Antibody-drug conjugate of Anti-EGFR / Anti-cmet bispecific antibody

Abstract

An antibody-drug conjugate of an anti-EGFR / anti-cMET bispecific antibody. The antibody-drug conjugate comprises: a bispecific antibody or antigen-binding fragment thereof specifically binding to EGFR and cMET, and a conjugation moiety. The bispecific antibody or antigen-binding fragment thereof specifically binding to EGFR and cMET comprises: a first protein functional region which targets the EGFR antigen and a second protein functional region which targets cMET.

Description

Antibody-drug conjugates of anti-EGFR and cMET bispecific antibodies Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, this invention relates to antibody-drug conjugates of anti-EGFR and cMET bispecific antibodies. Background Technology

[0002] EGFR (Epidermal Growth Factor Receptor) is the receptor for epidermal growth factor (EGF) and belongs to the ErbB receptor family. EGFR is a transmembrane glycoprotein with a molecular weight of 170 kDa. It is a receptor-type tyrosine kinase. Under the action of related ligands such as epidermal growth factor (EGF) and transforming growth factor-α (TGFα), EGFR is activated by converting from a monomer to a dimer, thereby further activating downstream signaling pathways, such as phosphorylation of kinases like Akt and ERK, and 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 highly expressed or abnormally expressed in most tumors, such as glial carcinoma, renal cell carcinoma, 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 tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of 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 tissue, and the other is the persistent activation of EGFR mutants in tumor cells (without 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. The cMET receptor binds to its ligand, hepatocyte growth factor (HGF), inducing cMET dimerization and activating it. This activation then activates downstream signaling pathways, such as the phosphorylation of Akt and ERK kinases. cMET plays a crucial role in embryonic development, organ growth, and wound healing, and is typically expressed only in stem cells and progenitor cells. In cancer, aberrant cMET activation due to cMET mutations promotes angiogenesis and cancer metastasis.

[0004] Lung cancer is the leading cause of cancer morbidity and mortality, with non-small cell lung cancer (NSCLC) accounting for 90% of lung cancer cases. EGFR mutation is a major driving factor in NSCLC, affecting over 40% of NSCLC patients in Asia. EGFR kinase inhibitors (EGFR-TKIs) are the primary treatment; however, drug resistance rates are high and the mechanisms are complex, with 7-15% of patients developing cMET amplification. Furthermore, cMET exon 14 skipping mutations, cMET fusions, cMET amplification, and overexpression are also primary driving factors in NSCLC. Bispecific antibodies targeting both EGFR and cMET can simultaneously inhibit both signaling pathways, offering promise for patients who are unresponsive or resistant to EGFR-TKIs.

[0005] Currently, several EGFR×cMET bispecific antibodies have been publicly reported. US9328173B2 (Eli Lilly and Company), WO2018221969A1 (Chungken Takagi Biopharmaceutical Co., Ltd.), and WO2022104236A2 (Ab Theraputics) disclose the construction methods of "2+2" type EGFR×cMET bispecific antibodies, where both the anti-EGFR and anti-cMET ends are bivalent. Because EGFR antibodies have skin toxicity, "2+2" type EGFR×cMET bispecific antibodies have potentially higher safety risks. Johnson & Johnson's Amivantamab (US2017275367A1) discloses the construction method of a "1+1" type EGFR×cMET bispecific antibody, where both the anti-EGFR and anti-cMET ends are monovalent. This antibody has been approved for marketing and has shown good safety.

[0006] Constructing a "1+1" type EGFR×cMET bispecific antibody presents significant technical challenges, requiring solutions to the correct pairing of light and heavy chains and the heterodimerization of the heavy chain. As reported in US2017275367A1, Amivantamab employs Fab arm exchange technology, which eliminates light-heavy chain mismatches. However, this method cannot utilize a single cell line for production; it necessitates the separate preparation of anti-EGFR and anti-cMET antibodies, followed by in vitro recombination to obtain the bispecific antibody, increasing production costs. WO2023186092A1 discloses a method for constructing a "1+1" type asymmetric EGFR×cMET bispecific antibody using a CH1 / CL biased mutation (patent number: WO2021067404A2) to reduce light-heavy chain mismatches. While this method allows for the production of bispecific antibodies using a single cell line, it cannot completely eliminate light-heavy chain mismatches.

[0007] Antibody-drug conjugates (ADCs) are targeted chemotherapy drugs that use chemical linkers to conjugate small cytotoxic molecules to monoclonal antibodies. Utilizing the antibody's targeting ability, they deliver therapeutic drugs to antigen-positive cancer cells. Developing ADC drugs based on EGFR×cMET bispecific antibodies can superimpose the cell-killing mechanisms of chemotherapy drugs onto anti-tumor mechanisms such as signal blocking, receptor downregulation, and Fc effects, potentially leading to better anti-tumor efficacy. However, currently, no EGFR×cMET bispecific antibody ADC drugs are on the market.

[0008] WO2023083846A1 and WO2024002235A1 disclose two "1+1" type EGFR×cMET bispecific antibody ADCs. Compared with Amivantamab, the two disclosed bispecific antibody ADCs have weaker affinity for human antigens. For example, the EGFR antibody RAA22 reported in WO2023083846A1 has an affinity of 45 nM; the EGFR antibody 6C4 reported in WO2024002235A1 has an affinity of 62.7 nM, and the cMET antibody 2F11 has an affinity of 54.2 nM. Weaker affinity weakens the antibody's signal blocking function and affects its antitumor activity.

[0009] Therefore, there is an urgent need in this field to develop an effective EGFR×cMET bispecific antibody-drug conjugate (ADC) drug, while also improving the affinity of the ADC drug for EGFR to enhance its anti-tumor activity. Summary of the Invention

[0010] The purpose of this invention is to provide antibody-drug conjugates containing bispecific antibodies against EGFR and cMET.

[0011] In a first aspect of the present invention, an antibody-drug conjugate of a bispecific antibody against EGFR and cMET is provided, comprising a bispecific antibody or an antigen-binding fragment thereof that specifically binds to EGFR and cMET and a conjugation portion, wherein the bispecific antibody or the antigen-binding fragment thereof that specifically binds to EGFR and cMET comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0012] The first protein functional region targeting EGFR includes:

[0013] HCDR1, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, or differs from the amino acid sequence shown in SEQ ID NO:25 or 26 by 1, 2 or 3 amino acids;

[0014] HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:27, or differs from the amino acid sequence shown in SEQ ID NO:27 by 1, 2 or 3 amino acids;

[0015] HCDR3, whose amino acid sequence is shown in SEQ ID NO:37 (ARVSX1YX2DSX3FD), 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;

[0016] LCDR1 has the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2 or 3 amino acids.

[0017] LCDR2, whose amino acid sequence is the same as that shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:14 by 1, 2, or 3 amino acids; and

[0018] LCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:15, 20 or 21, or differs from the amino acid sequence shown in SEQ ID NO:15, 20 or 21 by 1, 2 or 3 amino acids; and / or,

[0019] The second protein functional region targeting cMET includes:

[0020] HCDR1 has the amino acid sequence shown in SEQ ID NO:22, or differs from the amino acid sequence shown in SEQ ID NO:22 by 1, 2 or 3 amino acids.

[0021] HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:23, or differs from the amino acid sequence shown in SEQ ID NO:23 by 1, 2 or 3 amino acids;

[0022] HCDR3 has the amino acid sequence shown in SEQ ID NO:24, or differs from the amino acid sequence shown in SEQ ID NO:24 by 1, 2 or 3 amino acids.

[0023] LCDR1 has the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2 or 3 amino acids.

[0024] LCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:14 by 1, 2 or 3 amino acids.

[0025] And LCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:15, 20 or 21, or differs from the amino acid sequence shown in SEQ ID NO:15, 20 or 21 by 1, 2 or 3 amino acids.

[0026] In one or more embodiments, the first protein functional region targeting EGFR includes:

[0027] HCDR1, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, or differs from the amino acid sequence shown in SEQ ID NO:25 or 26 by 1, 2 or 3 amino acids;

[0028] HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:27, or differs from the amino acid sequence shown in SEQ ID NO:27 by 1, 2 or 3 amino acids;

[0029] HCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35 or 36, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35 or 36;

[0030] LCDR1 has the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2 or 3 amino acids.

[0031] LCDR2, whose amino acid sequence is the same as that shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:14 by 1, 2, or 3 amino acids; and

[0032] LCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:15, 20 or 21, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO:15, 20 or 21.

[0033] In one or more embodiments, the first protein functional region targeting EGFR includes a heavy chain variable region, the heavy chain variable region comprising:

[0034] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:35, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:35 by 1, 2, or 3 amino acids; or

[0035] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:28, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:28 by 1, 2, or 3 amino acids; or

[0036] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:31, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:31; or

[0037] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29 by 1, 2, or 3 amino acids; or

[0038] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:30, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:30; or

[0039] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:32, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:32; or

[0040] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:33, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:33; or

[0041] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:33, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:33; or

[0042] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:34, respectively; or HCDR1, HCDR2, and HCDR3 having a difference of 1, 2, or 3 amino acids from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:34; or

[0043] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:36, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:36.

[0044] In one or more embodiments, the first protein functional region targeting EGFR includes a light chain variable region, the light chain variable region comprising:

[0045] The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; or

[0046] The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20; or

[0047] The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21.

[0048] In one or more embodiments, the first protein functional region targeting EGFR includes a heavy chain variable region and a light chain variable region, wherein:

[0049] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, the amino acid sequence of HCDR2 is the amino acid sequence shown in SEQ ID NO:27, and the amino acid sequence of HCDR3 is selected from the amino acid sequences shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, or 36; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is the amino acid sequence shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is the amino acid sequence shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is selected from the amino acid sequences shown in SEQ ID NO:15, 20, or 21.

[0050] In one or more embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:15; or

[0051] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:28; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0052] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:31; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0053] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:20; or

[0054] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:29; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0055] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:30; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0056] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:32; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0057] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in 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:33; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0058] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:33; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0059] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:34; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0060] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0061] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:36; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21.

[0062] In one or more embodiments, the second protein functional region targeting cMET comprises a heavy chain variable region and a light chain variable region, wherein:

[0063] The heavy chain variable region comprises: HCDR1, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:22, or differs from the amino acid sequence shown in SEQ ID NO:22 by 1, 2, or 3 amino acids; HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:23, or differs from the amino acid sequence shown in SEQ ID NO:23 by 1, 2, or 3 amino acids; and HCDR3, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:24, or differs from the amino acid sequence shown in SEQ ID NO:24 by 1, 2, or 3 amino acids.

[0064] The light chain variable region comprises: LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; or

[0065] The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21; or

[0066] The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20.

[0067] In one or more embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:24; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:15; or

[0068] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:24; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:20; or

[0069] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:24; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21.

[0070] In one or more embodiments, the first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 4, 9, 2, 3, 5, 6, 7, 8, 10, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1, 4, 9, 2, 3, 5, 6, 7, 8, 10; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 39, 42, or 40, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39, 42, or 40; and / or,

[0071] The second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:41, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:41. The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:39, 42, or 40, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:39, 42, or 40.

[0072] In one or more embodiments, the first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 1, 4, 9, 2, 3, 5, 6, 7, 8, 10; and the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 39, 42, or 40; and / or, the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 41; and the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 39, 42, or 40.

[0073] In one or more embodiments, the first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:39, 40, or 42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:39, 40, or 42; or

[0074] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO:1, 4, 9, 2, 3, 5, 6, 7, 8, 10; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:41; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:42.

[0075] In one or more embodiments, the first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO:18, 52, 55, 53, 54, 56, 57, 58, 59, 60, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence shown in any one of SEQ ID NO:18, 52, 55, 53, 54, 56, 57, 58, 59, 60; the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12, or 38, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence shown in any one of SEQ ID NO:11, 12, or 38; and / or,

[0076] The second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain. The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19. The light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12, or 38, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:11, 12, or 38.

[0077] In one or more embodiments, the first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO:18, and the light chain comprising the amino acid sequence shown in any one of SEQ ID NO:11, 12, or 38; or the first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in any one of SEQ ID NO:52, 55, 53, 54, 56, 57, 58, 59, or 60, and the light chain comprising the amino acid sequence shown in any one of SEQ ID NO:38; and / or,

[0078] The second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19, and the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12, or 38.

[0079] In one or more embodiments, the first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO:18, and the light chain comprising the amino acid sequence shown in SEQ ID NO:11; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO:19, and the light chain comprising the amino acid sequence shown in SEQ ID NO:11; or

[0080] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:52 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0081] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:55 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0082] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:18, and the light chain comprises the amino acid sequence shown in SEQ ID NO:12; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:12; or

[0083] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:18 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0084] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:53 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0085] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0086] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:56 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0087] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:57 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0088] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:58, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0089] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:59 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0090] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:60 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38.

[0091] In one or more embodiments, the conjugation portion includes or is selected from: antitumor agents, immunomodulators, cytotoxic drugs, fluorescent substances, luminescent substances, enzymes, small nucleic acid molecules, and any combination thereof; preferably, the small nucleic acid is siRNA or antisense oligonucleotide (ASO).

[0092] In one or more embodiments, the antibody-drug conjugate has an Ab-(LD)m structure, wherein Ab is a bispecific antibody or its antigen-binding fragment that specifically binds EGFR and cMET as described in any embodiment of the present invention; L is a linker; D is a therapeutically active substance or a pharmaceutically active ingredient; m represents the average number of LD units conjugated to Ab, and m ranges from 1 to 10, preferably from 2 to 8, and more preferably from 4 to 8.

[0093] In one or more implementations, the L structure is as shown in equation (A):

[0094] in:

[0095] L1 represents the functional group that can react with the thiol group and the group or bond formed after the reaction with the thiol group;

[0096] L2 and Y are each independently selected from -(CH2). p -、-(OCH2CH2) p - and -(CH2CH2O) p -;

[0097] L3 is a hydrophilic group;

[0098] L4 is an amino acid group;

[0099] R6 is selected from H, C1-C6 alkoxy, and -(OCH2CH2). p O-C1-C6 alkyl groups and -(CH2CH2O) p -C1-C6 alkyl;

[0100] X is selected from -NH- and -NH(CH2). n C(=O)-、-C(=O)(CH2) n NH- and -(CH2) n C(=O)-;

[0101] Z is selected from C and S;

[0102] o is selected from 0 and 1;

[0103] n is selected from 0, 1, and 2;

[0104] p is an integer from 1 to 10.

[0105] In one or more embodiments, the functional group that can react with thiol group as described in L1 is selected from maleimide, halogen, halogen-substituted functional group (such as halogen-substituted aldehyde group, halogen-substituted -S(O)2-), aldehyde group, alkenyl group, alkynyl group, ketone group, sulfonyl group, silane, isocyanate group and norbornene group.

[0106] In one or more embodiments, the functional group in L1 that can react with a thiol group is selected from: and their derivatives; wherein: A is a halogen; the wavy line indicates the position where L1 and L2 are connected; preferably, the functional group of L1 is:

[0107] In one or more implementations, L2 is -(CH2). p - and p is an integer from 1 to 4; preferably, L2 is -CH2CH2- or -CH2CH2CH2-.

[0108] In one or more embodiments, X is -NH- or -NH(CH2). n C(=O)-; preferably, X is -NH- or -NHC(=O)-.

[0109] In one or more implementations, Y is -(CH2). p -, p is 1, 2 or 3; preferably, Y is methylene.

[0110] In one or more implementations, Z is C.

[0111] In one or more embodiments, L3 is selected from monosaccharides, disaccharides, and five- or six-membered saturated heterocycles containing 1-2 nitrogen atoms, as well as their derivatives, which are divalent hydrophilic groups with two monovalent base centers obtained by removing two hydrogen atoms.

[0112] In one or more embodiments, the monosaccharide is selected from trioses, tetraoses, pentoses, hexoses, and heptaoses.

[0113] In one or more embodiments, the disaccharide is selected from maltose, sucrose, and lactose.

[0114] In one or more embodiments, the five- or six-membered saturated heterocycle containing 1-2 nitrogen atoms or its derivatives are selected from piperazine, piperidinyl, and pyrrolidinyl.

[0115] In one or more embodiments, L3 is selected from glucose, galactose, mannose, glucuronic acid, galactobionic acid, mannouronic acid, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, and N-acetylmucolonic acid, which are divalent hydrophilic groups with two monovalent centers obtained by removing two hydrogen atoms.

[0116] In one or more embodiments, L3 has the structure shown in formula L3-1:

[0117] In the formula:

[0118] R1 is selected from -O(CH2) p - and -C(=O)-, p is an integer from 1 to 6, preferably 1, 2, 3 or 4;

[0119] R2, R3, and R4 are independently selected from H, C1-C6 alkyl, -(CH2CH2O)t-C1-C6 alkyl, sulfonic acid group, and phosphoric acid group;

[0120] t is an integer from 1 to 20;

[0121] The wavy line indicates the position where L3 connects to X and Y.

[0122] In one or more embodiments, L4 is selected from the following amino acid residues: valine, citrulline, alanine, glycine, phenylalanine, asparagine, glutamic acid, lysine, serine, threonine, cysteine, and tyrosine; said amino acid residues are optionally separated by one or more -OR 20 Replaced, of which R 20 It is selected from H, C1-C6 alkyl, -(CH2CH2O)t-C1-C6 alkyl, sulfonic acid group and phosphoric acid group, wherein t is an integer from 1 to 20.

[0123] In one or more embodiments, L4 is selected from the following peptides: valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), glycine-glycine-phenylalanine-glycine, glycine-glycine-phenylalanine-glycine, citrulline-valine, alanine-valine, alanine-alanine, citrulline-alanine, asparagine-citrulline, citrulline-asparagine, citrulline-citrulline, phenylalanine-lysine, and lysine-phenylalanine, preferably selected from the following peptides: valine-citrulline (VC), valine-alanine (VA), and glycine-glycine-phenylalanine-glycine (GGFG); each peptide is optionally substituted with one or more substituents selected from the group consisting of -OR20 and -(OCH2CH2). p O-C1-C6 alkyl, wherein R20 is selected from H, C1-C6 alkyl, -(CH2CH2O)t-C1-C6 alkyl, sulfonic acid group and phosphate group, t is an integer from 1 to 20, and p is an integer from 1 to 6.

[0124] In one or more implementations, L4 is selected from:

[0125] In the formula, the wavy line indicates the position where L4 connects to Z and NH.

[0126] In one or more embodiments, R6 is H, C1-C3 alkoxy, or -(OCH2CH2). p O-C1-C3 alkyl, wherein p is an integer from 1 to 6; preferably, R6 is H, methoxy, or -(OCH2CH2). p OCH3, where p is an integer from 1 to 6 or from 1 to 4.

[0127] In one or more embodiments, the structure of the connector L is shown in any of the following configurations:

[0128] In each of the formulas, R2, R3 and R4 are as described in any embodiment of the present invention, R6 is as described in any embodiment of the present invention, and R” is H or C1-C6 alkyl.

[0129] In one or more embodiments, the pharmaceutical portion D includes a tubulin inhibitor, a DNA damaging agent, a vitamin A precursor, and folic acid.

[0130] In one or more embodiments, the microtubule inhibitors include dolastatin and auristatin and maytansine; the DNA damaging agents include calicheamicin, duocarmycin, pyrrolobenzodiazepine (PBD), and camptothecin.

[0131] In one or more embodiments, the auristatin class of drugs includes monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF) or their derivatives; the maytansine class of drugs includes DM1, DM3, DM4 or their derivatives; and the camptothecin class of drugs includes exatecan, deruxtecan (Dxd), SN38, camptothecin, topotecan, irinotecan, belotetane, letopotecan, rubitecan, cilatecan, cocinotecan, gemmatotecan, eczetane, topotecan, and 9-nitrocamptothecin.

[0132] In one or more embodiments, the connector-drug (LD) portion has a structure as shown in the following formula: VCMMAE, GGFG-Deruxtecan, CPD2-Exatecan, CPD4-Exatecan, CPD5-Exatecan, CPD6-Exatecan, or SN-38Comp5.

[0133] In one or more embodiments, the antibody-drug conjugate is selected from the structures represented by formula ADC-I, ADC-II, ADC-III, ADC-IV, or ADC-V:

[0134] In the formula, m represents the average number of linker-drug (LD) units coupled to Ab, and m ranges from 1 to 10, preferably from 2 to 8, and more preferably from 4 to 8.

[0135] In one or more embodiments, the Ab is a bispecific antibody or antigen-binding fragment thereof that specifically binds EGFR and cMET as described in any embodiment of the present invention.

[0136] A second aspect of the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate of an anti-EGFR and cMET bispecific antibody as described in any embodiment of the present invention, and a pharmaceutically acceptable carrier or excipient.

[0137] A third aspect of the invention provides the use of an antibody-drug conjugate of an anti-EGFR and cMET bispecific antibody as described in any embodiment of the invention, or a pharmaceutical composition as described in any embodiment of the invention, in the preparation of a medicament for treating and / or preventing EGFR and / or cMET-mediated diseases or conditions.

[0138] In one or more embodiments, the disease or condition is a tumor or cancer.

[0139] In one or more embodiments, the tumor or cancer is selected from lung cancer, gastric cancer, glial cell carcinoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, bladder cancer, colon cancer, rectal cancer, liver cancer, brain cancer, thyroid cancer, and head and neck cancer.

[0140] In one or more embodiments, the lung cancer includes non-small cell lung cancer and small cell lung cancer, the gastric cancer includes gastric cancer and gastroesophageal junction cancer, and the head and neck cancer includes head and neck squamous cell carcinoma.

[0141] A fourth aspect of the present invention provides a method for treating or preventing tumors or cancer, comprising the step of administering to a subject in need an effective amount of an antibody-drug conjugate of an anti-EGFR and cMET bispecific antibody as described in any embodiment of the present invention, or a pharmaceutical composition as described in any embodiment of the present invention.

[0142] In one or more embodiments, the tumor or cancer is a tumor that highly expresses EGFR and / or cMET.

[0143] In one or more embodiments, the tumor or cancer is selected from lung cancer, gastric cancer, glial cell carcinoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, bladder cancer, colon cancer, rectal cancer, liver cancer, brain cancer, thyroid cancer, and head and neck cancer.

[0144] In one or more embodiments, the lung cancer includes non-small cell lung cancer and small cell lung cancer, the gastric cancer includes gastric cancer and gastroesophageal junction cancer, and the head and neck cancer includes head and neck squamous cell carcinoma.

[0145] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0146] Figure 1. Killing activity of bsAb1-CPD2-Exatecan-DAR8, bsAb1-CPD6-Exatecan-DAR8, and bsAb1-GGFG-Dxd-DAR8 against MKN45 cells (Figure 1A) and EBC-1 cells (Figure 1B).

[0147] Figure 2. Bystander killing effect (bypassive killing effect) of EGFR×cMET bispecific antibody ADC.

[0148] Figure 3. Inhibitory effect of EGFR×cMET bispecific antibody ADC on NCI-H292 xenograft model.

[0149] Figure 4. Inhibitory effect of EGFR×cMET bispecific antibody on MKN45 xenograft model.

[0150] Figure 5. Inhibitory effect of EGFR×cMET bispecific antibody ADC on MKN45 xenograft model.

[0151] Figure 6. Inhibitory effect of EGFR×cMET bispecific antibody on HT29 xenograft model.

[0152] Figure 7. Inhibitory effect of EGFR×cMET bispecific antibody ADC on HT29 xenograft model. Detailed Implementation

[0153] This invention provides antibodies or functional fragments thereof that specifically bind to EGFR and cMET, antibody-drug conjugates (ADCs) or pharmaceutical compositions thereof, the use of said antibodies, functional fragments, pharmaceutical compositions, and ADCs in the treatment and / or prevention of EGFR and / or cMET-mediated diseases, and methods for treating or preventing EGFR and / or cMET-mediated diseases using said antibodies, functional fragments, pharmaceutical compositions, and ADCs.

[0154] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new technical solutions.

[0155] definition

[0156] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0157] To facilitate a better understanding of this invention, certain technical terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For specific definitions and terms in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular forms used herein (including the claims) include their corresponding plural forms unless otherwise expressly specified herein.

[0158] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0159] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0160] As used herein, the term “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted inclusively, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, such as “only one” or “exactly one” or when “consisting of” is used in the claims, will it refer to only one number or one element of the list.

[0161] Unless the opposite is explicitly stated, the words “one” and “a” as used in this article should be understood as “at least one”.

[0162] The terms “EGFR”, “epidermal growth factor receptor”, or “epidermal growth factor receptor” refer to the cell surface receptors of extracellular protein ligands of the epidermal growth factor family (EGF-family). For EGFR protein, please refer to NCBI accession number: P00533.2.

[0163] The terms "MET" or "cMET" refer to mesenchymal-to-epidermal transition factor (MET), also known as cellular-mesenchymal-to-epidermal transition factor (c-Met). For cMET protein information, please refer to NCBI accession number: P08581.4. The terms "MET" and "cMET" are used interchangeably.

[0164] As used herein, when referring to the amino acid sequence of an EGFR or cMET protein, it includes the full-length EGFR or cMET protein, a fragment containing an EGFR ECD or cMET ECD, and also a fusion protein of the full-length EGFR or cMET protein or a fusion protein of an EGFR ECD or cMET ECD, such as a fragment fused to a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of an EGFR or cMET protein without affecting its biological function. Therefore, in this invention, the term "protein" should include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of an EGFR or cMET protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0165] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0166] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, which results in selective damage, destruction, or clearance from the body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation.

[0167] The terms "signal transduction pathway" or "signal transduction activity" refer to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of growth factors to receptors, that results in the transmission of a signal from one part of the cell to another. Generally, this transmission involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that induce signal transduction. The penultimate process typically involves nuclear events that lead to changes in gene expression.

[0168] The terms “activity” or “bioactivity”, or “biological property” or “biocharacteristic”, are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize EGFR or cMET activity in vivo or in vitro, IC50, etc. 50 The in vivo stability and immunogenic properties of antibodies. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics are observed, measured, or evaluated using techniques known in the art, including but not limited to ELISA, FACS, or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or any other source).

[0169] The term "antibody" refers to an immunoglobulin molecule, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, fat, or a combination thereof, that recognizes and specifically binds to an antigen (or target) through at least one antigen recognition site within a variant region of the immunoglobulin molecule. As used herein, "antibody" is used in the broadest sense and includes complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-domain antibodies (VHH), single-chain Fv (scFv), multispecific antibodies (e.g., bispecific antibodies generated from at least two complete antibodies), chimeric antibodies, humanized antibodies, fully human antibodies, camel-derived single-domain antibodies, fusion proteins containing the antigen-determining portion of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity. Antibodies can be any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (subtypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu) based on the recognition of their heavy chain constant regions. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated to other molecules such as toxins, radioactive isotopes, etc.

[0170] The term "bispecific antibody" refers to an antibody that has binding specificity to at least two independent antigens (or targets) or different epitopes within the same antigen. Exemplary bispecific antibodies may bind to two different epitopes of a single target, or they may bind to two different targets. Other such antibodies may combine a first binding site of one target with a second binding site of another target.

[0171] The terms "maternal antibody," "parental antibody," or simply "parental antibody" refer to the original antibody used as a template or reference in antibody engineering. It typically binds to a specific antigen (or target) and serves as a reference in later antibody design, where its amino acid sequence is modified to generate altered antibodies with different properties. Such modifications include, but are not limited to, the addition, substitution, deletion, or mutation of one or more amino acids, or the addition, substitution, deletion, or mutation of one or more domains or fragments. These modifications typically do not occur in the CDR region of the antibody. In specific embodiments, the modifications occur within the constant region of the antibody.

[0172] The term "isolated" refers to the purified state of the bound compound, and in this context means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. The term "isolated" does not mean the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that would significantly interfere with the experimental or therapeutic application of the bound compound described herein.

[0173] The term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific. The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies by any particular method.

[0174] The term "full-length antibody" refers to an immunoglobulin molecule that, in its natural state, contains at least four peptide chains: two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region (abbreviated as CH in this document). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and regions separated by more conserved regions called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at www.bioinf.org.uk / abs / index.html#kabatnum. For example, the CDR of the antibody used herein can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242).

[0175] An antibody's "antigen-binding fragment" comprises a fragment or derivative of the antibody, typically including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When the antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding affinity of the parent antibody. It is also anticipated that the antibody's antigen-binding fragment may include conserved or non-conserved amino acid substitutions that do not significantly alter its biological activity (referred to as "conserved variants" or "functionally conserved variants" of the antibody). The term "binding compound" refers to both the antibody and its binding fragment.

[0176] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment containing both VH and VL domains, which are located within a single polypeptide chain. Fv polypeptides typically also include a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired structure for antigen binding.

[0177] The term "domain antibody" refers to an immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0178] The term "bivalent antibody" refers to an antibody with two antigen-binding sites. In some cases, both binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific.

[0179] The term "dual antibody" refers to a small antibody fragment having two antigen-binding sites, wherein the fragment contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) within the same polypeptide chain (VH-VL or VL-VH). By using a linker too short to allow pairing between two domains on the same chain, the linker is forced to pair with a complementary domain of the other chain, creating two antigen-binding sites.

[0180] The terms "mouse antibody" or "hybridoma antibody" in this disclosure refer to monoclonal antibodies against EGFR or MET prepared in accordance with the knowledge and skills in the art. Preparation involves injecting the test subject with EGFR or MET antigen, followed by isolation of a hybridoma expressing an antibody with the desired sequence or functional characteristics.

[0181] The term "chimeric antibody" refers to an antibody possessing a variable domain of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies originate from different species. Typically, the variable domain is derived from antibodies from rodents, etc. ("parental antibodies"), while the constant domain sequence is derived from human antibodies, making the resulting chimeric antibody less likely to induce an adverse immune response in human subjects compared to parental rodent antibodies.

[0182] The term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, humanized antibodies contain at least one, and usually two, variable domains, where all or almost all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or almost all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Humanized antibodies may optionally contain at least a portion of the constant region (Fc) of human immunoglobulins.

[0183] The term "fully human antibody" refers to an antibody that contains only the sequence of human immunoglobulin proteins. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, a fully human antibody may contain mouse glycans. Similarly, a "mouse antibody" refers to an antibody that contains only the sequence of mouse immunoglobulins. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, a fully human antibody may contain rat glycans. Likewise, a "rat antibody" refers to an antibody that contains only the sequence of rat immunoglobulins.

[0184] "Isotype" antibodies refer to antibody classes provided by heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where modifications have been generated to alter Fc function, such as to enhance or weaken effector function or binding to the Fc receptor.

[0185] The term "Fc region" is used in this document to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is based on the EU numbering system, also known as the EU index, e.g., Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0186] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes are typically composed of various chemically active surface molecules such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational and non-conformational epitopes lies in the fact that, in the presence of denaturing solvents, the former loses binding, rather than the latter.

[0187] Methods for producing and purifying antibodies and antigen-binding fragments are well-known and readily available in existing technologies, such as Cold Spring Harbor's Guide to Antibody Experimentation, Chapters 5-8 and 15. For example, mice can be immunized with human ROR1 or fragments thereof, and the resulting antibodies can be refolded, purified, and sequenced amino acids using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described herein are prepared by adding one or more human FR regions to non-human CDR regions using genetic engineering methods. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351.

[0188] The term "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its pairmate Y can usually be determined by the equilibrium dissociation constant (K). D The equilibrium dissociation constant is represented by the dissociation rate constant and the binding rate constant (k, k ... dis and k on The ratio of affinity to kinetic binding affinity. Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay described in this paper.

[0189] The term "non-binding" protein or cell refers to proteins or cells that do not bind to each other, or do not bind to them with high affinity; that is, K, the protein or cell that binds to. D 1.0×10 -6 M or higher, more preferably 1.0 × 10 -5 M or higher, more preferably 1.0 × 10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0 × 10 -2 M or higher.

[0190] For IgG antibodies, the term "high affinity" refers to the affinity for the antigen's K+. D 1.0×10 -6 M or lower, preferably 5.0 × 10 -8 M or lower, more preferably 1.0 × 10-8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0 × 10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding in the IgM subtype refers to K... D 10 -6 M or lower, preferably 10 -7 M or lower, preferably 10 -8 M or lower.

[0191] The terms “nucleic acid,” “polynucleotide,” “nucleic acid molecule,” and “polynucleotide molecule” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see U.S. Patent No. 8,278,036, belonging to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0192] "Construction" refers to any recombinant polynucleotide molecule (such as plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecules), derived from any source, capable of integrating with or autonomously replicating the genome, constituting a polynucleotide molecule in which one or more polynucleotide molecules are functionally linked (i.e., operably linked). Recombinant constructs typically contain polynucleotides of the present invention operably linked to transcription initiation regulatory sequences that guide the transcription of the polynucleotide in the host cell. Expression of the nucleic acids of the present invention can be guided using both heterologous and non-heterologous (i.e., endogenous) promoters.

[0193] "Vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is the viral vector, into which an additional DNA segment can be ligated into the viral genome. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). After introduction into the host cell, other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors can guide the expression of operatively linked genes. These vectors are referred to herein as "expression vectors."

[0194] As used in this article, "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a target gene when transformed, transfected, or transduced into host cells. Expression vectors contain one or more phenotypic selection markers and origins of replication to ensure the maintenance of the vector and to provide amplification within the host when needed.

[0195] The term "conjugate" refers to an antibody linked to a moiety having the desired activity via a linker unit or directly. The linker can be covalent or non-covalent. The desired activity can be, for example, cytotoxic activity or detection (e.g., luminescence) activity. The moiety having the desired activity can be an antitumor agent, an immunomodulator, a cytotoxic drug, a fluorescent substance, a luminescent substance, an enzyme, a small nucleic acid molecule, or any combination thereof. Suitable small nucleic acids may include siRNA or antisense oligonucleotides (ASOs), etc. In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) as described in any embodiment herein, or an antibody or its antigen-binding fragment labeled with a radioactive atom or spin label as described in any embodiment herein.

[0196] The terms “antibody-drug conjugate”, “antibody-drug conjugate”, and “ADC” are used interchangeably and generally refer to one or more therapeutic compounds (e.g., Exatecan, Dxd) linked to one or more antibody or antigen-binding fragments and defined by the following general formula: Ab-(LD)m, where Ab = antibody or antigen-binding fragment, L = linker, D = drug unit, and m = number of drug portions of each antibody or antigen-binding fragment, which can be any number from 1 to 10. In some embodiments, the linker L may include a cleavable portion between the antibody or antigen-binding fragment and the drug unit.

[0197] The term "drug" in this invention refers broadly to any compound having the desired biological activity and reactive functional groups for the preparation of the conjugates described herein. The desired biological activity includes the diagnosis, cure, relief, treatment, and prevention of diseases in humans or other animals. As new drugs are continuously discovered and developed, these new drugs should also be included in the drugs described in this invention. Specifically, the drugs include, but are not limited to, cytotoxic drugs, cell differentiation factors, stem cell nutrient factors, steroid drugs, drugs for treating autoimmune diseases, anti-inflammatory drugs, or drugs for infectious diseases. More specifically, the drugs include, but are not limited to, microtubule inhibitors or DNA / RNA damaging agents.

[0198] The term "cytotoxic drug" or "cytotoxin" refers to substances that inhibit cell function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of selectivity, they can also induce apoptosis in normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, radioactive isotopes, chemotherapy drugs, antibiotics, and ribolysins, among others.

[0199] The term "microtubule inhibitor" refers to a class of compounds that exert antitumor effects by interfering with cell mitosis by inhibiting the polymerization of tubulin or promoting its aggregation. Non-limiting examples include: dolastatin, calichiomycin, taxanes, vincristine, colchicine, and dolastatin / auristatin / monomethylauristatin E (MMAE) / monomethylauristatin F (MMAF). In some embodiments, the microtubule inhibitors include, but are not limited to, dolastatin and auristatin derivatives, and maytansine derivatives. In some embodiments, the auristatin class of drugs includes, but is not limited to: monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF) or their derivatives, and the maytansine class of drugs includes, but is not limited to, DM1, DM3, DM4 or their derivatives (“Research Progress on Warhead Molecules of Antibody-Drug Conjugates”, Hu Xinyue et al., *Chinese Journal of Pharmaceutical Biotechnology*, December 2017, Vol. 12, No. 6; “Research Progress on Maytansine Antibody-Drug Conjugates”, Zhou Lei et al., *Chinese Journal of New Drugs*, 2016, Vol. 25, No. 22, pp. 2521-2530).

[0200] Non-limiting examples of the DNA and RNA damaging agents include, but are not limited to, calicheamicin derivatives, duocarmycin derivatives, pyrrolobenzodiazepine (PBD), and camptothecin derivatives. In some embodiments, the camptothecin drugs include, but are not limited to: exatecan (e.g., CAS Nr: 171335-80-1), Deruxtecan (Dxd), SN38, camptothecin, topotecan, irinotecan, belotetane, letopotecan, rubitecan, cilatecan, cocinotecan, gemmatotecan, eczetane, topotecan, and 9-nitrocamptothecin.

[0201] In this invention, the terms "linker," "connector," "linker," or "L" generally refer to any chemical part capable of covalently attaching a compound (typically a pharmaceutical unit) to another part (such as an antibody or antigen-binding fragment). A linker may be readily resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and / or disulfide bond cleavage under conditions that keep the compound or antibody active.

[0202] Based on their properties, the adapters suitable for use in this invention can be cathepsin-cleavable adapters, such as valine-citrulline (val-cit) adapters, cBu-Cit adapters, and CX adapters; non-cleavable adapters such as SMCC adapters or MD adapters; MC-GGFG adapters, TRX adapters, galactoside-containing adapters, etc.

[0203] In specific implementations, the connectors include, but are not limited to, maleimide-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimide hexanoic acid (MC), maleimide propionyl (MP), valine-citrulline (val-cit, vc), and alanine-phenylalanine (ala-phe). p-Aminobenzyloxycarbonyl (PAB), N-succinimide-4-(2-pyridinylthio)valerate (SPP), N-succinimide-4-(N-maleimide-methyl)-cyclohexane-1-carboxylate (SMCC), N-succinimide-(4-iodo-acetyl)aminobenzoate (SIAB), N-succinimide-4-(2-pyridinyl dithio)butyrate (SPDB), and N-succinimide-3-(pyridin-2-yl dithio)propionate (SPDP).

[0204] The terms “load” or “drug load” or “effective load” refer to the average effective load of each antibody within the ADC molecule (in this document, “effective load” may be used interchangeably with “therapeutic active substance or active pharmaceutical ingredient”). Drug load can range from 1 to 20 therapeutic active substances or active pharmaceutical ingredients per antibody.

[0205] The term "drug / antibody ratio" or "DAR" refers to the ratio of a therapeutically active substance or active pharmaceutical ingredient (D) conjugated to an antibody to the antibody. ADCs described herein typically have a DAR of 1-10, and in some specific embodiments, DARs of 1-8, 2-8, 2-6, 2-5, 3-8, 4-6, and 2-4. Representative DAR values ​​are 2, 3, 4, 5, 6, 7, 8, 9, and 10, and are typically expressed as a combination of the letter DAR and a number, where the number represents the numerical value of the DAR; for example, DAR8 indicates a drug / antibody ratio with a DAR value of 8. In some implementations, DAR is average DAR, which is characterized by detection methods (e.g., conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA, and HPLC). Quantitative DAR values ​​can also be determined. DAR may be limited by the number of binding sites on the antibody. For example, in the case where the binding site is cysteine ​​thiol, the antibody may have only one or a few cysteine ​​thiol groups or only one or a few sufficiently reactive thiol groups (through which linking units can be connected).

[0206] The terms “activation,” “stimulation,” and “treatment” used for cells or receptors can have the same meaning, such as activating, stimulating, or treating cells or receptors with ligands, unless the context otherwise specifies. “Ligand” includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, mutant proteins, and antibody-derived binding compounds. “Ligand” also includes small molecules, such as peptide mimics of cytokines and peptide mimics of antibodies. “Activation” can refer to cell activation regulated by internal mechanisms as well as external or environmental factors. “Response” refers to responses of cells, tissues, organs, or organisms, including changes in biochemical or physiological behaviors (e.g., concentrations, densities, adhesion or migration, gene expression rates, or differentiation states within biological compartments) that are related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

[0207] As used herein, the term “treatment” or “cure” for any disease or condition, in some embodiments, means improving the disease or condition (i.e., slowing or halting or reducing the progression of the disease or at least one of its clinical symptoms). In other embodiments, “treatment” or “cure” means alleviating or improving at least one bodily parameter, including those physical parameters that may not be identifiable by the patient. In still other embodiments, “treatment” or “cure” means regulating the disease or condition physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of bodily parameters), or in both ways. Unless explicitly described herein, methods for assessing the treatment and / or prevention of disease are generally known in the art.

[0208] "Subjects" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cyno-eating macaque" refers to the cyno-eating macaque.

[0209] "Combined" administration of one or more other therapeutic agents includes simultaneous (co-) administration and consecutive administration in any order.

[0210] "Therapeutic effective amount," "therapeutic effective dose," and "effective amount" refer to the amount of the FGFR2b antibody or its antigen-binding fragment of the present invention, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions, or the development of such diseases or conditions. Therapeutic effective dose also refers to the amount of antibody or its antigen-binding fragment sufficient to cause symptom improvement, such as the amount that treats, cures, prevents, or improves the associated medical condition, or increases the rate of treatment, cure, prevention, or improvement of such conditions. When administered to an individual as a single active ingredient, the therapeutic effective dose refers only to that ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether administered in combination, sequentially, or simultaneously. The effective amount of the therapeutic agent will result in an increase of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in diagnostic criteria or parameters.

[0211] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation or composition that are non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0212] The term “cancer” is used herein to refer to a group of cells exhibiting an abnormally high level of proliferation and growth. Cancer can be benign (also called a benign tumor), pre-malignant, or malignant. Cancer cells can be solid cancer cells or leukemia cancer cells. The term “tumor” as used herein refers to one or more cells containing cancer. The term “tumor growth” is used herein to refer to the proliferation or growth of one or more cells containing cancer, resulting in a corresponding increase in the size or extent of the cancer.

[0213] Anti-EGFR and cMET bispecific antibodies

[0214] In one aspect, the present invention provides a bispecific antibody or antigen-binding fragment thereof that specifically binds to EGFR and cMET. The terms “anti-EGFR and cMET bispecific antibody,” “anti-EGFR and cMET,” “EGFR / cMET bispecific antibody,” or “antibody binding to EGFR and cMET” refer to antibodies capable of binding with sufficient affinity to EGFR protein or fragment thereof and cMET protein or fragment thereof, such that the antibody can be used as a diagnostic and / or therapeutic agent targeting EGFR and cMET.

[0215] In some embodiments, the present invention provides antibodies that bind to EGFR and cMET proteins. In some embodiments, the present invention provides antibodies that block the EGFR and cMET signaling pathways.

[0216] In some embodiments, the antibody of the present invention binds to human EGFR protein and human cMET protein. In some embodiments, the antibody of the present invention binds to human EGFR and blocks the interaction between human EGF and EGFR protein, and the antibody of the present invention binds to human cMET and blocks the interaction between HGF and cMET.

[0217] In some embodiments, the bispecific antibody of the present invention that specifically binds to EGFR and cMET, or its antigen-binding fragment, comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0218] In some embodiments, the first protein functional region targeting EGFR comprises: HCDR1, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, or differs from the amino acid sequence shown in SEQ ID NO:25 or 26 by 1, 2, or 3 amino acids; HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:27, or differs from the amino acid sequence shown in SEQ ID NO:27 by 1, 2, or 3 amino acids; and HCDR3, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:37 (ARVSX1YX2DSX3FD), 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.

[0219] In some embodiments, the first protein functional region targeting EGFR comprises: HCDR1, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, or differs from the amino acid sequence shown in SEQ ID NO:25 or 26 by 1, 2, or 3 amino acids; HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:27, or differs from the amino acid sequence shown in SEQ ID NO:27 by 1, 2, or 3 amino acids; and HCDR3, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:37 (ARVSX1YX2DSX3FD), 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; LCDR1, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2, or 3 amino acids; and LCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:25 or 26 by 1, 2, or 3 amino acids; The amino acid sequence shown in NO:14 has 1, 2 or 3 amino acid differences; and LCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:15, 20 or 21, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO:15, 20 or 21.

[0220] In some embodiments, the first protein functional region targeting EGFR comprises: HCDR1, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, or differs from the amino acid sequence shown in SEQ ID NO:25 or 26 by 1, 2, or 3 amino acids; HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:27, or differs from the amino acid sequence shown in SEQ ID NO:27 by 1, 2, or 3 amino acids; HCDR3, whose amino acid sequence is selected from the amino acid sequences shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, or 36, or differs from the amino acid sequences shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, or 36 by 1, 2, or 3 amino acids; LCDR1, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2, or 3 amino acids; LCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:25 or 26 by 1, 2, or 3 amino acids; HCDR3, whose amino acid sequence is selected from the amino acid sequences shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, or 36 by 1, 2, or 3 amino acids; LCDR1, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2, or 3 amino acids; and LCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:1 The amino acid sequence shown in NO:14 has 1, 2 or 3 amino acid differences; and LCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:15, 20 or 21, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO:15, 20 or 21.

[0221] In some embodiments, the first protein functional region targeting EGFR includes a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO:25 or 26, SEQ ID NO:27, and SEQ ID NO:37 (ARVSX1YX2DSX3FD), respectively; 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.

[0222] In some implementations, the first protein functional region targeting EGFR includes a heavy chain variable region, which comprises:

[0223] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:35, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:35 by 1, 2, or 3 amino acids; or

[0224] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:31, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:31; or

[0225] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:28, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:28 by 1, 2, or 3 amino acids; or

[0226] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29 by 1, 2, or 3 amino acids; or

[0227] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:30, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:30; or

[0228] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:32, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:32; or

[0229] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:33, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:33; or

[0230] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:33, respectively; or HCDR1, HCDR2, and HCDR3 having a difference of 1, 2, or 3 amino acids from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:33; or

[0231] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:34, respectively; or HCDR1, HCDR2, and HCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:34; or

[0232] The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:36, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:36.

[0233] In some embodiments, the first protein functional region targeting EGFR comprises: LCDR1, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2, or 3 amino acids; LCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:14 by 1, 2, or 3 amino acids; and LCDR3, whose amino acid sequence is selected from the amino acid sequences shown in SEQ ID NO:15, 20, or 21, or differs from the amino acid sequences shown in SEQ ID NO:15, 20, or 21 by 1, 2, or 3 amino acids.

[0234] In some implementations, the first protein functional region targeting EGFR includes a light chain variable region, which comprises:

[0235] The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; or

[0236] The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21; or

[0237] The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20.

[0238] In some implementations, the first protein functional region targeting EGFR includes a heavy chain variable region and a light chain variable region, wherein:

[0239] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, the amino acid sequence of HCDR2 is the amino acid sequence shown in SEQ ID NO:27, and the amino acid sequence of HCDR3 is selected from the amino acid sequences shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, or 36; and

[0240] The light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is the amino acid sequence shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is the amino acid sequence shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is selected from the amino acid sequences shown in SEQ ID NO:15, 20 or 21.

[0241] In some implementations, the first protein functional region targeting EGFR includes a heavy chain variable region and a light chain variable region, wherein:

[0242] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:15; or

[0243] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:28; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0244] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:31; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0245] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:20; or

[0246] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:29; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0247] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:30; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0248] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:32; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0249] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in 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:33; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0250] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:33; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0251] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:34; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0252] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or

[0253] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:36; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21.

[0254] In some embodiments, the second protein functional region targeting cMET comprises: HCDR1, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:22, or differs from the amino acid sequence shown in SEQ ID NO:22 by 1, 2, or 3 amino acids; HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:23, or differs from the amino acid sequence shown in SEQ ID NO:23 by 1, 2, or 3 amino acids; and HCDR3, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:24, or differs from the amino acid sequence shown in SEQ ID NO:24 by 1, 2, or 3 amino acids.

[0255] In some embodiments, the second protein functional region targeting cMET comprises: LCDR1, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2, or 3 amino acids; LCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:14 by 1, 2, or 3 amino acids; and LCDR3, whose amino acid sequence is selected from the amino acid sequences shown in SEQ ID NO:15, 20, or 21, or differs from the amino acid sequences shown in SEQ ID NO:15, 20, or 21 by 1, 2, or 3 amino acids.

[0256] In some embodiments, the second protein functional region targeting cMET includes a light chain variable region, which comprises:

[0257] The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; or

[0258] The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20; or

[0259] The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21.

[0260] In some implementations, the second protein functional region targeting cMET includes a heavy chain variable region and a light chain variable region, wherein:

[0261] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is the amino acid sequence shown in SEQ ID NO:22, or differs from the amino acid sequence shown in SEQ ID NO:22 by 1, 2, or 3 amino acids; the amino acid sequence of HCDR2 is the amino acid sequence shown in SEQ ID NO:23, or differs from the amino acid sequence shown in SEQ ID NO:23 by 1, 2, or 3 amino acids; the amino acid sequence of HCDR3 is the amino acid sequence shown in SEQ ID NO:24, or differs from the amino acid sequence shown in SEQ ID NO:24 by 1, 2, or 3 amino acids.

[0262] The light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2, or 3 amino acids. The amino acid sequence of LCDR2 is the amino acid sequence shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:14 by 1, 2, or 3 amino acids. The amino acid sequence of LCDR3 is selected from the amino acid sequences shown in SEQ ID NO:15, 20, or 21, or differs from the amino acid sequences shown in SEQ ID NO:15, 20, or 21 by 1, 2, or 3 amino acids.

[0263] In some implementations, the second protein functional region targeting cMET includes a heavy chain variable region and a light chain variable region, wherein:

[0264] The heavy chain variable region comprises: HCDR1, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:22, or differs from the amino acid sequence shown in SEQ ID NO:22 by 1, 2, or 3 amino acids; HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:23, or differs from the amino acid sequence shown in SEQ ID NO:23 by 1, 2, or 3 amino acids; and HCDR3, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:24, or differs from the amino acid sequence shown in SEQ ID NO:24 by 1, 2, or 3 amino acids.

[0265] The light chain variable region comprises: LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20; or

[0266] The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20; or

[0267] The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21.

[0268] In some implementations, the second protein functional region targeting cMET includes a heavy chain variable region and a light chain variable region, wherein:

[0269] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:24; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:15; or

[0270] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:24; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:20; or

[0271] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:24; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21.

[0272] In some embodiments, the bispecific antibody or antigen-binding fragment of the present invention that specifically binds to EGFR and cMET comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0273] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 4, 9, 2, 3, 5, 6, 7, 8, 10, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1, 4, 9, 2, 3, 5, 6, 7, 8, 10; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 39, 40, or 42, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39, 40, or 42.

[0274] The second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:41, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:41. The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:39, 40, or 42, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:39, 40, or 42.

[0275] In some embodiments, the bispecific antibody or antigen-binding fragment of the present invention that specifically binds to EGFR and cMET comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0276] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 4, 9, 2, 3, 5, 6, 7, 8, 10; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 39, 40, or 42.

[0277] The second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:41; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:39, 40 or 42.

[0278] In some embodiments, the bispecific antibody or antigen-binding fragment of the present invention that specifically binds to EGFR and cMET comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0279] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, 40, or 42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, 40, or 42.

[0280] In some embodiments, the bispecific antibody or antigen-binding fragment of the present invention that specifically binds to EGFR and cMET comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0281] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO:1, 4, 9, 2, 3, 5, 6, 7, 8, 10; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:41; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:42.

[0282] In some embodiments, the bispecific antibody or antigen-binding fragment of the present invention that specifically binds to EGFR and cMET comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0283] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39; or

[0284] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or

[0285] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or

[0286] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:40; or

[0287] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:2; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or

[0288] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:3; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or

[0289] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or

[0290] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:6; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or

[0291] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or

[0292] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:8; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or

[0293] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; or

[0294] The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:42.

[0295] In some embodiments, the bispecific antibody or antigen-binding fragment of the present invention that specifically binds to EGFR and cMET comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0296] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain. The heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO:18, 52, 55, 53, 54, 56, 57, 58, 59, 60, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:18, 52-60. The light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12, or 38, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:11, 12, or 38.

[0297] The second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain. The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:19. The light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12, or 38, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:11, 12, or 38.

[0298] In some embodiments, the bispecific antibody or antigen-binding fragment of the present invention that specifically binds to EGFR and cMET comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0299] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:18, and the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12, or 38; or the first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO:52-60, and the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:38;

[0300] The second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19, and the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12, or 38.

[0301] In some embodiments, the bispecific antibody or antigen-binding fragment of the present invention that specifically binds to EGFR and cMET comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET.

[0302] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:18, and the light chain comprises the amino acid sequence shown in SEQ ID NO:11; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:11; or

[0303] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:52 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0304] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:55 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0305] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:18, and the light chain comprises the amino acid sequence shown in SEQ ID NO:12; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:12; or

[0306] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:18 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0307] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:53 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0308] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0309] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:56 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0310] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:57 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0311] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:58, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0312] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:59 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or

[0313] The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:60 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38.

[0314] In some embodiments, amino acid differences include amino acid deletions, insertions, or substitutions. In some embodiments, the bispecific antibodies of the present invention include those bispecific antibodies having amino acid sequences that have been mutated by amino acid deletions, insertions, or substitutions, but still possess at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the first protein functional region targeting EGFR and / or the second protein functional region targeting cMET (particularly in the CDR region depicted in the above sequence). In some embodiments, when compared to the CDR region depicted in the specific sequence, the bispecific antibodies of the present invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR region that have been mutated by amino acid deletions, insertions, or substitutions. In some embodiments, when compared to the frame region in the specific sequence, the antibodies of the present invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the frame region that have been mutated by amino acid deletions, insertions, or substitutions.

[0315] In some embodiments, the polynucleotide molecule encoding the antibody of the present invention includes a polynucleotide molecule that has been mutated by nucleotide deletion, insertion or substitution, but still has at least about 60, 70, 80, 90, 95 or 100% identity with the coding region corresponding to the CDR depicted in the sequence described above.

[0316] The bispecific antibodies of the present invention can be generated using any suitable method for generating bispecific antibodies. Any suitable form of EGFR and cMET can be used as an immunogen (antigen) for antibody generation. By way of example and not limitation, any EGFR and cMET variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells that generate murine anti-EGFR and cMET bispecific antibodies can be generated by methods known in the art.

[0317] Antibodies derived from rodents (such as mice) can cause unwanted antibody immunogenicity when used as therapeutic agents in vivo. Repeated use can lead to an immune response against the therapeutic antibody, which at least results in loss of therapeutic efficacy, and in severe cases, can cause potentially fatal allergic reactions. One approach to reducing the immunogenicity of rodent antibodies includes the production of chimeric bispecific antibodies, in which mouse variable regions are fused with human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, the preservation of the intact rodent variable region in chimeric antibodies can still cause harmful immunogenicity in patients. The transplantation of the complementarity-determining region (CDR) loop of the rodent variable domain onto a human scaffold (i.e., humanization) has been used to further minimize rodent sequences (Jones et al. (1986)). n nature 321:522; Verhoeyen et al. (1988) Science 239:1534).

[0318] In some embodiments, the chimeric or humanized bispecific antibody of the present invention can be prepared based on the sequence of the prepared mouse monoclonal hybridoma antibody. The DNA encoding heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to include non-mouse (e.g., human) immunoglobulin sequences.

[0319] In some embodiments, the chimeric bispecific antibody of the present invention can be prepared by effectively linking the variable regions of the heavy and light chains of hybridoma-derived immunoglobulins to the constant regions of human IgG using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.), to obtain chimeric heavy and light chains. In some embodiments, the constant regions included in the chimeric antibody of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG1.

[0320] In some embodiments, the chimeric bispecific antibody of the present invention can be obtained by transfecting expression cells with a chimeric light chain and a chimeric heavy chain expression plasmid in a “mixed and matched” manner. The EGFR or cMET binding of such “mixed and matched” bispecific antibodies can be tested using the binding assays described above and other conventional binding assays (e.g., ELISA).

[0321] The bispecific antibody of the present invention is preferably a "1+1" type bispecific antibody, that is, a monovalent antibody targeting the first protein functional region of EGFR + a monovalent antibody targeting the second protein functional region of cMET. In the art, a monovalent antibody generally refers to an antibody molecule having one antigen-binding site, and a divalent antibody generally indicates that an antibody molecule has two antigen-binding sites. For Y-shaped IgG monoclonal antibodies, they are usually divalent.

[0322] Bispecific antibodies can be generated using a common light chain technique. That is, both the first protein functional region targeting EGFR and the second protein functional region targeting cMET use light chains with identical sequences. Since only one light chain is used, there is no light-heavy chain mismatch; and since a single cell line can be used for production, this method greatly simplifies the preparation of bispecific antibodies. In some specific embodiments, the bispecific antibody of the present invention consists of a first protein functional region targeting EGFR and a second protein functional region targeting cMET, wherein the first protein functional region targeting EGFR includes a heavy chain targeting EGFR, the second protein functional region targeting cMET includes a heavy chain targeting cMET, and the bispecific antibody also contains two light chains with identical sequences.

[0323] Bispecific antibodies can be produced using heavy chain heterodimerization methods. These methods include "bulges in the pores" and those that alter the charge polarity of the entire Fc dimer interface. Such methods are described in further detail here, and are further described in, for example, U.S. Patent No. 7,183,076; Merchant et al. (1998) *Nature Biotech* 16:677-681; Ridgway et al. (1996) *Protein Engineering* 9:617-621; Davis et al. (2010) *Prot. Eng. Design & Selection* 23:195-202; WO2007 / 110205; WO2007 / 147901; Gunasekaran et al. (2010) *Journal of Biochemistry* 285:19637-46. In these methods, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell cultures. In the “bulge in the cavity” approach, a “bulge” is created by replacing one or more small amino acid side chains at the interface of the first antibody molecule with a larger side chain (e.g., tyrosine or tryptophan). Compensating “cavities” of the same or similar size as the one or more large side chains are created at the interface of the second antibody molecule by replacing amino acids with larger side chains with amino acids with smaller side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers beyond other unwanted end products that resemble dimers.

[0324] In some embodiments, the bispecific antibody is an IgG antibody, such as IgG1, IgG2, IgG3 or IgG4 antibodies or modified forms thereof, as described in the following sections.

[0325] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to produce an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0326] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0327] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0328] antibody expression

[0329] In another aspect, the present invention provides a polynucleotide molecule encoding the bispecific antibody or its antigen-binding fragment described herein. The polynucleotide molecule may comprise a polynucleotide molecule containing an amino acid sequence encoding the light chain variable region and / or the heavy chain variable region of a bispecific antibody, or a polynucleotide molecule containing an amino acid sequence encoding the light chain and / or the heavy chain of a bispecific antibody.

[0330] In another aspect, the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably a eukaryotic expression vector. In some embodiments, the polynucleotide molecule as described herein is contained in one or more expression vectors.

[0331] In another aspect, the present invention provides a host cell comprising a polynucleotide molecule as described herein or an expression vector as described herein, preferably a eukaryotic cell, more preferably a mammalian cell.

[0332] In another aspect, the present invention provides a method for preparing a bispecific antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or the antigen-binding fragment thereof in a host cell as described herein under conditions suitable for expression of the bispecific antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

[0333] This invention provides mammalian host cells for expressing the recombinant antibodies of this invention, including a variety of immortalized cell lines available from the American Type Culture Collection (ATCC). These particularly include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells, and many other cell lines. Mammal host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines exhibit high expression levels.

[0334] In one embodiment, the present invention provides a method for preparing bispecific antibodies, wherein the method includes, when an expression vector is introduced into a mammalian host cell, producing antibodies by culturing the host cells for a sufficient period of time to allow the antibodies to be expressed in the host cells, or more preferably by secreting the antibodies into the culture medium in which the host cells are grown. The antibodies can be recovered from the culture medium using standard protein purification methods.

[0335] Bispecific antibodies expressed in different cell lines or in transgenic animals may have different glycosylations. However, all antibodies encoded by nucleic acid molecules provided herein or containing amino acid sequences provided herein are part of the invention, regardless of antibody glycosylation. Similarly, in some embodiments, non-fucosylated antibodies are advantageous because they generally have stronger efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic because their sugar structure is a normal component of natural human serum IgG.

[0336] Antibody-drug conjugates (ADCs)

[0337] This article also provides an antibody-drug conjugate (ADC) comprising a bispecific antibody or antigen-binding fragment thereof that specifically binds to EGFR and cMET as described in any embodiment of the present invention, and a conjugation portion.

[0338] In some implementations, the conjugated portion includes: antitumor agents, immunomodulators, cytotoxic agents, fluorescent substances, luminescent substances, enzymes, small nucleic acid molecules (including siRNA or antisense oligonucleotides (ASO)) and any combination thereof.

[0339] In some embodiments, the antibody-drug conjugate comprises multiple conjugation portions, which may be a combination of different therapeutic active substances or pharmaceutical active ingredients, or a combination of the same therapeutic active substance or pharmaceutical active ingredient.

[0340] In some implementations, the conjugation portion is covalently linked, either non-site-specifically or site-specifically, to a bispecific antibody or its antigen-binding fragment that specifically binds to EGFR and cMET via a linker.

[0341] Based on the bispecific antibodies or antigen-binding fragments that specifically bind to EGFR and cMET, the inventors prepared a series of antibody-drug conjugates (ADCs) using different linkers and cytotoxic drug components, and verified their activity. They found that the ADCs of the present invention have good inhibitory effects on the proliferation of human gastric cancer cells MKN45 cells and human lung cancer squamous cell carcinoma cells EBC-1 cells that endogenously express human EGFR and human cMET, and have good inhibitory effects on tumor growth in transplanted tumor models (including transplanted human lung adenocarcinoma cells NCI-H292 model, transplanted human gastric cancer cells MKN45 model, and transplanted human colon cancer cells HT29 model).

[0342] In some embodiments, the present invention provides an antibody-drug conjugate having the structure Ab-(LD)m, where Ab is a bispecific antibody or antigen-binding fragment thereof that specifically binds to EGFR and MET as described in any embodiment of the present invention; L is a linker; D is a therapeutically active substance or pharmaceutically active ingredient; and m represents the average number of LD units conjugated to Ab, and m ranges from 1 to 8, preferably 4, and more preferably about 3.0, 3.5, 3.6, 3.7, 3.8, 4.0, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8 or 8.0.

[0343] In some embodiments, the bispecific antibody that specifically binds to EGFR and cMET is the bispecific antibody that specifically binds to EGFR and cMET described in any embodiment of the present invention. In some preferred embodiments, the bispecific antibody that specifically binds to EGFR and cMET is bsAb1, bsAb2, or bsAb3 as described herein.

[0344] In some implementations, antibodies can be conjugated to drugs directly or via a connector.

[0345] In some implementations, the drug fraction D is covalently linked, in a non-site-specific or site-specific manner, to a bispecific antibody or its antigen-binding fragment that specifically binds to EGFR and cMET via a linker L.

[0346] The joint may comprise one or more joint components. In some embodiments, the L structure is as shown in formula (A):

[0347] in:

[0348] L1 represents the functional group that can react with the thiol group and the group or bond formed after the reaction with the thiol group;

[0349] L2 and Y are each independently selected from -(CH2). p -、-(OCH2CH2) p - and -(CH2CH2O) p -;

[0350] L3 is a hydrophilic group;

[0351] L4 is an amino acid group;

[0352] R6 is selected from H, C1-C6 alkoxy, and -(OCH2CH2). p O-C1-C6 alkyl groups and -(CH2CH2O) p -C1-C6 alkyl;

[0353] X is selected from -NH- and -NH(CH2). n C(=O)-、-C(=O)(CH2) n NH- and -(CH2) n C(=O)-;

[0354] Z is selected from C and S;

[0355] n is selected from 0, 1, and 2;

[0356] p is an integer from 1 to 10.

[0357] In some embodiments, the functional group that can react with thiol as described in L1 is selected from maleimide, halogen, halogen-substituted functional groups (such as halogen-substituted aldehydes, halogen-substituted -S(O)2-), aldehyde, alkenyl, alkynyl, ketone, sulfonyl, silane, isocyanate, and norbornene.

[0358] In some embodiments, the functional group that can react with a thiol group, as described in L1, is selected from: And their derivatives; where: A is a halogen; the wavy line indicates the position where L1 and L2 are connected.

[0359] Preferably, the functional group of L1 is:

[0360] In some implementations, L2 is -(CH2). p - and p is an integer from 1 to 4; preferably, L2 is -CH2CH2- or -CH2CH2CH2-.

[0361] In some implementations, X is -NH- or -NH(CH2). nC(=O)-; preferably, X is -NH- or -NHC(=O)-.

[0362] In some implementations, Y is -(CH2). p -, p is 1, 2 or 3; preferably, Y is methylene.

[0363] In some implementations, Z is C.

[0364] In some embodiments, L3 is selected from monosaccharides, disaccharides, and five- or six-membered saturated heterocycles containing 1-2 nitrogen atoms and their derivatives, which are divalent hydrophilic groups with two monovalent centers obtained by removing two hydrogen atoms; preferably, the monosaccharide is selected from trioses, tetraoses, pentoses, hexoses, and heptaoses; preferably, the disaccharide is selected from maltose, sucrose, and lactose; preferably, the five- or six-membered saturated heterocycle containing 1-2 nitrogen atoms or its derivatives are selected from piperazine, piperidinyl, and pyrrolidinyl.

[0365] In some embodiments, the antibody-drug conjugate according to the present invention is characterized in that L3 is selected from glucose, galactose, mannose, glucuronic acid, galactobionic acid, mannuronic acid, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, and N-acetylmuramic acid, which are divalent hydrophilic groups with two monovalent centers obtained by removing two hydrogen atoms.

[0366] In some embodiments, the antibody-drug conjugate according to the present invention is characterized in that L3 has the structure shown in formula L3-1:

[0367] In the formula:

[0368] R1 is selected from -O(CH2) p - and -C(=O)-, p is an integer from 1 to 6, preferably 1, 2, 3 or 4;

[0369] R2, R3, and R4 are each independently selected from H, C1-C6 alkyl groups, and -(CH2CH2O). t -C1-C6 alkyl groups, sulfonic acid groups, and phosphate groups;

[0370] t is an integer from 1 to 20;

[0371] The wavy line indicates the position where L3 connects to X and Y.

[0372] In some embodiments, L4 is selected from the following amino acid residues: valine, citrulline, alanine, glycine, phenylalanine, asparagine, glutamic acid, lysine, serine, threonine, cysteine, and tyrosine; the amino acid group is optionally surrounded by one or more -OR groups. 20 Replaced, of which R20 Selected from H, C1-C6 alkyl groups, and -(CH2CH2O). t -C1-C6 alkyl, sulfonic acid and phosphate groups, where t is an integer from 1 to 20.

[0373] In some embodiments, L4 is selected from the following peptides: valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), glycine-glycine-phenylalanine-glycine, glycine-glycine-phenylalanine-glycine, citrulline-valine, alanine-valine, alanine-alanine, citrulline-alanine, asparagine-citrulline, citrulline-asparagine, citrulline-citrulline, phenylalanine-lysine, and lysine-phenylalanine, preferably from the following peptides: valine-citrulline (VC), valine-alanine (VA), and glycine-glycine-phenylalanine-glycine (GGFG); each peptide is optionally substituted with one or more substituents selected from the group consisting of: -OR 20 and -(OCH2CH2) p O-C1-C6 alkyl, wherein R 20 Selected from H, C1-C6 alkyl groups, and -(CH2CH2O). t -C1-C6 alkyl, sulfonic acid and phosphate groups, t is an integer from 1 to 20, p is an integer from 1 to 6.

[0374] In some implementations, L4 is selected from:

[0375] The wavy line indicates the position where L4 connects to the rest of Equation (A).

[0376] In some embodiments, R6 is H, C1-C3 alkoxy, or -(OCH2CH2). p O-C1-C3 alkyl, wherein p is an integer from 1 to 6; preferably, R6 is H, methoxy, or -(OCH2CH2). p OCH3, where p is an integer from 1 to 6 or from 1 to 4.

[0377] In some embodiments, the structure of the connector L is as described in any of the following formulas:

[0378] In each formula, R2, R3, R4 and R6 are as described in any embodiment of this document, R” is H or C1-C6 alkyl; the wavy line indicates the position where L is connected to the rest of the ADC.

[0379] In some embodiments, the connector is selected from maleimide-hexanoyl-valine-citrulline- p-Aminobenzyloxy (mc-vc-PAB), Acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimide hexanoic acid (mc), maleimide propionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimide 4-(2-pyridinylthio)valerate (SPP), N-succinimide 4-(N-maleimide methyl)-cyclohexane-1-carboxylate (SMCC), N-succinimide (4-iodo-acetyl)aminobenzoate (SIAB), N-succinimide 4-(2-pyridinyl dithio)butyrate (SPDB), N-succinimide 3-(pyridin-2-yl dithio)propionate (SPDP).

[0380] In some embodiments, the drug component D includes cytotoxins, plant toxins, small molecule toxins, radioactive isotopes, etc. In some embodiments, the drug component D includes microtubule inhibitors and DNA damaging agents.

[0381] In some preferred embodiments, the microtubule inhibitors include dolastatin and auristatin and maytansine; the DNA damaging agents include calicheamicin, duocarmycin, pyrrolobenzodiazepine (PBD), and camptothecin.

[0382] In some preferred embodiments, the auristatin class of drugs includes monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF) or their derivatives; the maytansine class of drugs includes, but is not limited to, DM1, DM3, DM4 or their derivatives; the camptothecin class of drugs includes, but is not limited to: Exatecan (e.g., CAS Nr:171335-80-1), Deruxtecan (Dxd), SN38, camptothecin, topotecan, irinotecan, belotetane, letopotecan, rubitecan, cilatecan, cocinotecan, gemmatotecan, eczetane, topotecan, and 9-nitrocamptothecin.

[0383] In some embodiments, the drug component D may also be a vitamin A precursor, folic acid, etc. The drug component D is not limited to the above categories, but also includes all drugs that can be used in ADCs.

[0384] In some embodiments, the connector-drug (LD) portion is selected from the structures shown in the following formulas: VC-MMAE, GGFG-Deruxtecan, CPD2-Exatecan, CPD4-Exatecan, CPD5-Exatecan, CPD6-Exatecan, or SN-38Comp5.

[0385] It should be understood that the structures shown above, such as MMAE, Deruxtecan, CPD2, CPD4, CPD5, CPD6, or SN-38Comp5, are coupled to the antibody via the reaction of their maleimide groups with the thiol groups of the antibody.

[0386] In some embodiments, the antibody-drug conjugate is selected from the structures represented by formula ADC-I, ADC-II, ADC-III, ADC-IV, or ADC-V:

[0387] In the formula, m represents the average number of linker-drug (LD) units coupled to Ab, and m ranges from 1 to 10, preferably from 4 to 8; preferably, the Ab is bsAb1, bsAb2 or bsAb3 as described herein.

[0388] It should be understood that in the antibody-drug conjugates described in this invention, the connection position between the drug portion D and the remaining portion of the linker-drug conjugate generally does not affect the biological activity of drug D itself. Those skilled in the art can easily determine the active site of drug D based on existing technology and covalently link it to the remaining portion of the linker-drug conjugate at a location far from the active site. Covalent linking methods are well known in the art, including via amide groups (-NRCO-, where R is H or C1-C4 alkyl), ester bonds (-COO-), -S-, etc.

[0389] In some implementations, the ADC described herein is selected from bsAb1-GGFG-Dxd-DAR8, bsAb1-CPD2-Exatecan-DAR8, bsAb1-CPD6-Exatecan-DAR8, bsAb2-MMAF-DAR4, bsAb2-GGFG-Dxd-DAR4, bsAb2-CPD6-Exatecan-DAR4, and bsAb3-CPD6-Exatecan-DAR4.

[0390] The ADC described herein can be prepared via several pathways using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) reacting the nucleophilic group of an antibody with a divalent linker reagent via a covalent bond to form an antibody-linker, followed by a reaction with a drug; and (2) reacting the nucleophilic group of a drug module with a divalent linker reagent to form a drug-linker, followed by a reaction with the nucleophilic group of an antibody.

[0391] Pharmaceutical compositions and pharmaceutical preparations

[0392] In another aspect, the present invention provides a pharmaceutical composition comprising a bispecific antibody or antigen-binding fragment thereof that specifically binds to EGFR and MET as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell or an immunoconjugate as described herein, and a pharmaceutically acceptable carrier or excipient. It should be understood that the bispecific antibody or pharmaceutical composition thereof that specifically binds to EGFR and MET provided by the present invention can integrate suitable carriers, excipients, and other agents in a formulation for co-administration, thereby providing improved transfer, delivery, tolerability, etc.

[0393] The term "pharmaceutical composition" refers to a formulation that allows the biologically effective form of the active ingredient contained therein to be present, and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation.

[0394] Pharmaceutical formulations containing the anti-FGFR2b antibody described herein can be prepared by mixing the bispecific antibody of the present invention, which specifically binds to EGFR and MET and has the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. editor (1980)). Preferably, the formulation is in the form of an aqueous solution or a lyophilized preparation.

[0395] In another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, a pharmaceutical composition or an immunoconjugate described herein, and one or more additional therapeutic agents.

[0396] Medical uses and treatment methods

[0397] Any bispecific antibody that specifically binds to EGFR and MET provided herein can be used for therapeutic purposes. It should also be understood that, when discussing "antibody," compositions containing antibodies are also included. The bispecific antibody that specifically binds to EGFR and MET of the present invention can be used in therapeutic or preventative amounts in any embodiment of the therapeutic or preventative method described herein.

[0398] In another aspect, the present invention provides the use of the bispecific antibodies or antigen-binding fragments thereof that specifically bind EGFR and MET as described herein, the polynucleotide molecules described herein, the expression vectors described herein, the host cells described herein, the antibody-drug conjugates described herein, or the pharmaceutical compositions described herein in the preparation of medicaments for treating and / or preventing EGFR and / or MET-mediated diseases or conditions, preferably said diseases or conditions being cancer, more preferably said cancers being selected from lung cancer (including non-small cell lung cancer, small cell lung cancer), gastric cancer (including gastric cancer, gastroesophageal junction cancer), glial carcinoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, bladder cancer, colon cancer, rectal cancer, liver cancer, brain cancer, thyroid cancer, and head and neck cancer (including head and neck squamous cell carcinoma).

[0399] In another aspect, the present invention provides antibodies or antigen-binding fragments thereof described herein, polynucleotide molecules described herein, expression vectors described herein, host cells described herein, immunoconjugates described herein, or pharmaceutical compositions described herein for the treatment and / or prevention of EGFR and / or MET-mediated diseases or conditions, preferably said diseases or conditions being cancer, more preferably said cancers selected from lung cancer (including non-small cell lung cancer, small cell lung cancer), gastric cancer (including gastric cancer, gastroesophageal junction cancer), glial carcinoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, bladder cancer, colon cancer, rectal cancer, liver cancer, brain cancer, thyroid cancer, and head and neck cancer (including head and neck squamous cell carcinoma).

[0400] In another aspect, the present invention provides a method for treating and / or preventing EGFR and / or MET-mediated diseases or conditions, comprising administering to a subject in need an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, an immunoconjugate described herein, or a pharmaceutical composition or combination described herein, preferably the disease or condition being cancer, more preferably the cancer being selected from lung cancer (including non-small cell lung cancer, small cell lung cancer), gastric cancer (including gastric cancer, gastroesophageal junction cancer), glial carcinoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, bladder cancer, colon cancer, rectal cancer, liver cancer, brain cancer, thyroid cancer, and head and neck cancer (including head and neck squamous cell carcinoma). In some embodiments, the present invention provides a method for treating diseases and / or conditions requiring blocking EGFR and / or MET, the method comprising administering to a subject in need a bispecific antibody that specifically binds to EGFR and MET as described herein, or an antigen-binding fragment thereof, a polynucleotide molecule as described herein, an expression vector as described herein, a host cell as described herein, an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein.

[0401] In some embodiments, the cancers or tumors described herein may be selected from lung cancer (including non-small cell lung cancer and small cell lung cancer), gastric cancer (including gastric cancer and gastroesophageal junction cancer), glial cell carcinoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, bladder cancer, colon cancer, rectal cancer, liver cancer, brain cancer, thyroid cancer, and head and neck cancer (including head and neck squamous cell carcinoma).

[0402] In some embodiments, the administration methods of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intra-articular, intra-articular (e.g., in arthritic joints), inhalation, aerosol delivery, or intratumoral administration.

[0403] Methods for diagnosis and detection

[0404] In another aspect, the present invention provides a method for detecting the presence of EGFR and / or MET in a sample using an antibody or antigen-binding fragment thereof described herein. The term "detection" as used herein includes quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. The method includes contacting the sample with an antibody or antigen-binding fragment thereof described herein, or a detection composition containing a bispecific antibody or antigen-binding fragment thereof that specifically binds to EGFR and MET, and detecting the presence of a conjugate or binding signal generated by the bispecific antibody or antigen-binding fragment thereof that specifically binds to EGFR and / or MET. When used for detection purposes, the antibody or antigen-binding fragment thereof described herein may be labeled to indicate whether the conjugate has been formed.

[0405] sequence

[0406] The sequences involved in this invention are as follows (the underlined parts are CDRs of the Kabat numbering scheme):

[0407] (1)E2-M1-VH

[0408] (2)E2-M2-VH

[0409] (3)E2-M5-VH

[0410] (4)E2-M12-VH

[0411] (5)E2-M13-VH

[0412] (6)E2-M16-VH

[0413] (7)E2-M17-VH

[0414] (8)E2-M25-VH

[0415] (9)E2-M34-VH

[0416] (10)E2-M38-VH

[0417] (11)E2-10-LC-13-91A

[0418] The sequences of three light chain CDRs are as follows:

[0419] LCDR1:RASQSVSSWLA(SEQ ID NO:13)

[0420] LCDR2: GASNRAT (SEQ ID NO:14)

[0421] LCDR3:LQAGSTPLT(SEQ ID NO:15)

[0422] The light chain variable region (VL) sequence is as follows:

[0423] (12)E2-10-LC-69

[0424] The sequences of three light chain CDRs are as follows:

[0425] LCDR1:RASQSVSSWLA(SEQ ID NO:13)

[0426] LCDR2: GASNRAT (SEQ ID NO:14)

[0427] LCDR3:LQAGSTPLT(SEQ ID NO:20)

[0428] The light chain variable region (VL) sequence is as follows:

[0429] (13)M5-HC-K409R (where the italicized part is the heavy chain variable region)

[0430] The sequences of the three heavy chain CDRs are as follows:

[0431] HCDR1: SSVYYWS (SEQ ID NO:22)

[0432] HCDR2:VIYPSGNTYYSPSLKS(SEQ ID NO:23)

[0433] HCDR3:TIYDLFDI(SEQ ID NO:24)

[0434] The VH sequence of the heavy chain variable region is as follows:

[0435] (14)E2mut34-HC-F405L (where the italicized part is the variable region of the heavy chain)

[0436] The sequences of the three heavy chain CDRs are as follows:

[0437] HCDR1: SGDYYWS (SEQ ID NO:25)

[0438] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0439] HCDR3:ARVSIYEDSGFDY(SEQ ID NO:35)

[0440] The sequence of the heavy chain variable region VH is as follows:

[0441] (15)E2mut34-HC-hole (where the italicized part is the heavy chain variable region)

[0442] The sequences of the three heavy chain CDRs are as follows:

[0443] HCDR1: SGDYYWS (SEQ ID NO:25)

[0444] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0445] HCDR3: ARVSIYEDSGFDY (SEQ ID NO:35); The sequence of the heavy chain variable region VH is as follows:

[0446] (16)M5-HC-knob (where the italicized part is the heavy chain variable region)

[0447] The sequences of the three heavy chain CDRs are as follows:

[0448] HCDR1: SSVYYWS (SEQ ID NO:22)

[0449] HCDR2:VIYPSGNTYYSPSLKS(SEQ ID NO:23)

[0450] HCDR3:TIYDLFDI(SEQ ID NO:24)

[0451] The VH sequence of the heavy chain variable region is as follows:

[0452] (17)E2-10-LC-13-91Q

[0453] The sequences of three light chain CDRs are as follows:

[0454] LCDR1:RASQSVSSWLA(SEQ ID NO:13)

[0455] LCDR2: GASNRAT (SEQ ID NO:14)

[0456] LCDR3:LQQGSTPLT(SEQ ID NO:21)

[0457] The light chain variable region (VL) sequence is as follows:

[0458] (18)E2mut1-HC-F405L (where the italicized part is the heavy chain variable region)

[0459] The sequences of the three heavy chain CDRs are as follows:

[0460] HCDR1: SGDYYWS (SEQ ID NO:25)

[0461] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0462] HCDR3:ARVSIYKDSGFDY(SEQ ID NO:28)

[0463] The VH sequence of the heavy chain variable region is as follows:

[0464] (19)E2mut2-HC-F405L (where the italicized part is the variable region of the heavy chain)

[0465] The sequences of the three heavy chain CDRs are as follows:

[0466] HCDR1: SGDYYWS (SEQ ID NO:25)

[0467] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0468] HCDR3:ARVSVYKDSGFDY(SEQ ID NO:29)

[0469] The VH sequence of the heavy chain variable region is as follows:

[0470] (20)E2mut5-HC-F405L (where the italicized part is the heavy chain variable region)

[0471] The sequences of the three heavy chain CDRs are as follows:

[0472] HCDR1: SGDYYWS (SEQ ID NO:25)

[0473] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0474] HCDR3:ARVSVYQDSGFDY(SEQ ID NO:30)

[0475] The VH sequence of the heavy chain variable region is as follows:

[0476] (21)E2mut12-HC-F405L (where the italicized part is the variable region of the heavy chain)

[0477] The sequences of the three heavy chain CDRs are as follows:

[0478] HCDR1: SGDYYWS (SEQ ID NO:25)

[0479] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0480] HCDR3:ARVSVYEDSRFDY(SEQ ID NO:31)

[0481] The VH sequence of the heavy chain variable region is as follows:

[0482] (22)E2mut13-HC-F405L (where the italicized part is the variable region of the heavy chain)

[0483] The sequences of the three heavy chain CDRs are as follows:

[0484] HCDR1: SGDYYWS (SEQ ID NO:25)

[0485] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0486] HCDR3:ARVSVYSDSMFDY(SEQ ID NO:32)

[0487] The VH sequence of the heavy chain variable region is as follows:

[0488] (23)E2mut16-HC-F405L (where the italicized part is the variable region of the heavy chain)

[0489] The sequences of the three heavy chain CDRs are as follows:

[0490] HCDR1: NGDYYWS (SEQ ID NO:26)

[0491] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0492] HCDR3:ARVSLYKDSRFDY(SEQ ID NO:33)

[0493] The VH sequence of the heavy chain variable region is as follows:

[0494] (24)E2mut17-HC-F405L (where the italicized part is the heavy chain variable region)

[0495] The sequences of the three heavy chain CDRs are as follows:

[0496] HCDR1: SGDYYWS (SEQ ID NO:25)

[0497] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0498] HCDR3:ARVSLYKDSRFDY(SEQ ID NO:33)

[0499] The VH sequence of the heavy chain variable region is as follows:

[0500] (25)E2mut25-HC-F405L (where the italicized part is the variable region of the heavy chain)

[0501] The sequences of the three heavy chain CDRs are as follows:

[0502] HCDR1: SGDYYWS (SEQ ID NO:25)

[0503] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0504] HCDR3:ARVSVYKDSRFDY(SEQ ID NO:34)

[0505] The VH sequence of the heavy chain variable region is as follows:

[0506] (26)E2mut38-HC-F405L (where the italicized part is the heavy chain variable region)

[0507] The sequences of the three heavy chain CDRs are as follows:

[0508] HCDR1: SGDYYWS (SEQ ID NO:25)

[0509] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0510] HCDR3:ARVSLYEDSGFDY(SEQ ID NO:36)

[0511] The VH sequence of the heavy chain variable region is as follows:

[0512] (27)E2mut1-HC-hole (where the italicized part is the heavy chain variable region)

[0513] The sequences of the three heavy chain CDRs are as follows:

[0514] HCDR1: SGDYYWS (SEQ ID NO:25)

[0515] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0516] HCDR3:ARVSIYKDSGFDY(SEQ ID NO:28)

[0517] The VH sequence of the heavy chain variable region is as follows:

[0518] (28)E2mut2-HC-hole (where the italicized part is the variable region of the heavy chain)

[0519] The sequences of the three heavy chain CDRs are as follows:

[0520] HCDR1: SGDYYWS (SEQ ID NO:25)

[0521] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0522] HCDR3:ARVSVYKDSGFDY(SEQ ID NO:29)

[0523] The VH sequence of the heavy chain variable region is as follows:

[0524] (29)E2mut5-HC-hole (where the italicized part is the heavy chain variable region)

[0525] The sequences of the three heavy chain CDRs are as follows:

[0526] HCDR1: SGDYYWS (SEQ ID NO:25)

[0527] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0528] HCDR3:ARVSVYQDSGFDY(SEQ ID NO:30)

[0529] The VH sequence of the heavy chain variable region is as follows:

[0530] (30)E2mut12-HC-hole (where the italicized part is the heavy chain variable region)

[0531] The sequences of the three heavy chain CDRs are as follows:

[0532] HCDR1: SGDYYWS (SEQ ID NO:25)

[0533] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0534] HCDR3:ARVSVYEDSRFDY(SEQ ID NO:31)

[0535] The VH sequence of the heavy chain variable region is as follows:

[0536] (31)E2mut13-HC-hole (where the italicized part is the heavy chain variable region)

[0537] The sequences of the three heavy chain CDRs are as follows:

[0538] HCDR1: SGDYYWS (SEQ ID NO:25)

[0539] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0540] HCDR3:ARVSVYSDSMFDY(SEQ ID NO:32)

[0541] The VH sequence of the heavy chain variable region is as follows:

[0542] (32)E2mut16-HC-hole (where the italicized part is the heavy chain variable region)

[0543] The sequences of the three heavy chain CDRs are as follows:

[0544] HCDR1: NGDYYWS (SEQ ID NO:26)

[0545] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0546] HCDR3:ARVSLYKDSRFDY(SEQ ID NO:33)

[0547] The VH sequence of the heavy chain variable region is as follows:

[0548] (33)E2mut17-HC-hole (where the italicized part is the heavy chain variable region)

[0549] The sequences of the three heavy chain CDRs are as follows:

[0550] HCDR1: SGDYYWS (SEQ ID NO:25)

[0551] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0552] HCDR3:ARVSLYKDSRFDY(SEQ ID NO:33)

[0553] The VH sequence of the heavy chain variable region is as follows:

[0554] (34)E2mut25-HC-hole (where the italicized part is the heavy chain variable region)

[0555] The sequences of the three heavy chain CDRs are as follows:

[0556] HCDR1: SGDYYWS (SEQ ID NO:25)

[0557] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0558] HCDR3:ARVSVYKDSRFDY(SEQ ID NO:34)

[0559] The VH sequence of the heavy chain variable region is as follows:

[0560] (35)E2mut38-HC-hole (where the italicized part is the heavy chain variable region)

[0561] The sequences of the three heavy chain CDRs are as follows:

[0562] HCDR1: SGDYYWS (SEQ ID NO:25)

[0563] HCDR2:YIYYSGSTDYNPSLKS(SEQ ID NO:27)

[0564] HCDR3:ARVSLYEDSGFDY(SEQ ID NO:36)

[0565] The VH sequence of the heavy chain variable region is as follows:

[0566] The advantages of this invention include:

[0567] This invention develops an antibody-drug conjugate targeting a bispecific antibody against EGFR and cMET. While retaining the activity of blocking EGF and HGF signaling pathways, it also enhances the killing mechanism of small molecule chemotherapeutic drugs. It can be used to treat cancer patients with EGFR and cMET overexpression (including but not limited to non-small cell lung cancer, colorectal cancer, advanced gastric / gastroesophageal junction cancer, head and neck squamous cell carcinoma, etc.), effectively improving the quality of life and prolonging the survival of cancer patients.

[0568] This invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific embodiments indicate preferred embodiments of the invention, these descriptions and embodiments are provided by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For all purposes, all disclosures, patents, and patent applications cited herein, including in citations, are incorporated herein by reference in their entirety.

[0569] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0570] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0571] Preparation Example 1: Sequence Design of Heavy Chain for Anti-EGFR Monoclonal Antibody

[0572] Ten anti-EGFR monoclonal antibodies were designed and screened, named E2-M1, E2-M2, E2-M5, E2-M12, E2-M13, E2-M16, E2-M17, E2-M25, E2-M34, and E2-M38. These ten antibodies belong to a mutant of the same parent antibody E2. The amino acid sequences of the heavy chain variable region of these ten anti-EGFR monoclonal antibodies are shown in SEQ ID NO:1-10. HCDR was determined using the Kabat numbering system, as shown in Table A below.

[0573] Table A

[0574] The structure of HCDR3 in the above antibody is shown in SEQ ID NO:37 below: ARVSX1YX2DSX3FD (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.

[0575] Preparation Example 2: Design and Preparation of EGFR×cMET Bispecific Antibody

[0576] 1) Construction of bispecific antibody molecules

[0577] The 10 high-affinity EGFR antibody heavy chains obtained in Preparation Example 1 above were used as the heavy chains of the EGFR-terminal parent antibody of the EGFR×cMET bispecific antibody. Alternatively, referring to the heavy chain of the published cMET antibody (SEQ ID NO: 410 in CN105705519A), after making appropriate modifications to the constant region according to the needs of preparing the bispecific antibody, it was used as the heavy chain of the cMET-terminal parent antibody of the EGFR×cMET bispecific antibody (see SEQ ID NO: 19). E2-10-LC-13-91A, E2-10-LC-69, or E2-10-LC-13-91Q were used as the common light chain.

[0578] The DNA sequences of light chains E2-10-LC-13-91A, E2-10-LC-69, or E2-10-LC-13-91Q, as shown in Table 1, were synthesized by Qingke Biotechnology. The DNA was digested with SapI (purchased from NEB, catalog number: R0569L) and ligated into the HXT2 vector (a vector independently modified by Junshi Biosciences, derived from pTT5).

[0579] The DNA sequences of EGFR antibodies E2mut34-HC-hole, E2mut1-HC-hole, E2mut2-HC-hole, E2mut5-HC-hole, E2mut12-HC-hole, E2mut13-HC-hole, E2mut16-HC-hole, E2mut17-HC-hole, E2mut25-HC-hole, and E2mut38-HC-hole, as well as the MET antibody M5-HC-knob heavy chain, were synthesized by Qingke Biotechnology. These sequences were then digested with SapI (purchased from NEB, catalog number: R0569L) and ligated into the HXT1S vector (a modified version developed by Junshi Biosciences). The vector was derived from pTT5, and the antibodies prepared were named E2mut34-91A×M5-91A, E2mut34-69×M5-69, E2mut1-91Q×M5-91Q, E2mut2-91Q×M5-91Q, E2mut5-91Q×M5-91Q, E2mut12-91Q×M5-91Q, E2mut13-91Q×M5-91Q, E2mut16-91Q×M5-91Q, E2mut17-91Q×M5-91Q, E2mut25-91Q×M5-91Q, E2mut34-91Q×M5-91Q, and E2mut38-91Q×M5-91Q.

[0580] Table 1 Heavy and light chains of EGFR×cMET bispecific antibodies

[0581] 2) Transient transtransfer protein expression and purification

[0582] The CHO-K1 cells being cultured (provided by Suzhou Junmeng) were counted, and when the cell density was 2-6 × 10⁻⁶... 6 At a density of 1.8-2.5 × 10⁶ cells / ml, the cells were passaged and amplified using CD CHO medium (purchased from Thermofisher, catalog number: 12490-001). The cell density was diluted to 1.8-2.5 × 10⁶ cells / ml one day before transfection. 6 / ml, the next day when the cell density reached approximately 3.5-5.0×10⁶. 6 Transfection was performed at a concentration of 1 / ml. First, one-tenth of the transfection volume of CD CHO medium was added, followed by 1-2 μg / ml of plasmid (prepared by the company) according to the combination shown in Table 2. Finally, 3-14 μg / ml of PEI (purchased from Polysciences, catalog number: 24765-1) was added, mixed well, and incubated at room temperature. The transfection mixture was then slowly added to the pre-treated cells, mixing constantly. The transfected mixture was placed in a shaker for incubation. On the first day after transfection, 4% Cell Boost 7a (purchased from Hyclone, catalog number: SH31026.05) and 0.4% Cell Boost 7b (purchased from Hyclone, catalog number: SH31027.04CN) were added as feed, followed by feeding every two days. Samples were collected 5-9 days post-transfection.

[0583] 3) Affinity capture of bispecific antibody molecules

[0584] After culture, the cells were centrifuged at 1000g for 5 min using a floor centrifuge (Thermo Fisher, R404A) to remove the precipitate. Then, the cells were centrifuged at 8000g for 30 min to collect the supernatant, which was then aseptically filtered using a 0.22μm filter cup (JET, FPE-214-000). Purification was performed using a protein purification system (GE, AKTA Avant). The Mabselect Sure LX column (Cytiva, 17547403) was equilibrated with PBS equilibration buffer (Wuxi Aorui Dongyuan Biotechnology Co., Ltd., ZLI-9061). After sample loading, first rinse with affinity chromatography elution buffer A (pH 5.5, 45 mM acetate-sodium acetate + 1 M sodium chloride system), then rinse with elution buffer B (pH 5.5, 45 mM acetate-sodium acetate system), and finally elute the target protein with affinity elution buffer (pH 3.6, 10 mM acetate-sodium acetate buffer). Neutralize the sample with 1 M Tris buffer (purchased from Merck, catalog number: E300016981946) and adjust the pH to 5.5-6.

[0585] 4) Purification of bispecific antibody molecules

[0586] Capto™ MMC Impres (Cytiva, 17371602) packing material was used for purification. The system was pre-equilibrated using washing buffer (pH 7.5, 20 mM Tris-HCl + 1 M NaCl buffer) and then equilibrated using equilibration buffer (pH 7.5, 20 mM Tris-HCl). After sample loading, equilibrate with 3–6 column volumes of equilibration buffer, and finally perform linear elution with elution buffer (pH 7.5, 20 mM Tris-HCl + 1 M NaCl buffer) to collect the target protein.

[0587] Preparation Example 3: Preparation of EGFR and cMET Monoclonal Antibodies

[0588] This preparation example prepared EGFR-terminal and cMET-terminal precursor antibodies for EGFR×cMET bispecific antibodies. Specifically, for the EGFR monoclonal antibody, the 10 high-affinity EGFR antibody heavy chains obtained in Preparation Example 1 were used as the heavy chain, and E2-10-LC-13-91A, E2-10-LC-69, or E2-10-LC-13-91Q were used as the light chain. For the cMET monoclonal antibody, the heavy chain of the published MET antibody (SEQ ID NO: 410 in CN105705519A) was used as the heavy chain, and E2-10-LC-13-91A, E2-10-LC-69, or E2-10-LC-13-91Q were used as the light chain. The constant region (Fc) of the antibody was further mutated to obtain the heavy and light chain sequences shown in Table 2.

[0589] The heavy and light chains in Table 2 were synthesized by Qingke Biotechnology. The plasmid construction and antibody expression methods were the same as those in Preparation Example 2.

[0590] Table 2 Heavy and light chains of EGFR monoclonal antibodies and cMET monoclonal antibodies

[0591] Example 1: Biacore detection combined with dissociation kinetics

[0592] The binding affinity of the EGFR-terminal progenitor antibody and the cMET-terminal progenitor antibody of the EGFR×cMET bispecific antibody to recombinant human EGFR and human cMET was detected using a Biacore T200 molecular interaction analyzer (GE Healthcare Life Sciences).

[0593] The method for determining the binding affinity of the EGFR-terminal precursor antibody of the EGFR×cMET bispecific antibody to human EGFR is as follows: 40 μg / mL of goat anti-human IgG-Fc fragment antibody (Jackson ImmunoResearch) was conjugated to the surface of a CM5 chip (Cytiva, catalog number BR-1005-30) for antibody capture. 1 μg / mL of ALK101 antibody was captured on the CM5 chip surface, and 40 nM and 10 nM human EGFR were injected to bind with the antibody. Binding and dissociation kinetics were detected using a Biacore T200 system (GE Healthcare). The affinity KD value was calculated using Biacore T200 Evaluation Software 3.0 to fit the binding and dissociation curves.

[0594] The method for determining the binding affinity between the cMET-terminal precursor antibody of the EGFR×cMET bispecific antibody and human cMET is as follows: 40 μg / mL of goat anti-human IgG-Fc fragment antibody (Jackson ImmunoResearch) was conjugated to the surface of a CM5 chip (Cytiva, catalog number BR-1005-30) for antibody capture. 1 μg / mL of ALK101 antibody was captured on the CM5 chip surface, and 40 nM and 10 nM of human cMET were injected to bind with the antibody. Binding and dissociation kinetics were detected using a Biacore T200 system (GE Healthcare). The affinity KD value was calculated using Biacore T200 Evaluation Software 3.0 to fit the binding and dissociation curves.

[0595] The results are shown in Table 3. E2mut34-91A-F405L-LF, E2mut34-69-F405L-LF, 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 with human EGFR, with affinity ranging from 38.5 nM to 1.09 nM. Based on affinity, E2mut34-91A-F405L-LF and E2mut34-69-F405L-LF were defined as high-affinity antibodies (K). DApproximately 1 nM); E2mut12-91Q-F405L-LF, E2mut13-91Q-F405L-LF, and E2mut34-91Q-F405L-LF were defined as intermediate affinity antibodies (K). D Approximately 5–8 nM); E2mut1-91Q-F405L-LF, E2mut2-91Q-F405L-LF, E2mut5-91Q-F405L-LF, E2mut16-91Q-F405L-LF, E2mut17-91Q-F405L-LF, E2mut25-91Q-F405L-LF, and E2mut38-91Q-F405L-LF are defined as low-affinity antibodies (K). D (Approximately 15–38 nM). M5-91Q-K409R-LF, M5-91A-K409R-LF, and M5-69-K409R-LF bind to active human cMET with high affinity and comparable affinity, ranging from 0.357 to 0.43 nM.

[0596] Table 3: Statistical Table of Affinity of EGFR Monoclonal Antibody and cMET Monoclonal Antibody Determined by Biacore

[0597] Example 2: Endocytosis Experiment

[0598] Internalization activity is a key indicator for ADC drugs. Example 2 yielded a series of EGFR×cMET bispecific antibodies. As shown in Example 1, these bispecific antibodies have similar MET-terminal affinities, while their EGFR-terminal affinities differ by approximately 35-fold. To assess the impact of EGFR-terminal affinity differences on the internalization activity of EGFR×MET bispecific antibodies, this example selected several representative bispecific antibody molecules to detect internalization activity. Among them, E2mut1-91Q×M5-91Q and E2mut25-91Q×M5-91Q are EGFR low-affinity antibodies (K... D Approximately 15–38 nM), E2mut12-91Q×M5-91Q and E2mut13-91Q×M5-91Q are EGFR intermediate affinity antibodies (K). D Approximately 5–8 nM), E2mut34-91A×M5-91A and E2mut34-69×M5-69 are high-affinity EGFR antibodies (K). D (Approximately 1 nM). The sample used in this embodiment adopts the technical solution disclosed in WO2011131746 and is prepared using Fab arm exchange technology.

[0599] After digesting and centrifuging MKN45 cells, resuspend them in staining buffer (PBS + 1 v / v % FBS), and add 2 × 10⁻⁶ cells per well. 5 Cells were seeded in 96-well round-bottom plates (Corning, catalog number: 3799). Antibodies E2mut12-91Q×M5-91Q, E2mut13-91Q×M5-91Q, and JNJ-372 (amivantamab, antibody preparation reference US2017275367A1) diluted with staining buffer (PBS + 1 v / v % FBS) (25 μg / ml) were added and incubated at 4°C for 30 min. Unbound antibodies were then washed away with staining buffer (PBS + 1 v / v % FBS). Cells were resuspended in 200 μl of complete culture medium (RPMI-1640 medium + 10 v / v % FBS), and then divided in half. One half was incubated at 4°C for 24 h, and the other at 37°C for 24 h. After incubation, the supernatant was discarded by centrifugation, and a fluorescent secondary antibody containing 5‰ (v / v) goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) prepared with staining buffer was added and incubated at 4°C in the dark for 30 minutes. Unbound antibodies were then washed away with staining buffer (PBS + 1v / v % FBS), fixed with PFA (Absin, catalog number: abs9179), and collected on a flow cytometer (BD, C6 PLUS model) to detect the fluorescent antibody bound to the cell surface. The raw data were analyzed using FlowJo to obtain the MFI value, and the Top value was obtained by fitting an antibody dose-dependent binding curve using GraphPad. The internalization rate was calculated as: Internalization% = (1 - Top_37℃ / Top_4℃) × 100. The results are shown in Table 4.

[0600] Table 4: Results of endocytic activity assay in MKN45 cells

[0601] The results showed that E2mut34-91A×M5-91A-FAE-LF, E2mut34-69A×M5-91A-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 the endocytosis activities were comparable, with endocytosis rates ranging from 72% to 84% over 24 hours.

[0602] NUGC4 cells (endogenously expressing human EGFR and human cMet) and BaF3-EGFR-cMet (overexpressing human EGFR and human cMet) were digested, centrifuged, and resuspended in staining buffer (PBS + 1 v / v % FBS). 2 × 10⁻⁶ cells were added to each well. 5 Cells were seeded in 96-well round-bottom plates (Corning, catalog number: 3799). Antibodies E2mut1-91Q×M5-91Q, E2mut12-91Q×M5-91Q, E2mut13-91Q×M5-91Q, E2mut13-91Q×M5-91Q, and JNJ-372 (100 μg / ml) diluted with staining buffer (PBS + 1 v / v % FBS) were added and incubated at 4°C for 30 min. Unbound antibodies were then washed away with staining buffer (PBS + 1 v / v % FBS). The cells were resuspended in 200 μl of complete culture medium (RPMI-1640 medium + 10 v / v % FBS), and then divided in half. One half was incubated at 4°C for 24 h, and the other at 37°C for 24 h. After incubation, the supernatant was discarded by centrifugation, and a fluorescent secondary antibody containing 5‰ (v / v) goat anti-human IgG PE (SouthernBiotech, Cat#2040-09) prepared with staining buffer was added and incubated at 4°C in the dark for 30 minutes. Unbound antibodies were then washed away with staining buffer (PBS + 1v / v % FBS), fixed with PFA (Absin, catalog number: abs9179), and collected on a flow cytometer (BD, C6 PLUS model) to detect the fluorescent antibody bound to the cell surface. The raw data were analyzed using FlowJo to obtain the MFI value, and the Top value was obtained by fitting an antibody dose-dependent binding curve using GraphPad. The internalization rate was calculated as: Internalization% = (1 - Top_37℃ / Top_4℃) × 100. The results are shown in Tables 5 and 6.

[0603] Table 5: Results of endocytic activity assay in NUGC4 cells

[0604] Table 6: Results of endocytic activity assay in BaF3-EGFR-cMET cells

[0605] 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, and the endocytic activity was comparable.

[0606] In summary, the inventors believe that the five EGFR×cMET bispecific antibodies used in this embodiment are representative. Therefore, the series of EGFR×cMET bispecific antibodies with different EGFR end affinities disclosed in this patent all have excellent endocytic activity, and the difference in EGFR end affinity does not affect the endocytic efficiency.

[0607] Example 3: Preparation of Antibody-Drug Conjugates (ADCs)

[0608] To evaluate the effect of EGFR×cMET bispecific antibodies with different EGFR-terminal affinities on the in vitro and in vivo activity of bispecific antibody-drug conjugates (ADCs), three EGFR×cMET bispecific antibodies with similar cMET-terminal affinities and low, medium, and high EGFR-terminal affinities were selected to prepare antibody-drug conjugates. The inventors believe that the three EGFR×cMET bispecific antibodies used in this embodiment are also representative.

[0609] The preparation of antibody-drug conjugates (ADCs) is as follows:

[0610] 1. The bsAb1 antibody (EGFR-termined high affinity) was conjugated to the adapter-cytotoxin Deruxtecan to prepare the conjugate bsAb1: E2mut34-91A×M5-91A.

[0611] Drug source: Deruxtecan (adapter-cytotoxin GGFG-Dxd, where Dxd CAS number: 1599440-13-7), purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0612] The method for preparing the conjugate by conjugating bsAb1 antibody to the adapter-cytotoxin Deruxtecan is as follows:

[0613] first step:

[0614] Step Two:

[0615] ADC fabrication

[0616] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 7.0 eq.) to the antibody in a buffer solution of pH 6.5, 50 mM PBS / 2 mM EDTA, and stir for 4 h at 25°C to obtain a solution after antibody reduction. Step 2: Add DMA (N,N-dimethylacetamide) and GGFG-DXD (14.0 eq.) (total DMA ratio 20%) to the solution after antibody reduction, and stir for 1 h at 25°C. Displace the reaction solution into a buffer solution (20 mM L-Histidine / pH 5.5), filter to obtain the antibody-drug conjugate ADC of this invention, and store at -60 to -90°C. Where m = 0-8. Free small molecules were detected by reversed-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The final conjugate was named bsAb1-GGFG-Dxd-DAR8.

[0617] 2. conjugation of bsAb1 antibody (EGFR-terminally high affinity) to the linker-cytotoxin CPD2 to prepare conjugates.

[0618] bsAb1: E2mut34-91A×M5-91A.

[0619] Drug source: CPD2 was purchased from Shanghai WuXi AppTec Co., Ltd.

[0620] The method for preparing the conjugate by conjugating bsAb1 antibody to the linker-cytotoxin CPD2 is as follows:

[0621] first step:

[0622] Step Two:

[0623] ADC fabrication

[0624] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 7.0 eq.) to the antibody in a buffer solution of pH 6.5, 50 mM PBS / 2 mM EDTA, and stir for 4 h at 25°C to obtain the antibody-reduced solution. Step 2: Add DMA (N,N-dimethylacetamide) and CPD2 (14.0 eq.) (total DMA ratio 20%) to the antibody-reduced solution, and stir for 1 h at 25°C. Displace the reaction solution into a buffer solution (20 mM L-Histidine / pH 5.5), filter to obtain the antibody-drug conjugate ADC of this invention, and store at -60 to -90°C. Where m = 0-8. Free small molecules were detected by reversed-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The final conjugate was named bsAb1-CPD2-Exatecan-DAR8.

[0625] 3. conjugation of bsAb1 antibody (EGFR-terminally high affinity) to the linker-cytotoxin CPD6 to prepare conjugates.

[0626] bsAb1: E2mut34-91A×M5-91A.

[0627] Drug source: CPD6 was purchased from Shanghai WuXi AppTec Co., Ltd.

[0628] The method for preparing the conjugate by conjugating bsAb1 antibody to the linker-cytotoxin CPD6 is as follows:

[0629] first step:

[0630] Step Two:

[0631] ADC fabrication

[0632] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 7.0 eq.) to the antibody in a buffer solution of pH 6.5, 50 mM PBS / 2 mM EDTA, and stir for 4 h at 25°C to obtain the antibody-reduced solution. Step 2: Add DMA (N,N-dimethylacetamide) and CPD6 (14.0 eq.) (total DMA ratio 20%) to the antibody-reduced solution, and stir for 1 h at 25°C. Displace the reaction solution into a buffer solution (20 mM L-Histidine / pH 5.5), filter to obtain the antibody-drug conjugate ADC of this invention, and store at -60 to -90°C. Where m = 0-8. Free small molecules were detected by reversed-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The final conjugate was named bsAb1-CPD6-Exatecan-DAR8.

[0633] 4. KLH antibody conjugated to adapter-cytotoxin Deruxtecan to prepare conjugates

[0634] KLH antibody: provided by Suzhou Junmeng.

[0635] Drug source: Deruxtecan (adapter-cytotoxin NC-Dxd, where Dxd's CAS number is 1599440-13-7), purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0636] The method for preparing the conjugate by conjugating KLH antibody to the adapter-cytotoxin Deruxtecan is as follows:

[0637] first step:

[0638] Step Two:

[0639] ADC fabrication

[0640] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 7.0 eq.) to the antibody in a buffer solution of pH 6.5, 50 mM PBS / 2 mM EDTA, and stir for 4 h at 25°C to obtain the antibody-reduced solution. Step 2: Add DMA (N,N-dimethylacetamide) and DXD (14.0 eq.) (total DMA ratio 20%) to the antibody-reduced solution, and stir for 1 h at 25°C. Displace the reaction solution into a buffer solution (20 mM L-Histidine / pH 5.5), filter to obtain the antibody-drug conjugate ADC of this invention, and store at -60 to -90°C. Where m = 0-8. Free small molecules were detected by reversed-phase high-performance liquid chromatography (RP-HPLC), and DAR was detected by reversed-phase chromatography (RP-LC). Purity was determined by size exclusion chromatography. The final conjugate was named KLH-NC-Dxd-DAR8.

[0641] 5. Prepare conjugates by conjugating bsAb2 (EGFR-terminal low affinity) antibody to the linker-cytotoxin McMMAF.

[0642] bsAb2:E2mut1-91Q×M5-91Q.

[0643] Drug source: McMMAF was purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0644] The method for preparing the conjugate by conjugating bsAb2 antibody to the adapter-cytotoxin McMMAF is as follows:

[0645] first step:

[0646] Step Two:

[0647] ADC fabrication

[0648] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 2.05 eq.) to the antibody buffer (pH 5.5, 20 mM His / 230 mM Sucrose / 2 mM EDTA), control the temperature at 25°C, and stir for 3.5 h to obtain the antibody-reduced solution. Step 2: Add DMA (N,N-dimethylacetamide) and McMMAF (7.0 eq.) (total DMA ratio 10%) to the antibody-reduced solution, control the temperature at 25°C, and react for 1 h. Displace the reaction solution into a buffer solution (20 mM L-Histidine / pH 5.5), filter to obtain the antibody-drug conjugate ADC of this invention, and store at -60 to -90°C. Where m = 0-8. Free small molecules were detected by reversed-phase high-performance liquid chromatography, and DAR was detected by hydrophobic interaction chromatography. Purity was determined by size exclusion chromatography. The final conjugate was named bsAb2MMAF-DAR4.

[0649] 6. conjugation of bsAb2 antibody (EGFR-terminally low affinity) to the linker-cytotoxin Deruxtecan to prepare conjugates.

[0650] bsAb2:E2mut1-91Q×M5-91Q.

[0651] Drug source: Deruxtecan (Dxd) was purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0652] The method for preparing the conjugate by conjugating bsAb2 antibody to the adapter-cytotoxin Deruxtecan is as follows:

[0653] first step:

[0654] Step Two:

[0655] ADC fabrication

[0656] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 2.1 eq.) to the antibody buffer (pH 5.5, 20 mM His / 220 mM Sucrose / 2 mM EDTA), control the temperature at 25°C, stir, and react for 3.5 h to obtain the antibody-reduced solution. Step 2: Add DMA (N,N-dimethylacetamide) and GGFG-DXD (7.0 eq.) (total DMA ratio 10%) to the antibody-reduced solution, control the temperature at 25°C, and react for 1 h. Displace the reaction solution into a buffer solution (20 mM Histidine / 230 mM sucrose / 0.02% (w / w) TW 80, pH = 5.5), filter to obtain the antibody-drug conjugate (ADC) of this invention, and store at -60 to -90°C. Where m = 0-8. Free small molecules are detected by reversed-phase high-performance liquid chromatography (RP-HPLC), and DAR is detected by hydrophobic interaction chromatography (HIPLC). Purity was determined using size exclusion chromatography. The final conjugate was named bsAb2-GGFG-Dxd-DAR4.

[0657] 7. conjugation of bsAb2 antibody (EGFR-terminally low affinity) to the linker-cytotoxin CPD6 to prepare conjugates.

[0658] bsAb2:E2mut1-91Q×M5-91Q.

[0659] Drug source: CPD6 was purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0660] The method for preparing the conjugate by conjugating bsAb2 antibody to the adapter-cytotoxin CPD6 is as follows:

[0661] first step:

[0662] Step Two:

[0663] ADC fabrication

[0664] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 2.05 eq.) to the antibody buffer (pH 5.5, 20 mM His / 220 mM Sucrose / 2 mM EDTA), control the temperature at 25°C, stir, and react for 3.5 h to obtain the antibody-reduced solution. Step 2: Add DMA (N,N-dimethylacetamide) and CPD6 (7.0 eq.) (total DMA ratio 10%) to the antibody-reduced solution, control the temperature at 25°C, and react for 1 h. Displace the reaction solution into a buffer solution (20 mM Histidine / 230 mM sucrose / 0.02% (w / w) TW 80, pH = 5.5), filter to obtain the antibody-drug conjugate (ADC) of this invention, and store at -60 to -90°C. Where m = 0-8. Free small molecules were detected by reversed-phase high-performance liquid chromatography (RP-HPLC), and DAR was detected by hydrophobic interaction chromatography (HIPLC). Purity was determined by size exclusion chromatography (SUC). The final conjugate was named bsAb2-CPD6-Exatecan-DAR4.

[0665] 8. Prepare conjugates by conjugating bsAb3 (EGFR-terminal mid-affinity) antibody to the linker-cytotoxin CPD6.

[0666] bsAb3: E2mut12-91Q×M5-91Q.

[0667] Drug source: CPD6 was purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0668] The method for preparing the conjugate by conjugating bsAb3 antibody to the linker-cytotoxin CPD6 is as follows:

[0669] first step:

[0670] Step Two:

[0671] ADC fabrication

[0672] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 2.15 eq.) to the antibody buffer (pH 5.5, 20 mM His / 220 mM Sucrose / 2 mM EDTA), control the temperature at 25°C, stir, and react for 3.5 h to obtain the antibody-reduced solution. Step 2: Add DMA (N,N-dimethylacetamide) and CPD6 (7.0 eq.) (total DMA ratio 10%) to the antibody-reduced solution, control the temperature at 25°C, and react for 1 h. Displace the reaction solution into a buffer solution (20 mM Histidine / 230 mM sucrose / 0.02% (w / w) TW 80, pH = 5.5), filter to obtain the antibody-drug conjugate (ADC) of this invention, and store at -60 to -90°C. Where m = 0-8. Free small molecules were detected by reversed-phase high-performance liquid chromatography (RP-HPLC), and DAR was detected by hydrophobic interaction chromatography (HIPLC). Purity was determined by size exclusion chromatography (SUC). The final conjugate was named bsAb3-CPD6-Exatecan-DAR4.

[0673] 9. Preparation of KLH antibody-drug conjugates (ADCs)

[0674] KLH antibody: provided by Suzhou Junmeng.

[0675] KLH antibody was conjugated to the adapter-cytotoxin CPD6 to prepare the conjugate.

[0676] Drug source: CPD6-Exatecan was purchased from Shanghai Haoyuan Pharmaceutical Co., Ltd.

[0677] The method for preparing the conjugate by conjugating KLH antibody to the adapter-cytotoxin CPD6-Exatecan is as follows:

[0678] first step:

[0679] Step Two:

[0680] ADC fabrication

[0681] Step 1: Add TCEP (tris(2-carbonylethyl)phosphine, 2.1 eq.) to the antibody in a buffer solution of pH 6.0, 20 mM PBS / 2 mM EDTA, and stir for 4 h at 25°C to obtain a solution after antibody reduction. Step 2: Add DMA (N,N-dimethylacetamide) and CPD6 (7.0 eq.) (total DMA ratio 20%) to the solution after antibody reduction, and stir for 1 h at 25°C. Displace the reaction solution into a buffer solution (20 mM L-Histidine / pH 5.5), filter to obtain the antibody-drug conjugate ADC of this invention, and store at -60 to -90°C. Where m = 0-8. Free small molecules were detected by reversed-phase high-performance liquid chromatography (RP-HPLC), and DAR was detected by reversed-phase reaction chromatography (RP-PDC). Purity was determined by size exclusion chromatography (SPC). The final conjugate was named Anti-KLH-CPD6-Exatecan-DAR4.

[0682] Example 4: In vitro cell killing experiment

[0683] To evaluate the effects of different adaptor-cytotoxins on ADC activity, MKN45 cells (human gastric cancer cells, endogenously expressing both human EGFR and human cMet) and EBC-1 cells (human lung cancer squamous cell carcinoma cells, endogenously expressing both human EGFR and human cMet) were incubated with different concentrations of bsAb1-CPD2-Exatecan-DAR8, bsAb1-CPD6-Exatecan-DAR8, bsAb1-GGFG-Dxd-DAR8, and KLH-NC-Dxd-DAR8 (starting at 2 μg / ml, 3-fold dilution, for a total of 9 concentration gradients) at 37°C for 120 hours. Finally, CellCounting-lite 2.0 luciferase assay kit (Vazyme) was added to the cell-antibody mixture, and the chemiluminescence signal was detected using a multi-mode microplate reader (TECAN M1000 pro). Four-parameter regression curves were fitted using GraphPad Prism software, and the EC50 value was calculated.

[0684] bsAb1-CPD2-Exatecan-DAR8 and bsAb1-CPD6-Exatecan-DAR8 are ADC drugs that covalently conjugate the small molecule toxin Exatecan to recombinant humanized anti-EGFR and anti-cMET bispecific antibodies; bsAb1-GGFG-Dxd-DAR8 is an ADC drug that covalently conjugates the small molecule toxin Dxd to recombinant humanized anti-EGFR and anti-cMET bispecific antibodies. As shown in Figures 1A and 1B, the EC50 values ​​of bsAb1-CPD2-Exatecan-DAR8, bsAb1-CPD6-Exatecan-DAR8, and bsAb1-GGFG-Dxd-DAR8 against MKN45 cells were 53.67 ng / ml, 56.87 ng / ml, and 55.29 ng / ml, respectively; and the EC50 values ​​against EBC-1 cells were 36.34 ng / ml, 41.78 ng / ml, and 19.95 ng / ml, respectively. The results showed that bsAb1-CPD2-Exatecan-DAR8, bsAb1-CPD6-Exatecan-DAR8, and bsAb1-GGFG-Dxd-DAR8 all had significant cytotoxic effects on MKN45 and EBC-1 cells, and the killing activities of the three ADC drugs were comparable, indicating that ADC drugs with CPD2-Exatecan, CPD6-Exatecan, and GGFG-Dxd as adaptor-cytotoxins have comparable killing activities against tumor cells.

[0685] Example 5: In vitro side-effect killing experiment

[0686] To evaluate the effect of EGFR×cMET bispecific antibodies with different affinities on the side-kill effect of ADCs, CellTrace was used as a reference. TM Violet (Product Code: Invitrogen) TM Pre-label EGFR / cMET double-positive EBC-1 cells according to the instructions (C34557). Stain with PBS at 37°C for 20 min, then add 5 volumes of complete culture medium (RPMI 1640 + 10% FBS) and incubate at 37°C for 5 min to terminate staining. Add 10,000 EBC-1 cells (100 μl) and 20,000 EGFR / cMET double-negative Jeko-1 cells (100 μl) to each well of a 24-well plate (Corning). Dilute the candidate antibody to 100 nM with complete culture medium, adding 50 μl to each well. Bring each well to a final volume of complete culture medium to 500 μl, gently mix, and incubate at 37°C for 5 days. After 5 days, gently shake the supernatant and collect the cells into flow cytometry tubes. Wash the culture medium with 300 μl of PBS per well, then transfer the PBS back to the corresponding flow cytometry tubes. Add 150 μl of TrypLE.TM (Product No.: Gibco-12604021) Digest at 37°C for 5 minutes, then terminate with the same volume of complete culture medium. Transfer the digested cells to the corresponding flow cytometry tubes. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 350 μl of buffer to each tube, containing 300 μl of FACS buffer (PBS + 2% FBS + 0.1% PI) and 50 μl of CountBright. TM Absolute Counting Beads (Catalog No.: Invitroge-C36950). Mix using a vortex mixer, and analyze using a Fortessa flow cytometer. Collect 20,000 beads per well as a stopping gate. Analyze EBC-1 and Jeko-1 PI-negative viable cells using FlowJo. (Based on CountBright) TM Absolute Counting Beads were used to count the number of viable EBC-1 and Jeko-1 PI-negative cells in the intact buffer volume. GraphPad Prism data were used for plotting. The experimental results in Figure 2 show that compared with bsAb2-GGFG-Dxd-DAR4, bsAb2-CPD6-exatecan-DAR4 and bsAb3-CPD6-exatecan-DAR4 not only killed EGFR / cMET double-positive EBC-1 cells, but also killed EGFR / cMET double-negative Jeko-1 cells better, indicating that they both have better side-effect killing effects.

[0687] Example 6: NCI-H292 xenograft model

[0688] This embodiment evaluates the inhibitory effect of the bsAb1-GGFG-Dxd-DAR8 of the present invention on the NCI-H292 human lung adenocarcinoma cell model transplanted into NDG mice.

[0689] 1. Testing Process

[0690] Six- to eight-week-old female NDG mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., Animal Qualification Certificate No.: 320726220100335212) were subcutaneously inoculated with 10 × 10 oz. 6 NCI-H292 cells. The average tumor volume was approximately 113 mm. 3 At that time, suitable animals were selected and randomly divided into two groups of six animals each, based on tumor volume. The groups were:

[0691] G1 saline control group (solvent control group);

[0692] G2 bsAb1-GGFG-Dxd-DAR8 (3mg / kg) group (treatment group).

[0693] The mice were administered the drug via tail vein injection, a single dose, and the experiment ended 27 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 growth rate was calculated as TGI(%) = [1-(Ti-T0) / (Vi-V0)]×100%. (Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration; V0: mean tumor volume in the solvent control group on day 0 of administration).

[0694] The results are shown in Table 7 and Figure 3.

[0695] Table 7: Efficacy analysis of each group in the NCI-H292 human lung cancer subcutaneous tumor model

[0696] Note: 1. Data are expressed as mean ± standard error;

[0697] 2.TGI%=[1-(Ti-T0) / (Vi-V0)]×100%;

[0698] 3. The p-value was obtained by comparing the tumor volume of each group using the t-test method.

[0699] The results showed that on day 27 after administration, the average tumor volume in the saline control group was 1148 mm. 3 The mean tumor volume in the bsAb1-GGFG-Dxd-DAR8 (3mg / kg) group was 79mm. 3 Compared with the saline control group, the tumor inhibition rate was 103.3%, significantly inhibiting tumor growth. These results indicate that bsAb1-GGFG-Dxd-DAR8 has a significant tumor-inhibiting effect at a dose level of 3 mg / kg in the NDG mouse NCI-H292 transplantation model.

[0700] Example 7-1: The role of EGFR×cMET bispecific antibodies in the MKN45 xenograft model

[0701] This embodiment evaluates the inhibitory effect of the EGFR×cMET bispecific antibody CB17-SCID on a mouse model transplanted with human gastric cancer cells MKN45. JNJ-372 was selected as a representative drug for testing.

[0702] 1. Testing Process

[0703] Female NDG mice aged 6-8 weeks (purchased from Shanghai Lingchang Biotechnology Co., Ltd., Animal Qualification Certificate No.: 20180003031585) were subcutaneously inoculated with 5×10⁶ mice on the right side.6 MKN45 cells. The average tumor volume was approximately 90 mm². 3 At that time, suitable animals were selected and randomly divided into 4 groups of 6 animals each, based on tumor volume. The groups were:

[0704] G1 saline control group (solvent control group);

[0705] G2 JNJ-372 (1mg / kg) group (treatment group);

[0706] G3 JNJ-372 (3mg / kg) group (treatment group);

[0707] G4 JNJ-372 (10mg / kg) group (treatment group).

[0708] The mice were administered the drug via intraperitoneal injection twice a week for a total of six administrations, ending the experiment after an initial 21 days. Tumor volume and body weight were measured twice weekly, and the mouse weight and tumor volume were recorded. At the end of the experiment, the mice were euthanized, and the relative tumor growth rate (TGI%) was calculated as: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%. (Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration; V0: mean tumor volume in the solvent control group on day 0 of administration).

[0709] The results are shown in Table 8 and Figure 4.

[0710] Table 8: Efficacy analysis of each group in the MKN45 human subcutaneous gastric cancer tumor model

[0711] Note: 1. Data are expressed as mean ± standard error;

[0712] 2.TGI%=[1-(Ti-T0) / (Vi-V0)]×100%;

[0713] 3. The p-value was obtained by comparing the tumor volume of each group using the t-test method.

[0714] The results showed that on day 21 after administration, the average tumor volume in the saline control group was 1085 mm. 3 The mean tumor volume in the JNJ-372 (1 mg / kg) group was 1061 mm. 3 Compared with the saline control group, the tumor inhibition rate was 2.4%, and no inhibition of tumor growth was observed; the average tumor volume in the JNJ-372 (3 mg / kg) group was 979 mm. 3Compared with the saline control group, the tumor inhibition rate was 10.7%, and no inhibition of tumor growth was observed. The average tumor volume in the JNJ-372 (10 mg / kg) group was 948 mm. 3 Compared with the saline control group, the tumor inhibition rate was 13.8%, and no inhibition of tumor growth was observed. These results indicate that JNJ-372 did not show tumor inhibition at doses ranging from 1 mg / kg to 10 mg / kg in the CD17SCID mouse MKN45 transplantation model. This suggests that the MKN45 model is an EGFR×cMET bispecific antibody resistance model.

[0715] Example 7-2: The role of EGFR×cMET bispecific antibody ADC in the MKN45 xenograft model

[0716] This embodiment evaluates the inhibitory effects of the present invention bsAb1-LF (i.e., E2mut34-91A×M5-91A-LF, prepared according to Preparation Example 2, with 6% glycoform regulator (purchased from Optimum Biotech, catalog number: R170026) added on the first day after transfection, and other steps being the same as described in Preparation Example 2), bsAb1-GGFG-Dxd-DAR8, bsAb1-CPD2-Dxd-DAR8 and bsAb1-CPD6-Dxd-DAR8 on the H292 human lung adenocarcinoma cell line transplanted in NDG mice.

[0717] 1. Testing Process

[0718] Female CB17 SCID mice aged 6-8 weeks (purchased from Shanghai Lingchang Biotechnology Co., Ltd., Animal Qualification Certificate No.: 20180003031585) were subcutaneously inoculated with 5×10⁶ mice on the right side. 6 MKN45 cells. The average tumor volume was approximately 93 mm. 3 At that time, suitable animals were selected and randomly divided into 5 groups of 6 animals each, based on tumor volume. The groups were:

[0719] G1 saline control group (solvent control group);

[0720] G2 bsAb1-LF (1 mg / kg) group (treatment group);

[0721] G3 bsAb1-GGFG-Dxd-DAR8 (1mg / kg) group (treatment group);

[0722] G4 bsAb1-CPD2-Dxd-DAR8 (1mg / kg) group (treatment group);

[0723] G5 bsAb1-CPD6-Dxd-DAR8 (1mg / kg) group (treatment group).

[0724] The mice were administered the drug via tail vein injection, a single dose, and the experiment ended 25 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 growth rate was calculated as TGI(%) = [1-(Ti-T0) / (Vi-V0)]×100%. (Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration; V0: mean tumor volume in the solvent control group on day 0 of administration).

[0725] The results are shown in Table 9 and Figure 5.

[0726] Table 9: Efficacy analysis of each group in the MKN45 human subcutaneous gastric cancer tumor model

[0727] Note: 1. Data are expressed as mean ± standard error;

[0728] 2.TGI%=[1-(Ti-T0) / (Vi-V0)]×100%;

[0729] 3. The p-value was obtained by comparing the tumor volume of each group using the t-test method.

[0730] The results showed that on day 25 after administration, the average tumor volume in the saline control group was 962 mm. 3 The mean tumor volume in the bsAb1-LF (1 mg / kg) group was 973 mm. 3 Compared with the saline control group, the tumor inhibition rate was -1%, showing no inhibition of tumor growth; the average tumor volume in the bsAb1-GGFG-Dxd-DAR8 (1 mg / kg) group was 465 mm. 3 Compared with the saline control group, the tumor inhibition rate was 57%, significantly inhibiting tumor growth. The average tumor volume in the bsAb1-CPD2-Dxd-DAR8 (1 mg / kg) group was 161 mm. 3 Compared with the saline control group, the tumor inhibition rate was 92%, significantly inhibiting tumor growth. The average tumor volume in the bsAb1-CPD6-Dxd-DAR8 (1 mg / kg) group was 274 mm. 3 Compared with the saline control group, the tumor inhibition rate was 79%, significantly inhibiting tumor growth. The results indicate that in the CB17 SCID mouse MKN45 transplantation model, bsAb1-GGFG-Dxd-DAR8, bsAb1-CPD2-Dxd-DAR8, and bsAb1-CPD6-Dxd-DAR8 exhibited significant tumor inhibitory effects at a dose level of 1 mg / kg.

[0731] Example 8-1: The role of EGFR×cMET bispecific antibody in HT29 xenograft model

[0732] This embodiment evaluates the inhibitory effects of the EGFR×cMET bispecific antibodies JNJ-372, bsAb1-LF, and the EGFR monoclonal antibody Cetuximab-analog (provided by Suzhou Junmeng) on ​​an NDG mouse model of transplanted human colon cancer cells HT29. Cetuximab-analog was used as a control.

[0733] 1. Testing Process

[0734] Female NDG mice aged 6-8 weeks (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., Animal Qualification Certificate No.: B202310120009) were subcutaneously inoculated with 3.5 × 10⁻⁶ oz. on the right side. 6 HT29 cells. The average tumor volume was approximately 127 mm. 3 At that time, suitable animals were selected and randomly divided into 4 groups of 5 animals each, based on tumor volume. The groups were:

[0735] G1 saline control group (solvent control group);

[0736] G2 JNJ-372 (10mg / kg) group (positive control group);

[0737] G3 bsAb1-LF (10mg / kg) group (treatment group);

[0738] G4 Cetuximab-analog (10 mg / kg) group (treatment group).

[0739] The mice were administered the drug via intraperitoneal injection twice a week for a total of three weeks, ending the experiment 34 days after the initial administration. Tumor volume and body weight were measured twice weekly, and the mouse body weight and tumor volume were recorded. At the end of the experiment, the mice were euthanized, and the relative tumor growth rate was calculated as TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%. (Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration; V0: mean tumor volume in the solvent control group on day 0 of administration).

[0740] The results are shown in Table 10 and Figure 6.

[0741] Table 10: Efficacy analysis of each group in the HT29 human subcutaneous colon cancer tumor model

[0742] Note: 1. Data are expressed as mean ± standard error;

[0743] 2.TGI%=[1-(Ti-T0) / (Vi-V0)]×100%;

[0744] 3. The p-value was obtained by comparing the tumor volume of each group using the t-test method.

[0745] The results showed that on day 34 after administration, the average tumor volume in the saline control group was 1503 mm. 3 The mean tumor volume in the JNJ-372 (10 mg / kg) group was 1803 mm. 3 Compared with the saline control group, the tumor inhibition rate was -22%, showing no inhibition of tumor growth; the average tumor volume in the bsAb1-LF (10 mg / kg) group was 1406 mm. 3 Compared with the saline control group, the tumor inhibition rate was 7%, and no inhibition of tumor growth was observed. The mean tumor volume in the Cetuximab-analog (10 mg / kg) group was 1113 mm. 3 Compared with the saline control group, the tumor inhibition rate was 28%, showing limited inhibition of tumor growth. The results indicate that in the NDG mouse transplantation HT29 model, JNJ-372, bsAb1-LF, and Cetuximab-analog did not have significant tumor-inhibiting effects at a dose level of 10 mg / kg, suggesting that the NDG mouse transplantation HT29 model is a resistance model to EGFR×cMET bispecific antibodies and EGFR monoclonal antibodies.

[0746] Example 8-2: The role of EGFR×cMET bispecific antibody ADC in HT29 xenograft model

[0747] This embodiment evaluates the inhibitory effects of bsAb3-CPD6-exatecan-DAR4, bsAb2-CPD6-exatecan-DAR4, Irinotecan (purchased from MCE, catalog number: HY-16562), and Cetuximab-analog (provided by Suzhou Junmeng) on ​​an NDG mouse model of transplanted human colon cancer cells HT29. Irinotecan and Cetuximab-analog were used as controls.

[0748] 1. Testing Process

[0749] Six- to eight-week-old female NDG mice (purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd., Animal Qualification Certificate No.: B202401110236) were subcutaneously inoculated with 3.5 × 10⁻⁶ oz. 6 HT29 cells. The average tumor volume was approximately 125 mm. 3 At that time, suitable animals were selected and randomly divided into 9 groups of 5 animals each, based on tumor volume. The groups were as follows:

[0750] G1 saline control group (solvent control group);

[0751] G2 bsAb3-CPD6-exatecan-DAR4 (10mg / kg) group (treatment group);

[0752] G3 bsAb3-CPD6-exatecan-DAR4 (3mg / kg) group (treatment group);

[0753] G4 bsAb2-CPD6-exatecan-DAR4 (10mg / kg) (treatment group);

[0754] G5 bsAb2-CPD6-exatecan-DAR4 (3mg / kg) (treatment group);

[0755] G6 Irinotecan (30 mg / kg) (treatment group);

[0756] G7 Cetuximab-analog (10 mg / kg) + Irinotecan (30 mg / kg) (treatment group).

[0757] Irinotecan was administered intraperitoneally twice weekly, and the remaining doses once weekly for a total of three weeks, ending the experiment after the initial 32 days of administration. Tumor volume and body weight were measured twice weekly, and mouse body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized, and the relative tumor growth rate (TGI) was calculated as: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%. (Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration; V0: mean tumor volume in the solvent control group on day 0 of administration).

[0758] The results are shown in Table 11 and Figure 7.

[0759] Table 11: Efficacy analysis of each group in the HT29 human subcutaneous colon cancer tumor model

[0760] Note: Data are expressed as mean ± standard error;

[0761] 2.TGI%=[1-(Ti-T0) / (Vi-V0)]×100%;

[0762] 3. The p-value was obtained by comparing the tumor volume of each group using the t-test method.

[0763] The results showed that on day 32 after administration, the average tumor volume in the saline control group was 1488 mm.3 The mean tumor volume in the bsAb3-CPD6-exatecan-DAR4 (10 mg / kg) group was 106 mm. 3 Compared with the saline control group, the tumor inhibition rate was 101%, significantly inhibiting tumor growth; the mean tumor volume in the bsAb3-CPD6-exatecan-DAR4 (3mg / kg) group was 725mm. 3 Compared with the saline control group, the tumor inhibition rate was 56%, significantly inhibiting tumor growth; the mean tumor volume in the bsAb2-CPD6-exatecan-DAR4 (10 mg / kg) group was 113 mm. 3 Compared with the saline control group, the tumor inhibition rate was 101%, significantly inhibiting tumor growth; the mean tumor volume in the bsAb2-CPD6-exatecan-DAR4 (3mg / kg) group was 1137 mm. 3 Compared with the saline control group, the tumor inhibition rate was 26%, and no inhibition of tumor growth was observed; the mean tumor volume in the Irinotecan 30 mg / kg group was 839 mm. 3 Compared with the saline control group, the tumor inhibition rate was 47%, significantly inhibiting tumor growth; the mean tumor volume in the Cetuximab-analog group (10 mg / kg + Irinotecan, 30 mg / kg) was 673 mm. 3 Compared with the saline control group, the tumor inhibition rate was 60%, significantly inhibiting tumor growth. The results indicate that in the NDG mouse HT29 transplantation model, bsAb3-CPD6-exatecan-DAR4 and bsAb2-CPD6-exatecan-DAR4 at a dose level of 10 mg / kg had significant tumor-inhibiting effects, and their tumor-inhibiting effects were superior to the Irinotecan, 30 mg / kg group and the Cetuximab-analog, 10 mg / kg + Irinotecan, 30 mg / kg group.

[0764] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. An antibody-drug conjugate containing a bispecific antibody against EGFR and cMET, comprising a bispecific antibody or its antigen-binding fragment that specifically binds to EGFR and cMET, and a conjugation moiety, characterized in that, The bispecific antibody that specifically binds to EGFR and cMET, or its antigen-binding fragment, comprises: a first protein functional region targeting the EGFR antigen and a second protein functional region targeting cMET. The first protein functional region targeting EGFR includes: HCDR1, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, or differs from the amino acid sequence shown in SEQ ID NO:25 or 26 by 1, 2 or 3 amino acids; HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:27, or differs from the amino acid sequence shown in SEQ ID NO:27 by 1, 2 or 3 amino acids; HCDR3, whose amino acid sequence is shown in SEQ ID NO:37 (ARVSX1YX2DSX3FD), 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; LCDR1 has the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2 or 3 amino acids. LCDR2, whose amino acid sequence is the same as that shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:14 by 1, 2, or 3 amino acids; and LCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:15, 20 or 21, or differs from the amino acid sequence shown in SEQ ID NO:15, 20 or 21 by 1, 2 or 3 amino acids; and / or, The second protein functional region targeting cMET includes: HCDR1 has the amino acid sequence shown in SEQ ID NO:22, or differs from the amino acid sequence shown in SEQ ID NO:22 by 1, 2 or 3 amino acids. HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:23, or differs from the amino acid sequence shown in SEQ ID NO:23 by 1, 2 or 3 amino acids; HCDR3 has the amino acid sequence shown in SEQ ID NO:24, or differs from the amino acid sequence shown in SEQ ID NO:24 by 1, 2 or 3 amino acids. LCDR1 has the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2 or 3 amino acids. LCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:14 by 1, 2 or 3 amino acids; And LCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:15, 20 or 21, or differs from the amino acid sequence shown in SEQ ID NO:15, 20 or 21 by 1, 2 or 3 amino acids.

2. The antibody-drug conjugate as described in claim 1, characterized in that, The first protein functional region targeting EGFR includes: HCDR1, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, or differs from the amino acid sequence shown in SEQ ID NO:25 or 26 by 1, 2 or 3 amino acids; HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:27, or differs from the amino acid sequence shown in SEQ ID NO:27 by 1, 2 or 3 amino acids; HCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35 or 36, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35 or 36; LCDR1 has the amino acid sequence shown in SEQ ID NO:13, or differs from the amino acid sequence shown in SEQ ID NO:13 by 1, 2 or 3 amino acids. LCDR2, whose amino acid sequence is the same as that shown in SEQ ID NO:14, or differs from the amino acid sequence shown in SEQ ID NO:14 by 1, 2, or 3 amino acids; and LCDR3, whose amino acid sequence is selected from the amino acid sequence shown in SEQ ID NO:15, 20 or 21, or has 1, 2 or 3 amino acid differences from the amino acid sequence shown in SEQ ID NO:15, 20 or 21; Preferably, the first protein functional region targeting EGFR includes a heavy chain variable region, which comprises: The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:35, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:35 by 1, 2, or 3 amino acids; or The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:28, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:28 by 1, 2, or 3 amino acids; or The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:31, respectively; or HCDR1, HCDR2, and HCDR3 having a difference of 1, 2, or 3 amino acids from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:31; or The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29 by 1, 2, or 3 amino acids; or The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:30, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:30; or The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:32, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:32; or The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:33, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:33; or The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:33, respectively; or HCDR1, HCDR2, and HCDR3 having a difference of 1, 2, or 3 amino acids from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:33; or The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:34, respectively; or HCDR1, HCDR2, and HCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:34; or The amino acid sequences are HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:36, respectively; or HCDR1, HCDR2, and HCDR3 that differ from the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:36 by 1, 2, or 3 amino acids; and / or, Preferably, the first protein functional region targeting EGFR includes a light chain variable region, which comprises: The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; or The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20; or The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:

21.

3. The antibody-drug conjugate as described in claim 1 or 2, characterized in that, The first protein functional region targeting EGFR includes a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is selected from the amino acid sequence shown in SEQ ID NO:25 or 26, the amino acid sequence of HCDR2 is the amino acid sequence shown in SEQ ID NO:27, and the amino acid sequence of HCDR3 is selected from the amino acid sequences shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, or 36; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is the amino acid sequence shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is the amino acid sequence shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is selected from the amino acid sequences shown in SEQ ID NO:15, 20, or 21. Preferably, The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:15; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:28; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:31; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:20; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:29; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:30; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:32; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in 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:33; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:33; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:34; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:35; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:21; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:25, 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:36; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

21.

4. The antibody-drug conjugate according to any one of claims 1-3, characterized in that, The second protein functional region targeting cMET includes a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises: HCDR1, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:22, or differs from the amino acid sequence shown in SEQ ID NO:22 by 1, 2, or 3 amino acids; HCDR2, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:23, or differs from the amino acid sequence shown in SEQ ID NO:23 by 1, 2, or 3 amino acids; and HCDR3, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO:24, or differs from the amino acid sequence shown in SEQ ID NO:24 by 1, 2, or 3 amino acids. The light chain variable region comprises: LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15; or The amino acid sequences are as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21, respectively; or LCDR1, LCDR2, and LCDR3 having one, two, or three amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:21; or The amino acid sequences are LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20, respectively; or LCDR1, LCDR2, and LCDR3 that differ from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:20 by 1, 2, or 3 amino acids. Preferably, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:24; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:15; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:24; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:20; or The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:22, the amino acid sequence of HCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:24; the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

21.

5. The antibody-drug conjugate according to any one of claims 1-4, characterized in that, The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 4, 9, 2, 3, 5, 6, 7, 8, 10, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1, 4, 9, 2, 3, 5, 6, 7, 8, 10; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 39, 42, or 40, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 39, 42, or 40; and / or, The second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:41, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

41. The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:39, 42, or 40, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:39, 42, or 40. Preferably, the first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 4, 9, 2, 3, 5, 6, 7, 8, 10; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 39, 42, or 40; and / or, the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 41; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 39, 42, or 40; More preferably, the first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, 40, or 42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, 40, or 42; or The first protein functional region targeting the EGFR antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO:1, 4, 9, 2, 3, 5, 6, 7, 8, 10; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:42; the second protein functional region targeting the cMET antigen comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:41; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:

42.

6. The antibody-drug conjugate according to any one of claims 1-5, characterized in that, The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain. The heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO:18, 52, 55, 53, 54, 56, 57, 58, 59, 60, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:18, 52, 55, 53, 54, 56, 57, 58, 59, 60; the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12, or 38, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:11, 12, or 38; and / or, The second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain. The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

19. The light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12, or 38, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:11, 12, or 38. Preferably, the first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:18, and the light chain comprises the amino acid sequence shown in any one of SEQ ID NO:11, 12, or 38; or the first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in any one of SEQ ID NO:52, 55, 53, 54, 56, 57, 58, 59, or 60, and the light chain comprises the amino acid sequence shown in any one of SEQ ID NO:38; and / or, The second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:19, and the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:11, 12 or 38. More preferably, the first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO:18, and the light chain comprising the amino acid sequence shown in SEQ ID NO:11; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO:19, and the light chain comprising the amino acid sequence shown in SEQ ID NO:11; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:52, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:55 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:18 and the light chain comprises the amino acid sequence shown in SEQ ID NO:12; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:12; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:18 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:53 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:54, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19, and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:56 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:57 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:58 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:59 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or The first protein functional region targeting the EGFR antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:60 and the light chain comprises the amino acid sequence shown in SEQ ID NO:38; the second protein functional region targeting the cMET antigen comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:19 and the light chain comprises the amino acid sequence shown in SEQ ID NO:

38.

7. The antibody-drug conjugate according to any one of claims 1-6, characterized in that, The conjugated portion includes or is selected from: antitumor agents, immunomodulators, cytotoxic drugs, fluorescent substances, luminescent substances, enzymes, small nucleic acid molecules, and any combination thereof; preferably, the small nucleic acid is siRNA or antisense oligonucleotide (ASO).

8. The antibody-drug conjugate as described in claim 7, characterized in that, The antibody-drug conjugate has an Ab-(LD)m structure, wherein Ab is a bispecific antibody or antigen-binding fragment thereof that specifically binds EGFR and cMET as described in any one of claims 1-6; L is a linker; D is a therapeutically active substance or a pharmaceutically active ingredient; m represents the average number of LD units conjugated to Ab, and m ranges from 1 to 10, preferably from 2 to 8, and more preferably from 4 to 8.

9. The antibody-drug conjugate as described in claim 8, characterized in that, The L structure is shown in equation (A): in: L1 represents the functional group that can react with the thiol group and the group or bond formed after the reaction with the thiol group; L2 and Y are each independently selected from -(CH2). p -、-(OCH2CH2) p - and -(CH2CH2O) p -; L3 is a hydrophilic group; L4 is an amino acid group; R6 is selected from H, C1-C6 alkoxy, and -(OCH2CH2). p O-C1-C6 alkyl groups and -(CH2CH2O) p -C1-C6 alkyl; X is selected from -NH- and -NH(CH2). n C(=O)-、-C(=O)(CH2) n NH- and -(CH2) n C(=O)-; Z is selected from C and S; o is selected from 0 and 1; n is selected from 0, 1, and 2; p is an integer from 1 to 10.

10. The antibody-drug conjugate as described in claim 9, characterized in that, The functional groups that can react with thiol groups as described in L1 are selected from maleimide, halogen, halogen-substituted functional groups (such as halogen-substituted aldehyde groups, halogen-substituted -S(O)2-), aldehyde groups, alkenyl groups, alkynyl groups, ketone groups, sulfonyl groups, silane groups, isocyanate groups, and norbornyl groups. Preferably, the functional group that can react with the thiol group in L1 is selected from: and their derivatives; where: A is a halogen; The wavy line indicates the position where L1 and L2 are connected; preferably, the functional group in L1 is:

11. The antibody-drug conjugate as described in claim 9 or 10, characterized in that: L2 is -(CH2) p - and p is an integer from 1 to 4; preferably, L2 is -CH2CH2- or -CH2CH2CH2-; and / or X is -NH- or -NH(CH2) n C(=O)-; preferably, X is -NH- or -NHC(=O)-; and / or Y is -(CH2) p - p is 1, 2 or 3; preferably, Y is methylene; and / or Z is C.

12. The antibody-drug conjugate according to any one of claims 9-11, characterized in that, L3 is selected from monosaccharides, disaccharides, and five- or six-membered saturated heterocycles containing 1-2 nitrogen atoms, as well as their derivatives, which are divalent hydrophilic groups with two monovalent groups obtained by removing two hydrogen atoms. Preferably, the monosaccharide is selected from trioses, tetraoses, pentoses, hexoses, and heptaoses; Preferably, the disaccharide is selected from maltose, sucrose, and lactose; Preferably, the five- or six-membered saturated heterocycle containing 1-2 nitrogen atoms or its derivatives are selected from piperazine, piperidinyl, and pyrrolidinyl. Preferably, L3 is selected from glucose, galactose, mannose, glucuronic acid, galactobionic acid, mannouronic acid, N-acetylglucosamine, N-acetylglucosamine, N-acetylglucosamine, and N-acetylmuramic acid, which are divalent hydrophilic groups with two monovalent centers produced by removing two hydrogen atoms. More preferably, L3 has the structure shown in formula L3-1: In the formula: R1 is selected from -O(CH2) p - and -C(=O)-, p is an integer from 1 to 6, preferably 1, 2, 3 or 4; R2, R3, and R4 are independently selected from H, C1-C6 alkyl, -(CH2CH2O)t-C1-C6 alkyl, sulfonic acid group, and phosphoric acid group; t is an integer from 1 to 20; The wavy line indicates the position where L3 connects to X and Y.

13. The antibody-drug conjugate according to any one of claims 9-12, characterized in that: L4 is selected from the following amino acid residues: valine, citrulline, alanine, glycine, phenylalanine, asparagine, glutamic acid, lysine, serine, threonine, cysteine, and tyrosine; wherein the amino acid residue is optionally separated by one or more -OR 20 Replaced, of which R 20 Selected from H, C1-C6 alkyl, -(CH2CH2O)t-C1-C6 alkyl, sulfonic acid group, and phosphoric acid group, wherein t is an integer from 1 to 20; or L4 is selected from the following peptides: valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), glycine-glycine-phenylalanine-glycine, glycine-glycine-phenylalanine-glycine, citrulline-valine, alanine-valine, alanine-alanine, citrulline-alanine, asparagine-citrulline, citrulline-asparagine, citrulline-citrulline, phenylalanine-lysine, and lysine-phenylalanine, preferably selected from the following peptides: valine-citrulline (VC), valine-alanine (VA), and glycine-glycine-phenylalanine-glycine (GGFG); said peptides are optionally substituted with one or more substituents selected from the group consisting of: -OR20 and -(OCH2CH2). p O-C1-C6 alkyl, wherein R20 is selected from H, C1-C6 alkyl, -(CH2CH2O)t-C1-C6 alkyl, sulfonic acid group and phosphoric acid group, t is an integer from 1 to 20, and p is an integer from 1 to 6; Preferably, L4 is selected from: In the formula, the wavy line indicates the position where L4 connects to Z and NH.

14. The antibody-drug conjugate according to any one of claims 9-13, characterized in that, R6 is H, C1-C3 alkoxy, or -(OCH2CH2). p O-C1-C3 alkyl, wherein p is an integer from 1 to 6; preferably, R6 is H, methoxy, or -(OCH2CH2). p OCH3, where p is an integer from 1 to 6 or from 1 to 4.

15. The antibody-drug conjugate according to any one of claims 9-14, characterized in that, The structure of the connector L is shown in any of the following configurations: In each formula, R2, R3 and R4 are as described in claim 12, R6 is as described in claim 9 or 14, and R” is H or C1-C6 alkyl.

16. The antibody-drug conjugate according to any one of claims 9-15, characterized in that, The drug component D includes a microtubule inhibitor, a DNA damaging agent, a vitamin A precursor, and folic acid; Preferably, the microtubule inhibitors include dolastatin and auristatin derivatives, and maytansine derivatives; the DNA damaging agents include calicheamicin derivatives, duocarmycin derivatives, pyrrolobenzodiazepine derivatives (PBD), and camptothecin derivatives; More preferably, the auristatin class of drugs includes monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF) or their derivatives; the maytansine class of drugs includes DM1, DM3, DM4 or their derivatives; and the camptothecin class of drugs includes: exatecan, deruxtecan (Dxd), SN38, camptothecin, topotecan, irinotecan, belotetane, letopotecan, rubitecan, cilatecan, cocinotecan, gemmatotecan, eczetane, topotecan, and 9-nitrocamptothecin.

17. The antibody-drug conjugate according to any one of claims 9-16, characterized in that, The connector-drug (LD) portion has the structure shown in the following formulas: VCMMAE, GGFG-Deruxtecan, CPD2-Exatecan, CPD4-Exatecan, CPD5-Exatecan, CPD6-Exatecan, or SN-38Comp5.

18. The antibody-drug conjugate according to any one of claims 1-17, characterized in that, The antibody-drug conjugate is selected from the structure shown in formula ADC-Ⅰ, ADC-Ⅱ, ADC-Ⅲ, ADC-Ⅳ or ADC-V: In the formula, m represents the average number of linker-drug (LD) units coupled to Ab, and m ranges from 1 to 10, preferably from 2 to 8, and more preferably from 4 to 8. Preferably, the Ab is a bispecific antibody or antigen-binding fragment thereof that specifically binds EGFR and cMET as described in any one of claims 1-6.

19. A pharmaceutical composition comprising an antibody-drug conjugate of the anti-EGFR and cMET bispecific antibody as described in any one of claims 1-18, and a pharmaceutically acceptable carrier or excipient.

20. Use of the antibody-drug conjugate of any one of claims 1-18, or the pharmaceutical composition of claim 19, in the preparation of a medicament for the treatment and / or prevention of EGFR and / or cMET-mediated diseases or conditions. Preferably, the disease or symptom is a tumor or cancer; More preferably, the tumor or cancer is selected from lung cancer, gastric cancer, glial cell carcinoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, bladder cancer, colon cancer, rectal cancer, liver cancer, brain cancer, thyroid cancer, and head and neck cancer. More preferably, the lung cancer includes non-small cell lung cancer and small cell lung cancer, the gastric cancer includes gastric cancer and gastroesophageal junction cancer, and the head and neck cancer includes head and neck squamous cell carcinoma.

21. A method of treating or preventing tumors or cancer, comprising administering to a subject in need an effective amount of any one of 1-18 of the antibody-drug conjugate of the anti-EGFR and cMET bispecific antibodies, or the pharmaceutical composition of claim 19. Preferably, the tumor or cancer is a tumor that highly expresses EGFR and / or cMET; More preferably, the tumor or cancer is selected from lung cancer, gastric cancer, glial cell carcinoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, biliary tract cancer, bronchial cancer, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, bladder cancer, colon cancer, rectal cancer, liver cancer, brain cancer, thyroid cancer, and head and neck cancer. More preferably, the lung cancer includes non-small cell lung cancer and small cell lung cancer, the gastric cancer includes gastric cancer and gastroesophageal junction cancer, and the head and neck cancer includes head and neck squamous cell carcinoma.

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