10 minutes, avoiding bubbles. Finally, 2 M hydrochloric acid solution was added to terminate the reaction at 100 μl / well. The plate was read on a microplate reader within 10 minutes (detection wavelength 450 nm; reference wavelength 620 nm). The IC values ​​were fitted using a four-parameter logistic regression (4PL) model. 50 As shown in Figure 6, the IC values ​​of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 for blocking EGFR 50 The values ​​were 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 was slightly better than that of JNJ-372.

[0349] 5.2 Met ELISA blocking assay

[0350] HGF-his-tag (purchased from Acro, batch number: HGF-H52H3) was diluted to 4.0 μg / ml with PBS (purchased from Hyclone, product number: SH30256.01), and 100 μl / well was added to the ELISA plate and coated overnight at 4°C; the plate was washed; 200 μl / well 2% BSA (purchased from Sigma, product number: B2064) was added to the plate, incubated at room temperature for 90 minutes, and the plate was washed; Met biotinylated (purchased from Suzhou Junmeng, batch number: 20220526) was diluted to 0.1 μg / ml with 2% BSA, and then 0.1 μg / ml Met The biotinylated sample was diluted to 20 μg / ml and serially diluted 2.5-fold to 0.84 ng / ml. 100 μl / well of the microplate was added and incubated at room temperature for 60 min. The plate was washed. Horseradish peroxidase (HRP)-conjugated streptavidin (purchased from Jackson ImmunoResearch, catalog number: 016-030-084) was diluted 5000-fold with 2% BSA and added to the microplate at 100 μl / well. The plate was incubated at room temperature for 60 min and washed. TMB (purchased from Sigma, catalog number: T2885) 0.1 mg / ml was added at 100 μl / well. The color was developed at room temperature in the dark for 10 min. Finally, 2 M hydrochloric acid solution was added to terminate the reaction at 100 μl / well. The plate was read on a microplate reader within 10 min (detection wavelength: 450 nm; reference wavelength: 620 nm). The IC values ​​were fitted using a four-parameter logistic regression (4PL) model. 50 As shown in Figure 7, E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 blocked the IC of Met 50 The values ​​were 216.7, 185.2 and 165 ng / ml respectively. The blocking ability of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF against Met was comparable to that of JNJ-372.

[0351] Example 4: Biacore affinity determination

[0352] The molecular interaction analyzer Biacore T200 (GE Healthcare Life Sciences) was used 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.

[0353] The binding affinity of antibodies to EGFR was determined as follows: 40 μg / ml goat anti-human IgG-Fc fragment antibody was coupled to a CM5 chip (Cytiva, Cat. No. BR-1005-30) to capture the antibody. 1 μg / ml of E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 antibodies were captured on the CM5 chip. EGFR was injected at 20 nM and 5 nM to bind to E2-mut34-91A×M5-91A-FAE-LF and E2-mut34-69×M5-69-FAE-LF, while EGFR was injected at 80 nM and 20 nM to bind to JNJ-372. Association and dissociation kinetics were measured using a Biacore T200 system (GE Healthcare). The affinity K was calculated by fitting the binding and dissociation curves using Biacore T200 Evaluation Software 3.0. D value.

[0354] The binding affinity of antibodies to Met was determined as follows: 40 μg / ml goat anti-human IgG-Fc fragment antibody (Jackson ImmunoResearch) was coupled to the surface of a CM5 chip (Cytiva, Cat. No. BR-1005-30) to capture the antibodies. 1 μg / ml of E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 antibodies were captured on the CM5 chip. Met was injected at 50 nM and 12.5 nM to bind to the three antibodies. Binding and dissociation kinetics were measured using a Biacore T200 system (GE Healthcare). Binding and dissociation curves were fitted using Biacore T200 Evaluation Software 3.0 to calculate affinity (KD) values.

[0355] The Biacore data are shown in Table 4, Figures 8A to 8C, and Figures 9A to 9C.

[0356] Table 4: Statistics of antibody affinity determined by Biacore

[0357] The results showed that the antibodies E2-mut34-91A×M5-91A-FAE-LF, E2-mut34-69×M5-69-FAE-LF, and JNJ-372 all showed binding activity to human EGFR and Met. The EGFR affinity of E2-mut34-91A×M5-91A-FAE-LF and E2-mut34-69×M5-69-FAE-LF was similar and approximately five-fold stronger than that of JNJ-372. The Met affinity of E2-mut34-91A×M5-91A-FAE-LF and E2-mut34-69×M5-69-FAE-LF was similar and approximately two-fold weaker than that of JNJ-372.

[0358] Example 5: Cell binding assay

[0359] 293F-EGFR and 293-cMet cells overexpressing human EGFR and human cMet, respectively (293F cells were purchased from ATCC cell bank, catalog number: CRL-1573. Based on these cells, stable cell lines overexpressing human EGFR and human cMet were independently constructed. These two cell lines were used to detect the binding affinity of the EGFR and cMet ends of the bispecific antibody, respectively. The amino acid sequence of human EGFR is from NCBI accession number NP_005219.2, and the amino acid sequence of human cMet is from NCBI accession number NP_000236.2) were co-infected with different concentrations of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, JNJ-372 and anti-KLH, respectively. IgG1 antibody (starting concentration of 25 μg / ml, 4-fold dilution, a total of 10 concentration gradients) was incubated at 4 ° C for 30 minutes, and then the unbound antibody was washed with staining buffer (PBS + 1v / v% FBS) and incubated with 5‰ (v / v) of fluorescent secondary antibody goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) prepared in staining buffer at 4 ° C for 30 minutes in the dark. Finally, the cells were collected using a flow cytometer (BD company, C6 PLUS model) to detect the fluorescent antibody bound to the cell surface. The raw data were analyzed with FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad and the EC was calculated. 50 .

[0360] NCI-H1975 cells (purchased from Shanghai No. 1 Biotechnology Co., Ltd., No. C01-HE, endogenously expressing human EGFR and human cMet, used to detect the synergistic 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 antibodies (starting at 100 μg / ml, 4-fold dilution, first 7 concentrations; 10-fold dilution, last 5 concentrations) at 4°C for 30 min. Unbound antibodies were then washed away with staining buffer (PBS + 1 v / v% FBS) and incubated with 5‰ (v / v) of fluorescent secondary antibody goat anti-human IgG PE (Southern Biotech, Cat#2040-09) prepared in staining buffer at 4°C in the dark for 30 min. Finally, cells were collected using a flow cytometer (BD, C6 PLUS model) to detect fluorescent antibodies bound to the cell surface. The raw data were analyzed using FlowJo to obtain the MFI value, and the antibody dose-dependent binding curve was fitted using GraphPad to calculate the EC 50 .

[0361] As shown in Figures 10A to 10C, the binding EC of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 to 293F-EGFR cells 50 The EC values ​​for binding to 293F-cMet cells were 100.7 ng / ml, 85.02 ng / ml, and 57.40 ng / ml, respectively. 50 The EC values ​​for binding to H1975 cells were 127.7 ng / ml, 87.39 ng / ml, and 46.65 ng / ml, respectively. 50 The binding affinity of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF to single antigen EGFR or cMet was slightly weaker than that of the positive control JNJ-372, but the binding affinity to dual antigen EGFR & cMet was significantly better than that of the positive control JNJ-372.

[0362] Example 6: Phosphorylation inhibition experiment

[0363] NCI-H1975 cells were digested and centrifuged and then resuspended in complete culture medium (RPMI-1640 medium + 10v / v% FBS) at a rate of 2×10 4Cells were seeded per well in a 96-well flat-bottom plate (Corning, Catalog No. 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 the cells were incubated in a CO2 incubator overnight. On the third day, 2× assay concentrations of antibodies E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 were prepared in serum-free RPMI-1640 medium (EGF (R&D, Catalog No. 236-EG-200) activation starting concentration 6 μM, three-fold dilutions, a total of 11 concentrations; HGF (R&D, Catalog No. 294-HG-100 / CF) activation starting concentration 660 nM, three-fold dilutions, a total of 12 concentrations). After discarding the culture medium from the 96-well plate, add 50 μl of the diluted antibody and preincubate at 37°C for 30 minutes. Prepare 2× the assay concentration of the agonist, EGF (8 ng / ml) or HGF (140 ng / ml), in serum-free RPMI-1640 medium and place in the incubator until ready for use. After preincubation, remove the 96-well flat-bottom plate from the incubator and add 50 μl of the diluted EGF or HGF agonist solution per well. Incubate at 37°C for an additional 30 minutes. After incubation, remove the 96-well flat-bottom plate, discard the supernatant, and add 50 μl of 1× lysis buffer according to the KITs instructions (phospho-AKT (Ser473) kit (Cisbio, Catalog No.: 64AKSPEH) & Advanced phospho-ERK1 / 2 (Thr202 / Tyr204) (Cisbio, Catalog No.: 64AERPEH)). After shaking at room temperature at 350 rpm for 30 min, add 16 μl of cell lysate and 4 μl of the mixed antibody in the KITs to HTRF 96-well low volume plates (Cisbio, Catalog No.: 66PL96025). After gently mixing with a pipette tip, seal the plate with a film and centrifuge at 1000 rpm for 30 s. Finally, the plate was incubated at room temperature (20°C-25°C) in the dark and read using a microplate reader (BioTek, Synergy H1 model) within 4-24 hours. The absorbance (A) at wavelengths of 655 nm and 620 nm was measured and their ratio (Ratio 665 / 620) was calculated using the formula: Ratio 665 / 620 = A655 / A620 × 10000. GraphPad Prism software was used to analyze the data for Ratio 665 / 620.

[0364] As shown in Figures 11A to 11E , under EGF activation conditions, E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 inhibited AKT phosphorylation at the IC 50 The values ​​were 4.370nM, 2.837nM, and 22.41nM, respectively; the IC values ​​for inhibition of ERK phosphorylation 50 The values ​​were 15.51nM, 10.49nM, and 53.58nM, respectively. The results show that under EGF activation conditions, E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF have better inhibitory activities on AKT and ERK phosphorylation than the positive control JNJ-372. Under HGF activation conditions, the IC values ​​of E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 for inhibiting AKT phosphorylation were 15.51nM, 10.49nM, and 53.58nM, respectively. 50 The values ​​were 0.5869nM, 0.3944nM, and 0.8861nM, respectively; the IC values ​​for inhibition of ERK phosphorylation were 50 The values ​​were 0.9124nM, 0.9498nM, and 1.617nM, respectively. The results show that under HGF activation conditions, 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.

[0365] Example 7: ADCC activity experiment

[0366] Reporter gene method: NCI-H1975 cells were cultured at a rate of 5×10 4 Cells were seeded into each well of a 96-well white plate (Costar, Cat. No. 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 antibodies E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 diluted in assay buffer (RPMI-1640 medium + 2v / v% FBS) were added to each well (starting concentration was 50 μg / ml, 3-fold dilution, first 3 concentration gradients; 5-fold dilution, last 7 concentration gradients) and pre-incubated with target cells for 30 minutes. After pre-incubation, 40 μl of effector cells Jurkat ADCC expressing NFAT-Luc and FcγRIIIa resuspended in assay buffer (RPMI-1640 medium + 2v / v% FBS) (1×10 per well) were added to each well. 5Cells were plated and incubated at 37°C for 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, M1000pro). Higher fluorescence values ​​indicate stronger ADCC activity. Dose-dependent ADCC curves were fitted using GraphPad.

[0367] LDH method: First, PBMC effector cells were revived and placed in a CO2 incubator for overnight recovery. Target cells NCI-H1975 were digested and centrifuged, the supernatant was discarded, and the cell density was adjusted to 4 × 10 cells / mL using phenol red-free RPMI Medium 1640 + 1 v / v% FBS. 5 cells / ml. Add 50 μl of target cells to each well of a 96-well low-adsorption plate (Costar, Cat. No. 7007). Then dilute the antibodies E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, JNJ-372 and anti-KLH IgG1 with phenol red-free RPMI Medium 1640 + 1% FBS medium to a 4× assay concentration (antibody starting concentration 66.67 μg / ml, 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 minutes. Take out the PBMCs that have been revived overnight and adjust the density of the PBMCs to 5×10 6 cells / ml (effector-target ratio of 25:1). 100 μl of PBMCs were added to each well of a 96-well low-adhesion plate and co-cultured in a CO2 incubator for 4 hours. Half an hour before the end of the co-culture period, 20 μl of cell lysis buffer from the LDH-cytotoxicity kit (BioVision, Cat. No. K311-400) was added to each well of the Tmax group. The 96-well low-adhesion plate was then returned to the incubator for further incubation. The 96-well low-adhesion plate was removed and centrifuged (1500 rpm for 5 minutes). 50 μl of the culture supernatant was aspirated into each well of a new 96-well flat-bottom plate (Corning, Cat. No. 3599). 50 μl of the diluted Dye Solution from the LDH-cytotoxicity kit was then added. The plate was incubated on a shaker in the dark for 10-30 minutes. The plate was then read using a multi-function microplate reader (TECAN, Model M1000 pro) and data were analyzed using GraphPad Prism software. The killing calculation formula is: ADCC%=[(Sample-Buffer) / (Tmax-Ts)]×100.

[0368] As shown in Figures 12A and 12B, in the reporter gene assay, both E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF had ADCC activity, and their EC 50 The ADCC activities of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF were stronger than that of the positive control JNJ-372.

[0369] As shown in Figure 12C, when PBMCs were used as effector cells and NCI-H1975 cells were used as target cells, E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 could induce significant ADCC effects, with their EC 50 The ADCC activity of E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF was stronger than that of the positive control JNJ-372.

[0370] Example 8: Endocytosis experiment

[0371] MKN45 cells (purchased from Shanghai Nuobai Biotechnology Co., Ltd., No. C01-1C, endogenously expressing human EGFR and human cMet) were digested and centrifuged, and then resuspended in staining buffer (PBS + 1v / v% FBS). 2×10 5Cells were seeded in a 96-well round-bottom plate (Corning, Cat. No. 3799) and incubated at 4°C for 30 min with antibodies E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, JNJ-372, and anti-KLH IgG1 (starting at 100 μg / ml, 4-fold dilution, 11 concentration steps) diluted in staining buffer (PBS + 1 v / v% FBS). Unbound antibodies were then washed away with staining buffer (PBS + 1 v / v% FBS), and the cells were resuspended in 200 μl of complete culture medium (RPMI-1640 medium + 10 v / v% FBS) and split into two, with one portion incubated at 4°C for another 24 h and the other incubated at 37°C for 24 h. After the incubation period, the supernatant was centrifuged and discarded. A fluorescent secondary antibody, goat anti-human IgG PE (Southern Biotech, Cat#2040-09), containing 5‰ (v / v) in staining buffer was added and incubated at 4°C in the dark for 30 minutes. Unbound antibody was then washed away with staining buffer (PBS + 1% v / v FBS). After fixation with PFA (Absin, Cat# abs9179), cells were collected on a flow cytometer (BD Biosciences, C6 PLUS model) to detect cell surface-bound fluorescent antibody. Raw data were analyzed using FlowJo to obtain MFI values, and GraphPad was used to fit the antibody dose-dependent binding curves to obtain Top values. The internalization rate was calculated as follows: Internalization rate (%) = (1 - Top_37°C / Top_4°C) × 100.

[0372] The results are shown in Table 5.

[0373] Table 5: Endocytosis activity detection results of MKN45 cells

[0374] The results showed that E2mut34-91A×M5-91A-FAE-LF, E2mut34-69×M5-69-FAE-LF, and JNJ-372 were all internalized by MKN45 cells, while the negative control anti-KLH IgG1 had almost no internalization activity. The results showed that E2mut34-91A×M5-91A-FAE-LF and E2mut34-69×M5-69-FAE-LF had comparable endocytic activity, with a 24-hour endocytic rate of 72%, which was superior to JNJ-372 (24-hour endocytic rate of 49%).

[0375] Example 9: H1975 transplanted tumor model

[0376] This example evaluates the inhibitory effect of E2mut34-91A×M5-91A-FAE-LF of the present invention on the CB-17 SCID mouse transplanted human lung adenocarcinoma cell NCI-H1975 model.

[0377] 1. Testing process

[0378] Female CB-17 SCID mice aged 6-8 weeks (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., animal qualification certificate number: 20170012022482) were inoculated subcutaneously on the right side with 5×10 6 NCI-H1975 cells. The average tumor volume is approximately 164 mm 3 At the same time, appropriate animals were selected and randomly divided into 5 groups according to tumor volume, with 6 animals in each group. They were:

[0379] G1 normal saline control group (solvent control group);

[0380] G2 JNJ-372 (5 mg / kg) group (positive control group);

[0381] G3 E2mut34-91A×M5-91A-FAE-LF (5 mg / kg) group (treatment group).

[0382] Intraperitoneal injection, twice a week, for 3 consecutive weeks, the experiment was terminated 3 days after the last dose. Tumor volume and body weight were measured twice a week, and the weight and tumor volume of the mice were recorded. At the end of the experiment, the mice were euthanized and the relative tumor growth rate was calculated, TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100%. (Ti: mean tumor volume of the treatment group on the i-th day of administration, T0: mean tumor volume of the treatment group on the 0th day of administration; Vi: mean tumor volume of the solvent control group on the i-th day of administration, V0: mean tumor volume of the solvent control group on the 0th day of administration).

[0383] The results are shown in Table 6 and Figure 13.

[0384] Table 6: Efficacy analysis of each group in the NCI-H1975 human lung cancer subcutaneous tumor model

[0385] Note: 1. Data are expressed as "mean ± standard error";

[0386] 2.TGI%=[1-(Ti-T0) / (Vi-V0)]×100%;

[0387] 3. P values ​​were obtained by comparing the tumor volumes of each group using the T test method.

[0388] The results showed that on the 20th day after administration, the average tumor volume of the saline control group was 3374 mm3 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%, which significantly inhibited 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 saline control group, the tumor inhibition rate was 83.0%, significantly inhibiting tumor growth. The results showed that in the CB-17 SCID mouse transplanted NCI-H1975 model, at a dose level of 5 mg / kg, E2mut34-91A×M5-91A-FAE-LF had a significant tumor inhibitory effect.

[0389] Preparation Example 3: Design and preparation of the second batch of EGFR×Met bispecific antibody mutants

[0390] 1. Construction of bispecific antibody molecules

[0391] Ten high-affinity EGFR antibody heavy chains prepared in Preparation Example 1 above were used as the heavy chains of the EGFR-terminal parent antibody of the EGFR×Met bispecific antibody. In addition, the heavy chain of a previously disclosed Met antibody (SEQ ID NO: 410 in CN105705519A) was modified in the constant region to meet the requirements of bispecific antibody preparation and 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.

[0392] The heavy and light chains in Code Table 7 were synthesized by Qingke Biotechnology, and the method was similar to Preparation Example 2.

[0393] Table 7: Names and combinations of mutant heavy and light chains

[0394] 2. Transient protein expression and purification

[0395] The required heavy chains and light chains are shown in Table 7. Among them:

[0396] 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 were used for subsequent preparation of bispecific antibodies (in vitro recombinant method);

[0397] E2mut1-91Q×M5-91Q-KIH-LF, E2mut2-91Q×M5-91Q-KIH-LF, E2mut5-91Q×M5-91Q-KIH-LF, E2m ut12-91Q×M5-91Q-KIH-LF, E2mut13-91Q×M5-91Q-KIH-LF, E2mut16-91Q×M5-91Q-KIH-LF, E2mu t17-91Q×M5-91Q-KIH-LF, E2mut25-91Q×M5-91Q-KIH-LF, E2mut34-91Q×M5-91Q-KIH-LF, and E2mut38-91Q×M5-91Q-KIH-LF used common light chains and "knob-into-hole" technology (see WO1996027011A1) to prepare bispecific antibodies.

[0398] 2.1 Expression of bispecific antibody molecules

[0399] Count the CHO-K1 cells in culture (owned by Suzhou Junmeng). When the cell density is 2-6*106 / ml, use CD CHO (purchased from Thermofisher, catalog number: 12490-001) medium for passage expansion. Dilute the cell density to 1.8-2.5*106 / ml the day before transfection. Transfection is performed the next day when the cell density reaches about 3.5-5.0*106 / ml. First, add one-tenth of the transfection volume of CD CHO medium, add 1-2μg / ml of plasmid (made by the company) according to the combination table shown in Table 2, and finally add 3-14μg / 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. The transfected mixture is placed in a shaker for culture. On the first day after transfection, 6% of glycoformulator (purchased from Aupromycin, catalog number: R170026) was added, and 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) were supplemented. Then, the culture medium was supplemented every two days, and samples were collected 5-9 days after transfection.

[0400] 3. In vitro Reconstitution and Purification

[0401] 3.1 Affinity capture of bispecific antibody molecules

[0402] After the incubation period, the cell supernatant was collected by centrifugation at 1000 g for 5 min in a floor-standing centrifuge (ThermoFisher, R404A) and the precipitate was discarded. The supernatant was then collected by centrifugation at 8000 g for 30 min and sterile filtered using a 0.22 μm filter cup (Jet, FPE-214-000). Purification was performed using a protein purifier (GE, AKTA Avant). A Mabselect Sure LX column (Cytiva, 17547403) was equilibrated with PBS equilibration buffer (Wuxi Aorui Dongyuan Biotechnology Co., Ltd., ZLI-9061). After the sample loading is completed, the sample is first eluted with affinity chromatography elution buffer A (pH 5.5, 45mM acetic acid-sodium acetate + 1M sodium chloride system), then eluted with eluent B (pH 5.5, 45mM acetic acid-sodium acetate system), and finally eluted with affinity elution buffer (pH 3.6, 10mM acetic acid-sodium acetate buffer). The sample is neutralized with 1M Tris (purchased from Merck, product number: E300016981946) buffer and the pH is adjusted to 5.5-6 for the next in vitro reconstitution.

[0403] 3.2 In vitro recombination of bispecific antibody molecules

[0404] Referring to the technical solution disclosed in WO2011131746, a bispecific antibody was prepared using Fab arm exchange technology as follows:

[0405] The two parent antibodies after affinity were concentrated and exchanged into PBS equilibration buffer (purchased from Wuxi Aorui Dongyuan Biotechnology Co., Ltd., ZLI-9061), and the protein concentration was set at 1±0.05 mg / ml.

[0406] 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, add PBS to quantify to 30 ml, then filter with a 0.22 μm filter (purchased from Sartorius, 16541-K), and finally wrap with aluminum foil and store in the dark.

[0407] To construct an incubation system: Prepare the parent antibody treated in the above steps, as shown in Table 3. Add 2 ml of the M parent antibody, 2.4 ml of the E parent antibody, and 0.489 ml of 750 mM cysteamine hydrochloride to a 15 ml centrifuge tube (Genemore, G3210015) at a molar ratio of 1:1.2. Seal the prepared incubation system with aluminum foil and incubate in a 31°C water bath (Shanghai Jinghong Laboratory Equipment Co., Ltd., DK-S28) for 3 hours, protected from light. After incubation, concentrate and replace the solution with PBS equilibration buffer and incubate at room temperature, protected from light, for 16–24 hours.

[0408] Table 8: In vitro recombination combinations of bispecific antibody mutants

[0409] 3.3 Purification of bispecific antibody molecules

[0410] Capto™ MMC Impres (Cytiva, 17371602) was used for purification. Pre-equilibration was performed using wash buffer (pH 7.5, 20 mM Tris-HCl + 1 M NaCl buffer) and equilibration was performed using equilibration buffer (pH 7.5, 20 mM Tris-HCl system). After loading, equilibration was performed using equilibration buffer for 3–6 column volumes. Finally, linear elution was performed using elution buffer (pH 7.5, 20 mM Tris-HCl + 1 M NaCl system) to collect the target protein.

[0411] Example 10: Biacore detection of binding and dissociation kinetics of dual antibody mutants

[0412] The binding affinity of the antibodies to recombinant human EGFR and human c-Met was detected using the molecular interaction analyzer Biacore T200 (GE Healthcare Life Sciences).

[0413] The method for determining the binding affinity of the antibody to human EGFR is as follows: 40 μg / mL goat anti-human IgG-Fc fragment antibody (Jackson ImmunoResearch) was coupled to the surface of a CM5 chip (Cytiva, Cat. No. BR-1005-30) to capture the antibody. 1 μg / mL ALK101 antibody was captured on the surface of the CM5 chip, and 40 nM and 10 nM human EGFR were injected to bind to the antibody. The Biacore T200 system (GE Healthcare) was used to detect the binding and dissociation kinetic signals. The affinity K was calculated by fitting the binding and dissociation curves using the Biacore T200 Evaluation Software 3.0 software. D value.

[0414] The binding affinity of the antibody to human c-Met was determined as follows: 40 μg / mL goat anti-human IgG-Fc fragment antibody (Jackson ImmunoResearch) was coupled to the surface of a CM5 chip (Cytiva, Cat. No. BR-1005-30) to capture the antibody. 1 μg / mL ALK101 antibody was captured on the CM5 chip surface, and 40 nM and 10 nM human c-Met were injected to bind to the antibody. The binding and dissociation kinetics were detected using a Biacore T200 system (GE Healthcare). The affinity K was calculated by fitting the binding and dissociation curves using Biacore T200 Evaluation Software 3.0. D value.

[0415] 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 showed binding activity to human EGFR with affinity comparable to or slightly weaker than that of JNJ-372. M5-91Q-K409R-LF showed binding activity to human c-Met with affinity comparable to that of JNJ-372. The affinities of four bivalent 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 also tested. The results showed that the affinity of the monovalent antibodies was consistent with that of the bivalent monoclonal antibodies.

[0416] Table 9: Biacore-based affinity analysis of antibody mutants

[0417] Example 11: Double-resistance mutant cell binding experiment

[0418] 293F-EGFR and 293-cMet cells were incubated 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 antibodies (starting concentration of 100 μg / ml, 5-fold dilution, a total of 12 concentration gradients) at 4°C for 30 min. Unbound antibodies were then washed away with staining buffer (PBS + 1 v / v% FBS) and incubated with 5‰ (v / v) fluorescent secondary antibody goat anti-human IgG PE (Southern Biotech, Cat#2040-09) prepared in staining buffer at 4°C in the dark for 30 min. Finally, cells were collected using a flow cytometer (BD, C6 PLUS model) and fluorescent antibodies bound to the cell surface were detected. Raw data were analyzed using FlowJo to obtain MFI values, and antibody dose-dependent binding curves were fitted using GraphPad to calculate EC50.

[0419] MKN45 cells (which endogenously express both human EGFR and human cMet to test the synergistic binding affinity of the bispecific antibody) were incubated with various concentrations of JNJ372, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and anti-KLH IgG1 (starting at 100 μg / ml, diluted fourfold over 11 concentrations) at 4°C for 30 min. Unbound antibodies were then washed away with staining buffer (PBS + 1% v / v FBS) and incubated with 5‰ (v / v) of the fluorescent secondary antibody, goat anti-human IgG PE (Southern Biotech, Cat#2040-09), in staining buffer at 4°C for 30 min in the dark. Cells were harvested and analyzed using a flow cytometer (BD Biosciences, C6 PLUS model) for cell surface-bound fluorescent antibodies. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curves were fitted using GraphPad to calculate the EC50.

[0420] PC-9 cells (endogenously expressing human EGFR and human cMet to test the synergistic binding affinity of the bispecific antibody) were incubated with various concentrations of JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and anti-KLH IgG1 (starting at 25 μg / ml, diluted fourfold over a total of eight concentrations) at 4°C for 30 min. Unbound antibodies were then washed away with staining buffer (PBS + 1% v / v FBS) and incubated with 5‰ (v / v) of the fluorescent secondary antibody, goat anti-human IgG PE (Southern Biotech, Cat#2040-09), in staining buffer at 4°C for 30 min in the dark. Cells were harvested and analyzed using a flow cytometer (BD Biosciences, C6 PLUS model) for cell surface-bound fluorescent antibodies. The raw data were analyzed by FlowJo to obtain the MFI value, and the antibody dose-dependent binding curves were fitted using GraphPad to calculate the EC50.

[0421] As shown in Figures 10D to 10G , the EC50s for binding 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 and 17708, respectively. , 23177, 19445, 23508; JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, E2mut25-91Q×M5-91Q-LF and 293F-CMet cells had binding EC50 values ​​of 24.13 ng / ml, 25.80 ng / ml, 26.95 ng / ml, 25.99 ng / ml and 28.22 ng / ml, respectively; JNJ372, E2mut The EC50s of the binding of E2mut1-91Q×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 EC50s of the binding 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 and 14 9.2ng / ml, 194.0ng / ml; 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; the binding affinity to the dual antigen EGFR&cMet was comparable to that of the positive control JNJ-372.

[0422] Example 12: Phosphorylation inhibition experiment of double-antibody mutants

[0423] NCI-H1975 cells were digested and centrifuged and then resuspended in complete culture medium (RPMI-1640 medium + 10v / v% FBS) at a rate of 2×10 4Cells were seeded 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 in a CO2 incubator overnight. 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 were prepared in serum-free RPMI-1640 medium at 2× the assay concentration (starting at 500 μg / ml, diluted 3-fold, for a total of 12 concentrations). After discarding the medium from the 96-well plate, 50 μl of the diluted antibodies were added and pre-incubated at 37°C for 30 min. Prepare 2× the assay concentration of EGF agonist (8 ng / ml) in serum-free RPMI-1640 medium and place in the incubator until ready for use. After preincubation, remove the 96-well flat-bottom plate from the incubator and add 50 μl of the diluted EGF agonist solution to each well. Incubate at 37°C for an additional 30 minutes. After incubation, remove the 96-well flat-bottom plate, discard the supernatant, and add 50 μl of 1× lysis buffer according to the KITs instructions (phospho-AKT (Ser473) kit (Cisbio, Catalog No.: 64AKSPEH) & Advanced phospho-ERK1 / 2 (Thr202 / Tyr204) (Cisbio, Catalog No.: 64AERPEH)). After shaking at room temperature at 350 rpm for 30 min, add 16 μl of cell lysate and 4 μl of the mixed antibody in the KITs to HTRF 96 well low volume plates (Cisbio, Catalog No.: 66PL96025). After gently mixing with a pipette tip, seal the plate with a film and centrifuge at 1000 rpm for 30 s. Finally, the plate was incubated at room temperature (20°C-25°C) in the dark and read using a microplate reader (BioTek, Synergy H1 model) within 4-24 hours. The absorbance (A) at wavelengths of 655 nm and 620 nm was measured and their ratio (Ratio 665 / 620) was calculated using the formula: Ratio 665 / 620 = A655 / A620 × 10000. GraphPad Prism software was used to analyze the data for Ratio 665 / 620.

[0424] BxPC-3 cells were digested and centrifuged and then resuspended in complete culture medium (RPMI-1640 medium + 10 v / v% FBS) at a rate of 2×10 4Cells were seeded 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 in a CO2 incubator overnight. 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 were prepared in serum-free RPMI-1640 medium at 2× the assay concentration (starting at 20 μg / ml, diluted 3-fold, for a total of 10 concentrations). After discarding the medium from the 96-well plate, 50 μl of the diluted antibodies were added and pre-incubated at 37°C for 30 min. Prepare 2× the assay concentration of HGF agonist (300 ng / ml) in serum-free RPMI-1640 medium and place in the incubator until ready for use. After pre-incubation, remove the 96-well flat-bottom plate from the incubator and add 50 μl of the diluted HGF agonist solution to each well. Incubate at 37°C for an additional 30 minutes. After incubation, remove the 96-well flat-bottom plate, discard the supernatant, and add 50 μl of 1× lysis buffer according to the KITs instructions (phospho-AKT (Ser473) kit (Cisbio, Catalog No.: 64AKSPEH) & Advanced phospho-ERK1 / 2 (Thr202 / Tyr204) (Cisbio, Catalog No.: 64AERPEH)). After shaking at room temperature at 350 rpm for 30 min, add 16 μl of cell lysate and 4 μl of the mixed antibody in the KITs to HTRF 96 well low volume plates (Cisbio, Catalog No.: 66PL96025). After gently mixing with a pipette tip, seal the plate with a film and centrifuge at 1000 rpm for 30 s. Finally, the plate was incubated at room temperature (20°C-25°C) in the dark and read using a microplate reader (BioTek, Synergy H1 model) within 4-24 hours. The absorbance (A) at wavelengths of 655 nm and 620 nm was measured and their ratio (Ratio 665 / 620) was calculated using the formula: Ratio 665 / 620 = A655 / A620 × 10000. GraphPad Prism software was used to analyze the data for Ratio 665 / 620.

[0425] As shown in Figures 11F to 11M , under EGF-activated conditions, JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and E2mut25-91Q×M5-91Q-LF inhibited AKT phosphorylation with the IC . 50 The values ​​were 1377ng / ml, 464713ng / ml, 2988ng / ml, 8899ng / ml, and 111137ng / ml, respectively; the IC values ​​for inhibition of ERK phosphorylation 50 The values ​​were 3383ng / ml, 4711189ng / ml, 8496ng / ml, 42459ng / ml, and 575652ng / ml, respectively. The results show that under EGF activation conditions, the inhibitory activity of E2mut12-91Q×M5-91Q-LF on AKT and ERK phosphorylation was slightly weaker than that of the positive control JNJ-372, while the inhibitory activities of E2mut1-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and E2mut25-91Q×M5-91Q-LF on AKT and ERK phosphorylation were significantly weaker than those of the positive control JNJ-372. Under HGF activation conditions, JNJ372, E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and E2mut25-91Q×M5-91Q-LF inhibited AKT phosphorylation at the IC 50 The values ​​were 296.9ng / ml, 410.4ng / ml, 268.3ng / ml, 404.5ng / ml, and 766.3ng / ml, respectively; the IC values ​​for inhibition of ERK phosphorylation 50 The values ​​were 457.8ng / ml, 547.6ng / ml, 517.9ng / ml, 535.0ng / ml, and 1312ng / ml, respectively. The results show that under HGF activation conditions, the inhibitory activities of E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, and E2mut13-91Q×M5-91Q on AKT and ERK phosphorylation were comparable to those of the positive control JNJ-372, while the inhibitory activities of E2mut25-91Q×M5-91Q-LF on AKT and ERK phosphorylation were slightly weaker than those of the positive control JNJ-372.

[0426] Example 13 ADCC activity experiment of dual-antibody mutant

[0427] Reporter gene method: NCI-H1975 cells were cultured at a rate of 5×10 4Cells were seeded into each well of a 96-well plate (Costar, Cat. No. 3917) and incubated overnight in a CO2 incubator. The next day, the 96-well plate was removed from the CO2 incubator, the culture supernatant was removed, and 40 μl of antibodies diluted in assay buffer (RPMI-1640 medium + 2% FBS) was added to each well. Antibodies included E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, JNJ-372, and anti-KLH IgG1 (starting at 10 μg / ml, four-fold dilutions, and 10 concentration steps) were preincubated with target cells for 30 minutes. After pre-incubation, 40 μl of Jurkat ADCC cells expressing NFAT-Luc and FcγRIIIa (1×10 per well) resuspended in assay buffer (RPMI-1640 medium + 2 v / v% FBS) were added to each well. 5 Cells were plated and incubated at 37°C for 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, M1000pro). Higher fluorescence values ​​indicate stronger ADCC activity. Dose-dependent ADCC curves were fitted using GraphPad.

[0428] LDH method: First, PBMC effector cells were revived and placed in a CO2 incubator for overnight recovery. Target cells NCI-H1975 were digested and centrifuged, the supernatant was discarded, and the cell density was adjusted to 4 × 10 cells / mL using phenol red-free RPMI Medium 1640 + 1 v / v% FBS. 5 cells / ml. Add 50 μl of target cells to each well of a 96-well low-adsorption plate (Costar, Cat. No. 7007). Then dilute the antibody with phenol red-free RPMI Medium 1640 + 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 to a 4× assay concentration (antibody starting concentration 66.67 μg / ml, 2 concentration gradients before 3-fold dilution; 7 concentration gradients after 5-fold dilution). Add 50 μl of diluted antibody solution to each well, gently pipet and mix several times, and incubate in the incubator for 30 minutes. Take out the PBMCs that have been revived overnight and adjust the density of PBMCs to 5×10 6cells / ml (effector-target ratio of 25:1). 100 μl of PBMCs were added to each well of a 96-well low-adhesion plate and co-cultured in a CO2 incubator for 4 hours. Half an hour before the end of the co-culture period, 20 μl of cell lysis buffer from the LDH-cytotoxicity kit (BioVision, Cat. No. K311-400) was added to each well of the Tmax group. The 96-well low-adhesion plate was then returned to the incubator for further incubation. The 96-well low-adhesion plate was removed and centrifuged (1500 rpm for 5 minutes). 50 μl of the culture supernatant was aspirated into each well of a new 96-well flat-bottom plate (Corning, Cat. No. 3599). 50 μl of the diluted Dye Solution from the LDH-cytotoxicity kit was then added. The plate was incubated on a shaker in the dark for 10-30 minutes. The plate was then read using a multi-function microplate reader (TECAN, Model M1000 pro) and data were analyzed using GraphPad Prism software. The killing calculation formula is: ADCC%=[(Sample-Buffer) / (Tmax-Ts)]×100.

[0429] As shown in Figure 12D , 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 EC 50 The ADCC activity was slightly weaker than that of the positive control JNJ-372.

[0430] As shown in Figure 12E , when PBMCs were used as effector cells and NCI-H1975 cells were used 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 The ADCC activity of the positive control JNJ-372 was comparable to that of the positive control anti-KLH IgG1, while the negative control anti-KLH IgG1 could not induce ADCC effect.

[0431] Example 14: Double-resistance mutant endocytosis experiment

[0432] MKN45 cells were digested and centrifuged, and then resuspended in staining buffer (PBS + 1v / v% FBS). 2×10 5Cells were seeded in a 96-well round-bottom plate (Corning, Cat. No. 3799) and incubated at 4°C for 30 min with antibodies E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and JNJ-372 (25 μg / ml) diluted in staining buffer (PBS + 1 v / v% FBS). Unbound antibodies were then washed away with staining buffer (PBS + 1 v / v% FBS), and the cells were resuspended in 200 μl of complete culture medium (RPMI-1640 medium + 10 v / v% FBS) and split in half. One aliquot was incubated at 4°C for another 24 h, while the other aliquot was incubated at 37°C for 24 h. After the incubation period, the supernatant was discarded by centrifugation, and a fluorescent secondary antibody, goat anti-human IgG PE (Southern Biotech, Cat#2040-09), containing 5‰ (v / v) in staining buffer was added and incubated at 4°C in the dark for 30 minutes. Unbound antibody was then washed away with staining buffer (PBS + 1% v / v FBS). After fixation with PFA (Absin, Cat# abs9179), cells were collected on a flow cytometer (BD Biosciences, C6 PLUS model) to detect cell surface-bound fluorescent antibody. Raw data were analyzed using FlowJo to obtain MFI values, and GraphPad was used to fit the antibody dose-dependent binding curve to obtain Top values. The internalization rate was calculated as follows: Internalization rate (%) = (1 - Top_37°C / Top_4°C) × 100.

[0433] The results are shown in Table 10.

[0434] Table 10: Endocytic activity detection results of MKN45 cells 2

[0435] The results showed that E2mut12-91Q×M5-91Q-FAE-LF, E2mut13-91Q×M5-91Q-FAE-LF and JNJ-372 could all be internalized by MKN45 cells, and their endocytic activities were comparable, with 24h endocytic rates of 75%, 84% and 80%, respectively.

[0436] PC-9 cells were digested and centrifuged, and then resuspended in staining buffer (PBS + 1v / v% FBS). 2×10 5Cells were seeded in a 96-well round-bottom plate (Corning, Cat. No. 3799) and incubated at 4°C for 30 min with antibodies E2mut1-91Q×M5-91Q-LF, E2mut12-91Q×M5-91Q-LF, E2mut13-91Q×M5-91Q-LF, and JNJ-372 (25 μg / ml) diluted in staining buffer (PBS + 1 v / v% FBS). Unbound antibodies were then washed away with staining buffer (PBS + 1 v / v% FBS), and the cells were resuspended in 200 μl of complete culture medium (RPMI-1640 medium + 10 v / v% FBS) and split into two, one portion incubated at 4°C for another 24 h, and the other portion incubated at 37°C for 24 h. After the incubation period, the supernatant was discarded by centrifugation, and a fluorescent secondary antibody, goat anti-human IgG PE (Southern Biotech, Cat#2040-09), containing 5‰ (v / v) in staining buffer was added and incubated at 4°C in the dark for 30 minutes. Unbound antibody was then washed away with staining buffer (PBS + 1% v / v FBS). After fixation with PFA (Absin, Cat# abs9179), cells were collected on a flow cytometer (BD Biosciences, C6 PLUS model) to detect cell surface-bound fluorescent antibody. Raw data were analyzed using FlowJo to obtain MFI values, and GraphPad was used to fit the antibody dose-dependent binding curve to obtain Top values. The internalization rate was calculated as follows: Internalization rate (%) = (1 - Top_37°C / Top_4°C) × 100.

[0437] The results are shown in Table 11.

[0438] Table 11: Endocytic activity detection results of PC-9 cells

[0439] 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 internalized by MKN45 cells, and their endocytic activities were comparable, with 24-h endocytic rates of 88%, 87%, 85% and 81%, respectively.

[0440] NUGC4 cells (endogenously expressing human EGFR and human cMet) and BaF3-EGFR-cMet (overexpressing human EGFR and human cMet) were digested and centrifuged, then resuspended in staining buffer (PBS + 1 v / v% FBS) and 2×10 5Cells were seeded in a 96-well round-bottom plate (Corning, Cat. No. 3799) and incubated at 4°C for 30 min with 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 in staining buffer (PBS + 1 v / v% FBS). Unbound antibodies were then washed away with staining buffer (PBS + 1 v / v% FBS), and the cells were resuspended in 200 μl of complete culture medium (RPMI-1640 medium + 10 v / v% FBS) and split into two, one portion incubated at 4°C for another 24 h, and the other portion incubated at 37°C for 24 h. After the incubation period, the supernatant was discarded by centrifugation, and a fluorescent secondary antibody, goat anti-human IgG PE (Southern Biotech, Cat#2040-09), containing 5‰ (v / v) in staining buffer was added and incubated at 4°C in the dark for 30 minutes. Unbound antibody was then washed away with staining buffer (PBS + 1% v / v FBS). After fixation with PFA (Absin, Cat# abs9179), cells were collected on a flow cytometer (BD Biosciences, C6 PLUS model) to detect cell surface-bound fluorescent antibody. Raw data were analyzed using FlowJo to obtain MFI values, and GraphPad was used to fit the antibody dose-dependent binding curve to obtain Top values. The internalization rate was calculated as follows: Internalization rate (%) = (1 - Top_37°C / Top_4°C) × 100.

[0441] The results are shown in Tables 12 and 13.

[0442] Table 12: Endocytosis activity detection results of NUGC4 cells

[0443] Table 13: Results of endocytic activity detection of BaF3-EGFR-cMet cells

[0444] 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 internalized by NUGC4 cells and BaF3-EGFR-cMet cells.

[0445] Example 15: Double-resistant mutant H1975 transplanted tumor model

[0446] This example evaluates the inhibitory effects of E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-LF, and ALK101-31Q-LF of the present invention on the NCI-H1975 human lung adenocarcinoma cell line transplanted in NDG mice. ALK101-15Q-LF, ALK101-17Q-LF, and ALK101-31Q-LF were selected from the bispecific antibodies listed in Table 8.

[0447] 1. Testing process

[0448] Female NDG mice aged 6-8 weeks (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., animal qualification certificate number: 20170012022482) were inoculated subcutaneously on the right side with 5×10 6 NCI-H1975 cells. The average tumor volume is about 125mm 3 At the same time, appropriate animals were selected and randomly divided into 6 groups according to tumor volume, with 6 animals in each group. They are:

[0449] G1 normal saline control group (solvent control group);

[0450] G2 JNJ-372 (3 mg / kg) group (positive control group);

[0451] G3 E2mut34-91A×M5-91A-FAE-LF (3 mg / kg) group (treatment group);

[0452] G4 ALK101-15Q-LF (3 mg / kg) group (treatment group);

[0453] G5 ALK101-31Q-LF (3 mg / kg) group (treatment group);

[0454] G6 ALK101-17Q-LF (3 mg / kg) group (treatment group).

[0455] Intraperitoneal injection, twice a week, for 2 consecutive weeks, the experiment was terminated 28 days after the initial administration. Tumor volume and body weight were measured twice a week, and the 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: mean tumor volume of the treatment group on the i-th day of administration, T0: mean tumor volume of the treatment group on the 0th day of administration; Vi: mean tumor volume of the solvent control group on the i-th day of administration, V0: mean tumor volume of the solvent control group on the 0th day of administration).

[0456] The results are shown in Table 14 and Figure 14.

[0457] Table 14: Efficacy analysis of each group in the NCI-H1975 human lung cancer subcutaneous tumor model

[0458] Note: 1. Data are expressed as "mean ± standard error";

[0459] 2.TGI%=[1-(Ti-T0) / (Vi-V0)]×100%;

[0460] 3. P values ​​were obtained by comparing the tumor volumes of each group using the T test method.

[0461] The results showed that on the 28th day after 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 Compared with the normal saline control group, the tumor inhibition rate was 92.5%, which significantly inhibited tumor growth; the average tumor volume of the E2mut34-91A×M5-91A-FAE-LF (3 mg / kg) group was 223 mm 3 Compared with the normal saline control group, the tumor inhibition rate was 95.0%, significantly inhibiting tumor growth. The average tumor volume in the ALK101-15Q-LF (3 mg / kg) group was 600 mm 3 Compared with the normal saline control group, the tumor inhibition rate was 75.6%, significantly inhibiting tumor growth. The average tumor volume in the ALK101-31Q-LF (3 mg / kg) group was 348 mm 3 Compared with the normal saline control group, the tumor inhibition rate was 88.5%, significantly inhibiting tumor growth. The average tumor volume in the ALK101-17Q-LF (3 mg / kg) group was 394 mm 3 Compared with the saline control group, the tumor inhibition rate was 86.1%, significantly inhibiting tumor growth. The results showed that in the NDG mouse transplantation NCI-H1975 model, E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-L, and ALK101-31Q-LF had a significant tumor inhibitory effect at a dose level of 3 mg / kg.

[0462] Example 16: Double-resistant mutant H1975 (L858R / T790M / C797S) transplant tumor model

[0463] This example evaluates the inhibitory effects of E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-LF and ALK101-31Q-LF of the present invention on the NDG mouse transplanted human lung adenocarcinoma cell NCI-H1975 (L858R / T790M / C797S) model.

[0464] 1. Testing process

[0465] Female NDG mice aged 6-8 weeks (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., animal qualification certificate number: 20170012022482) were inoculated subcutaneously on the right side with 5×10 6 NCI-H1975 cells. The average tumor volume is approximately 186 mm 3 At the same time, appropriate animals were selected and randomly divided into 6 groups according to tumor volume, with 5 animals in each group. They are:

[0466] G1 normal saline control group (solvent control group);

[0467] G2 JNJ-372 (2 mg / kg) group (positive control group);

[0468] G3 E2mut34-91A×M5-91A-FAE-LF (2 mg / kg) group (treatment group);

[0469] G4 ALK101-15Q-LF (2 mg / kg) group (treatment group);

[0470] G5 ALK101-31Q-LF (2 mg / kg) group (treatment group);

[0471] G6 ALK101-17Q-LF (2 mg / kg) group (treatment group).

[0472] Intraperitoneal injection, twice a week, a total of 5 doses, the experiment was terminated 28 days after the initial administration. Tumor volume and body weight were measured twice a week, and the 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: mean tumor volume of the treatment group on the i-th day of administration, T0: mean tumor volume of the treatment group on the 0th day of administration; Vi: mean tumor volume of the solvent control group on the i-th day of administration, V0: mean tumor volume of the solvent control group on the 0th day of administration).

[0473] The results are shown in Table 15 and Figure 15.

[0474] Table 15: Efficacy analysis of each group in the H1975 (L858R / T790M / C797S) human lung cancer subcutaneous tumor model

[0475] Note: 1. Data are expressed as "mean ± standard error";

[0476] 2.TGI%=[1-(Ti-T0) / (Vi-V0)]×100%;

[0477] 3. P values ​​were obtained by comparing the tumor volumes of each group using the T test method.

[0478] The results showed that on the 28th day after administration, the average tumor volume of the 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%, which significantly inhibited 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 in 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 in 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 in the ALK101-17Q-LF (2 mg / kg) group was 0 mm 3 Compared with the saline control group, the tumor inhibition rate was 112.5%, significantly inhibiting tumor growth. The results showed that in the NDG mouse transplant NCI-H1975 (L858R / T790M / C797S) model, E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-L, and ALK101-31Q-LF had significant tumor inhibitory effects at a dose level of 2 mg / kg.

[0479] Example 17: Double-resistance mutant H292 transplant tumor model

[0480] This example evaluates the inhibitory effects of E2mut34-91A×M5-91A-FAE-LF, ALK101-15Q-LF, ALK101-17Q-LF and ALK101-31Q-LF of the present invention on the NDG mouse transplanted human lung adenocarcinoma cell H292 model.

[0481] 1. Testing process

[0482] Female NDG mice aged 6-8 weeks (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., animal qualification certificate number: 20170012022482) were inoculated subcutaneously on the right side with 5×10 6 H292 cells. The average tumor volume is approximately 223 mm 3 At the same time, appropriate animals were selected and randomly divided into 6 groups according to tumor volume, with 5 animals in each group. They are:

[0483] G1 normal saline control group (solvent control group);

[0484] G2 JNJ-372 (1 mg / kg) group (positive control group);

[0485] G3 E2mut34-91A × M5-91A-FAE-LF (1 mg / kg) group (treatment group);

[0486] G4 ALK101-15Q-LF (1 mg / kg) group (treatment group);

[0487] G5 ALK101-31Q-LF (1 mg / kg) group (treatment group);

[0488] G6 ALK101-17Q-LF (1 mg / kg) group (treatment group).

[0489] Intraperitoneal injection, once in the first week, twice a week starting from the second week, for a total of 6 doses, the experiment was terminated 28 days after the initial administration. Tumor volume and body weight were measured twice a week, and the 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: mean tumor volume of the treatment group on the i-th day of administration, T0: mean tumor volume of the treatment group on the 0th day of administration; Vi: mean tumor volume of the solvent control group on the i-th day of administration, V0: mean tumor volume of the solvent control group on the 0th day of administration).

[0490] The results are shown in Table 16 and Figure 16.

[0491] Table 16: Efficacy analysis of each group in the H292 human lung cancer subcutaneous tumor model

[0492] Note: 1. Data are expressed as "mean ± standard error";

[0493] 2.TGI%=[1-(Ti-T0) / (Vi-V0)]×100%;

[0494] 3. P values ​​were obtained by comparing the tumor volumes of each group using the T test method.

[0495] The results showed that on the 28th day after administration, the average tumor volume of the 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%, which significantly inhibited 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 in 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 in 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 in the ALK101-17Q-LF (1 mg / kg) group was 152 mm 3 Compared with the saline control group, the tumor inhibition rate was 104.7%, significantly inhibiting tumor growth. The results showed that in the NDG mouse transplant H292 model, E2mut34-91A×M5-91A-FAE-LF, ALK101-17Q-L, and ALK101-31Q-LF had a significant tumor inhibitory effect at a dose level of 1 mg / kg.

[0496] Example 18: Thermal stability of bispecific antibodies

[0497] Differential Scanning Fluorescence (DSF) is a method for detecting protein denaturation based on changes in intrinsic fluorescence. When a protein is folded, the emission of 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, it unfolds and the tryptophan is exposed to the liquid phase. This shifts the maximum emission, ultimately increasing the emission at 350 nm to greater than that at 330 nm. Therefore, changes in fluorescence can be used to detect the unfolding of the protein caused by temperature or chemical denaturants, allowing the calculation of the half-melting temperature (Tm). The stability of the bispecific antibody was investigated in a buffer system (20 mM histidine-histidine hydrochloride buffer, 150 mM arginine hydrochloride, pH 6.0) using a starting temperature of 20°C and an ending temperature of 95°C, with a heating rate of 1°C / min.

[0498] The bispecific antibody sample was replaced in the above buffer system, with the sample concentration controlled at approximately 130 mg / mL, and then detected using nanoDSF. 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 herein were all above 62°C, demonstrating good thermal stability.

[0499] Table 17: Thermal stability of bispecific antibodies

[0500] Note: “ / ” means it does not exist

[0501] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full 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-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.

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. 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.

16. The pharmaceutical composition according to claim 15, further comprising 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.

17. The pharmaceutical composition according to any one of claims 15 to 16, 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.

18. 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 the 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.

19. The combined pharmaceutical product according to claim 18, 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.

20. The combined pharmaceutical product according to any one of claims 18 to 19, 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.

21. A 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 described 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 described in any one of claims 1 to 11.

22. The bispecific antibody according to claim 21, wherein, the second protein functional region comprises the heavy chain variable region of an anti-Met antibody or its antigen-binding fragment; preferably it comprises the heavy chain variable region and the light chain variable region of an 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.

23. The bispecific antibody according to any one of claims 21 to 22, 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, wherein 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.

24. The bispecific antibody according to any one of claims 21 to 23, wherein, Among them, the first protein functional region and the second protein functional region are independently a fusion protein of single-chain antibodies or a half-molecule type monovalent antibody (IgG half molecule, IgG-HM).

25. The bispecific antibody according to any one of claims 21 to 24, 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.

26. The bispecific antibody according to any one of claims 21 to 25, 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.

27. The bispecific antibody according to any one of claims 24 to 26, 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.

28. The bispecific antibody according to any one of claims 24 to 27, 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).

29. The bispecific antibody according to any one of claims 24 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 according to the EU Numbering System, the 435th position of one half-molecule type monovalent antibody heavy chain constant region is mutated to amino acid R, and the 436th position is mutated to amino acid F.

30. The bispecific antibody according to any one of claims 24 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 405th position of one half-molecule type monovalent antibody heavy chain constant region is mutated to amino acid L, and the 409th position of the other half-molecule type monovalent antibody heavy chain constant region is mutated to amino acid R.

31. The bispecific antibody according to any one of claims 21 to 30, 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) Peptide chains selected from SEQ ID NO: 16 and SEQ ID NO: 20, and (3) Peptide chains selected from SEQ ID NO: 11 to SEQ ID NO: 13, and 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 connected by one or more disulfide bonds (such as 2 or 3 disulfide bonds).

32. The bispecific antibody according to any one of claims 21 to 31, 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.

33. An isolated nucleic acid molecule encoding the bispecific antibody according to any one of claims 21 to 32.

34. A recombinant expression vector comprising the isolated nucleic acid molecule according to claim 33.

35. A recombinant host cell comprising the recombinant expression vector according to claim 34, preferably, the recombinant host cell is a recombinant CHO-K1 cell.

36. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 21 to 32, and one or more pharmaceutically acceptable excipients. Use of an anti-EGFR antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, or a bispecific antibody according to any one of claims 22 to 33, in the preparation of a medicament for treating or preventing a tumor; 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, hematologic 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 non-small cell lung cancer.

38. An anti-EGFR antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, or a bispecific antibody according to any one of claims 21 to 32, for use in treating or preventing a tumor; 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, hematologic 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 non-small cell lung cancer.

39. A method for treating or preventing a tumor, comprising the step of administering to a subject in need thereof an effective amount of an anti-EGFR antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, or a bispecific antibody according to any one of claims 21 to 32; 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, hematologic 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 non-small cell lung cancer.

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