Antibody-drug conjugates targeting egfr / c-met and methods of making and using the same

Abstract

Disclosed are antibody-drug conjugates (ADCs) useful for preventing, treating, and / or as an adjunct medication for c-MET and / or EGFR related diseases. The ADCs include a bispecific antibody or antigen-binding fragment thereof coupled to a cytotoxic drug, the bispecific antibody or antigen-binding fragment thereof including an arm that specifically binds EGFR and an arm that specifically binds c-MET.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese application 202311531642.X, filed November 15, 2023; Chinese application 202311596146.2, filed November 24, 2023; and Chinese application 202411591580.6, filed November 7, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to targeted therapy, and more specifically, to antibody-drug conjugates targeting EGFR and c-MET, methods for their preparation, and uses. Background Technology

[0004] EGFR (epidermal growth factor receptor, abbreviated as EGFR, ErbB-1, or HER1) is a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4). The HER family plays an important regulatory role in cellular physiological processes. EGFR is a type I transmembrane glycoprotein composed of 1210 amino acids, with a molecular weight of approximately 170 kDa. Its main structure includes a ligand-binding extracellular domain, a hydrophobic transmembrane region, and an intracellular region containing tyrosine kinases. EGFR is highly expressed in various tumors, including colorectal cancer, head and neck cancer, and non-small cell lung cancer, and is also expressed to some extent in normal epithelial tissues such as skin and lung. In tumor tissues, highly expressed EGFR homodimerizes or heterodimerizes by binding ligands such as EGF or TGFα. Dimerization leads to the activation of tyrosine kinases and protein phosphorylation in tumor cells, mediating gene transcription and the cell cycle. Activation of various cell signaling pathways plays an important role in promoting the survival, proliferation, migration, and angiogenesis of tumor cells.

[0005] Met (c-MET), also known as hepatocyte growth factor receptor (HGFR), is a heterodimeric transmembrane tyrosine kinase receptor encoded by the Met proto-oncogene. c-MET is a type I transmembrane glycoprotein composed of 1390 amino acids with a molecular weight of approximately 190 kDa. It ultimately forms two polypeptide chains linked by disulfide bonds through cleavage: an α chain (50 kDa) and a β chain (140 kDa). From the extracellular space to the intracellular space, the MET protein can be divided into the SEMA domain (SEMA), the Plexin-semaphorin-integrin domain (PSI), four immunoglobulin-plexins-transcription domains (IPT), a transmembrane region, a juxtamembrane region (JM), a tyrosine kinase domain (TK), and a carboxyl terminal region (CT). The natural ligand for the c-MET receptor is hepatocyte growth factor (HGF), an inactive protein that is converted into its active form through proteolytic cleavage. Aberrant regulation of c-MET has been reported in various cancers, including colorectal cancer, non-small cell lung cancer, gastric cancer, and breast cancer. Hyperactivation of c-MET and its downstream signaling pathways has been shown to trigger excessive proliferation, tumor invasion, and angiogenesis, and is associated with poor survival.

[0006] In tumors resistant to EGFR tyrosine kinase inhibitors (TKIs), nearly 60% showed increased c-MET expression, amplified c-MET, or increased c-MET ligand HGF (Turke et al., Cancer Cell, 17:77-88, 2010), indicating that the tumor c-MET signaling pathway compensates for the EGFR signaling pathway. Both EGFR and c-MET transmit signals through the same survival and anti-apoptotic pathways (ERK and AKT). Simultaneous targeting of EGFR and c-MET holds promise for inhibiting both signaling pathways, preventing tumor development and progression caused by c-MET signaling pathway activation after EGFR inhibition, thereby improving overall efficacy and safety.

[0007] Anti-c-MET and anti-EGFR antibodies are known in the art. For example, Telisotuzumab vedotin (Teliso-v or ABBV399) disclosed in U.S. Patent No. 10603389 is an antibody-drug conjugate of VC-MMAE (vedotin) conjugated with anti-c-MET antibody ABT-700, and MRG003 (ADC-3 or MYK-3) disclosed in WO2023040941 is an antibody-drug conjugate of vedotin conjugated with anti-EGFR antibody BA03.

[0008] Several EGFR / c-Met bispecific antibodies have been disclosed in the art. The bispecific antibody BSAB01, disclosed in WO2010115551, comprises an EGFR-binding VH / VL pair (Fab) of cetuximab and a c-Met-binding VH / VL pair (Fab) of onartuzumab, and is currently in Phase III trials. US Patent No. 9593164 discloses Amivantamab, a bispecific antibody marketed as RYBREVANT®. Amivantamab includes an anti-EGFR Fab comprising VH and VL of zalutusumab (see US Patent Nos. 7247301 and 7595378) and an anti-cMET Fab comprising VH and VL of antibody 069 disclosed in US Patent No. 9068011. Sellman et al. (J Biol Chem. 291(48): 25106-25119(2016)) disclosed EGFR / c-Met bispecific antibodies conjugated to cytotoxins, and also disclosed EGFR / c-Met bispecific antibodies conjugated to vedotin. WO2023083846 discloses AZD9592, in which an EGFR / c-Met bispecific antibody is conjugated to a camptothecin derivative. There is a need in the art for antibody-drug conjugates (ADCs) that bind to EGFR and c-MET and contain cytotoxic drugs. Invention Overview

[0010] This application relates to an antibody-drug conjugate for the prevention and / or treatment and / or as adjunctive therapy for c-MET and / or EGFR-related diseases. The ADC of the present invention comprises a bispecific antibody conjugated to a cytotoxic drug, said antibody comprising an arm specifically binding to EGFR and an arm specifically binding to c-MET. In some specific embodiments, the bispecific antibody comprises an EGFR-binding scFv and a c-MET-binding Fab. In other embodiments, the bispecific antibody comprises an EGFR-binding Fab and a c-MET-binding scFV, or comprises an EGFR-binding scFv and a c-MET-binding scFv. In still other embodiments, the anti-EGFR Fab and / or anti-c-MET Fab comprises a drug selected from CrossMab. Fab CrossMab VH-VL and CrossMab CH1-CL The CrossMab form. The ADC and its drugs can be used for the prevention and / or treatment and / or as adjunctive therapy for diseases associated with c-MET and / or EGFR (e.g., cancer).

[0011] Exemplary ADCs containing the bispecific antibodies 07B, 10B, 38B, 41B, 49B, 55B, or 56B conjugated with camptothecin derivatives disclosed herein exhibit affinity for EGFR and c-MET, as well as good affinity and in vivo and in vitro killing activity against EGFR and / or c-MET positive tumor cells, such as gastric cancer, non-small cell lung cancer (e.g., lung adenocarcinoma), and epidermal squamous cell carcinoma cells.

[0012] This invention provides an antibody-drug conjugate containing the structure shown below: Ab-[MLED]x in: Ab is a bispecific antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR; wherein, the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), the first VL and the first VH together forming a domain capable of specifically binding to c-MET; the second antigen-binding domain comprises a second VL and a second VH, the second VL and the second VH together forming a domain capable of specifically binding to EGFR. M is the linker site that is linked to the bispecific antibody or its antigen-binding fragment; L is a structural segment connecting the joint portions M and E; E is a structural segment connecting L and D; D is a cytotoxic drug or a fragment thereof; and x is any integer from 1 to 10.

[0013] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain and the second antigen-binding domain are each independently selected from scFv, Fab, and scFab.

[0014] In another embodiment of the antibody-drug conjugate, the bispecific antibody or its bispecific antigen-binding fragment further comprises an Fc domain, the Fc domain comprising a first Fc domain monomer and a second Fc domain monomer, and wherein the first and second Fc domain monomers comprise one or more modifications that promote heterodimerization of the Fc domain monomers.

[0015] In another embodiment of the antibody-drug conjugate, the Fc domain comprises a first Fc domain monomer and a second Fc domain monomer, the first Fc domain monomer comprising a modification to form a knock structure, and the second Fc domain monomer comprising a modification to form a hole structure, wherein the hole structure can pair with the knock structure to form a heterodimerized Fc domain.

[0016] In another embodiment of the antibody-drug conjugate, the first Fc domain monomer comprises an amino acid sequence as shown in SEQ ID NO: 49 or 51, and the second Fc domain monomer comprises an amino acid sequence as shown in SEQ ID NO: 50 or 52.

[0017] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain and the second antigen-binding domain are each linked to one of the first and second Fc domain monomers of the Fc domain.

[0018] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain is linked to the first Fc domain monomer, and the second antigen-binding domain is linked to the second Fc domain monomer; or the first antigen-binding domain is linked to the second Fc domain monomer, and the second antigen-binding domain is linked to the first Fc domain monomer.

[0019] In another embodiment of the antibody-drug conjugate, the first VL comprises the amino acid sequences of the complementarity-determining regions (CDRs)-L1, CDR-L2, and CDR-L3 of the VL amino acid sequence described in SEQ ID NO: 17 or SEQ ID NO: 59; and / or the first VH comprises the amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 of the VH amino acid sequence described in SEQ ID NO: 18 or SEQ ID NO: 60.

[0020] In another embodiment of the antibody-drug conjugate, the first VL comprises: (i) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38; or, (ii) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 35, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 37, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38; And / or, The first VH includes: (i) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 47; (ii) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 40, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 47; (iii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 42, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47; or, (iv) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 48.

[0021] In another embodiment of the antibody-drug conjugate, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17 or 59, and / or the first VH comprises the amino acid sequence shown in SEQ ID NO: 18 or 60; or

[0022] The first VL contains the amino acid sequence shown in SEQ ID NO:17, and the first VH contains the amino acid sequence shown in SEQ ID NO:18; or the first VL contains the amino acid sequence shown in SEQ ID NO:59, and the first VH contains the amino acid sequence shown in SEQ ID NO:60.

[0023] In another embodiment of the antibody-drug conjugate, the second VL comprises the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence described in SEQ ID NO: 15; and / or the second VH comprises the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence described in SEQ ID NO: 16.

[0024] In another embodiment of the antibody-drug conjugate, the second VL comprises: (i) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23; or, (ii) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 20, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 22, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 23; And / or, The second VH includes: (i) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 24, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 28, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 32; (ii) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 25, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 29, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 32; (iii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 27, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32; or, (iv) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 26, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 30, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 33.

[0025] In another embodiment of the antibody-drug conjugate, the second VL comprises an amino acid sequence as shown in SEQ ID NO: 15, and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 16.

[0026] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain is Fab and the second antigen-binding domain is scFv.

[0027] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises polypeptide chain IA, polypeptide chain IB, and polypeptide chain IC; wherein polypeptide chain IA comprises the first VL and a light chain constant region, polypeptide chain IB comprises the first VH, a heavy chain CH1 region, and a first Fc domain monomer (or a second Fc domain monomer), and polypeptide chain IC comprises the second VL, the second VH, and a second Fc domain monomer (the first Fc domain monomer).

[0028] In another embodiment of the antibody-drug conjugate, the polypeptide chain IA comprises the first VL and a light chain constant region from the N-terminus to the C-terminus, the polypeptide chain IB comprises the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or the polypeptide chain IC comprises (i) the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the second VH, the second VL and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus.

[0029] In another embodiment of the antibody-drug conjugate, adjacent domains of the polypeptide chain IA are optionally linked by or without a linker, adjacent domains of the polypeptide chain IB are optionally linked by or without a linker, and / or adjacent domains of the polypeptide chain IC are optionally linked by or without a linker.

[0030] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having a structure such as (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

[0031] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0032] In another embodiment of the antibody-drug conjugate, the polypeptide chain IA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB comprises the amino acid sequence shown in SEQ ID NO: 2 or 9, and / or the polypeptide chain IC comprises the amino acid sequence shown in SEQ ID NO: 3 or 10.

[0033] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain is scFv and the second antigen-binding domain is Fab.

[0034] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein polypeptide chain II-A comprises the second VL and a light chain constant region, polypeptide chain II-B comprises the second VH, a heavy chain CH1 region, and a first Fc domain monomer (or the second Fc domain monomer), and polypeptide chain II-C comprises the first VL, the first VH, and the second Fc domain monomer (or the first Fc domain monomer).

[0035] In another embodiment of the antibody-drug conjugate, polypeptide chain II-A comprises the second VL and a light chain constant region from the N-terminus to the C-terminus, polypeptide chain II-B comprises the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or polypeptide chain II-C comprises (i) the first VL, the first VH and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the first VH, the first VL and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus.

[0036] In another embodiment of the antibody-drug conjugate, adjacent domains of polypeptide chain II-A are optionally linked by or without a linker, adjacent domains of polypeptide chain II-B are optionally linked by or without a linker, and / or adjacent domains of polypeptide chain II-C are optionally linked by or without a linker.

[0037] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having a structure such as (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

[0038] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0039] In another embodiment of the antibody-drug conjugate, the polypeptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4, the polypeptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 5 or 7, and / or the polypeptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 6 or 8.

[0040] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain is Fab, and the second antigen-binding domain is scFab.

[0041] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises polypeptide chain III-A, polypeptide chain III-B, and polypeptide chain III-C; wherein polypeptide chain III-A comprises a first VL and a light chain constant region, polypeptide chain III-B comprises a first VH, a heavy chain CH1 region, and a first Fc domain monomer (or a second Fc domain monomer), and / or polypeptide chain III-C comprises a second VL, a light chain constant region, a second VH, a heavy chain CH1 region, and a second Fc domain monomer (or a first Fc domain monomer).

[0042] In another embodiment of the antibody-drug conjugate, polypeptide chain III-A includes a first VL and a light chain constant region from the N-terminus to the C-terminus, polypeptide chain III-B includes a first VH, the heavy chain CH1 region and a first Fc domain monomer (or a second Fc domain monomer) from the N-terminus to the C-terminus, and / or polypeptide chain III-C includes (i) the second VL, the light chain constant region, the second VH, the heavy chain CH1 region and a second Fc domain monomer (or a first Fc domain monomer), or (ii) the second VH, the heavy chain CH1 region, the second VL, the light chain constant region and a second Fc domain monomer (or a first Fc domain monomer).

[0043] In another embodiment of the antibody-drug conjugate, adjacent domains of polypeptide chain III-A are optionally linked by or without a linker, adjacent domains of polypeptide chain III-B are optionally linked by or without a linker, and / or adjacent domains of polypeptide chain III-C are optionally linked by or without a linker.

[0044] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

[0045] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0046] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain is scFab and the second antigen-binding domain is Fab.

[0047] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C; wherein polypeptide chain IV-A comprises the second VL and a light chain constant region, polypeptide chain IV-B comprises the second VH, a heavy chain CH1 region, and a first Fc domain monomer (or the second Fc domain monomer), and / or polypeptide chain IV-C comprises the first VL, a light chain constant region, the first VH, a heavy chain CH1 region, and a second Fc domain monomer (or the first Fc domain monomer).

[0048] In another embodiment of the antibody-drug conjugate, polypeptide chain IV-A comprises the second VL and a light chain constant region from the N-terminus to the C-terminus, polypeptide chain IV-B comprises the second VH, a heavy chain CH1 region, and a first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or polypeptide chain IV-C comprises (i) the first VL, the light chain constant region, the first VH, the heavy chain CH1 region, and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the first VH, the heavy chain CH1 region, the first VL, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer).

[0049] In another embodiment of the antibody-drug conjugate, adjacent domains of polypeptide chain IV-A are optionally linked by or without a linker, adjacent domains of polypeptide chain IV-B are optionally linked by or without a linker, and / or adjacent domains of polypeptide chain IV-C are optionally linked by or without a linker.

[0050] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having a structure such as (GGGGS). )n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

[0051] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0052] In another embodiment of the antibody-drug conjugate, the polypeptide chain IV-A comprises the amino acid sequence shown in SEQ ID NO: 4, the polypeptide chain IV-B comprises the amino acid sequence shown in SEQ ID NO: 7, and / or the polypeptide chain IV-C comprises the amino acid sequence shown in SEQ ID NO: 11.

[0053] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain and the second antigen-binding domain are Fab, and the Fab of the second antigen-binding domain contains domain exchanges of the form of CrossMab.

[0054] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises polypeptide chain VA, polypeptide chain VB, polypeptide chain VC, and polypeptide chain VD; wherein polypeptide chain VA comprises the first VL and a light chain constant region, polypeptide chain VB comprises the first VH, a heavy chain CH1 region, and a first Fc domain monomer (or a second Fc domain monomer), polypeptide chain VC comprises the second VH, a light chain constant region, and a second Fc domain monomer (or a first Fc domain monomer), and / or polypeptide chain VD comprises the second VL and a heavy chain CH1 region.

[0055] In another embodiment of the antibody-drug conjugate, the polypeptide chain VA comprises the first VL and the light chain constant region from the N-terminus to the C-terminus, the polypeptide chain VB comprises the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, the polypeptide chain VC comprises the second VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus, and / or the polypeptide chain VD comprises the second VL and the heavy chain CH1 region from the N-terminus to the C-terminus.

[0056] In another embodiment of the antibody-drug conjugate, adjacent domains of the polypeptide chain VA are optionally linked by or without a linker, adjacent domains of the polypeptide chain VB are optionally linked by or without a linker, adjacent domains of the polypeptide chain VC are optionally linked by or without a linker, and / or adjacent domains of the polypeptide chain VD are optionally linked by or without a linker.

[0057] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having (GGGGS).n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

[0058] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0059] In another embodiment of the antibody-drug conjugate, the polypeptide chain VA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain VB comprises the amino acid sequence shown in SEQ ID NO: 9, the polypeptide chain VC comprises the amino acid sequence shown in SEQ ID NO: 13, and / or the polypeptide chain VD comprises the amino acid sequence shown in SEQ ID NO: 12.

[0060] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain and the second antigen-binding domain are Fab, and the Fab of the first antigen-binding domain contains domain exchanges of the form of CrossMab.

[0061] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises polypeptide chain VI-A, polypeptide chain VI-B, polypeptide chain VI-C, and polypeptide chain VI-D; wherein polypeptide chain VI-A comprises the second VL and a light chain constant region, polypeptide chain VI-B comprises the second VH, a heavy chain CH1 region, and a monomer of the first Fc domain (or the second Fc domain monomer), polypeptide chain VI-C comprises the first VH, a light chain constant region, and a monomer of the second Fc domain (or the first Fc domain monomer); and polypeptide chain VI-D comprises the first VL and a heavy chain CH1 region.

[0062] In another embodiment of the antibody-drug conjugate, polypeptide chain VI-A comprises the second VL and the light chain constant region from the N-terminus to the C-terminus, polypeptide chain VI-B comprises the second VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, polypeptide chain VI-C comprises the first VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus, and / or polypeptide chain VI-D comprises the first VL and the heavy chain CH1 region from the N-terminus to the C-terminus.

[0063] In another embodiment of the antibody-drug conjugate, adjacent domains of polypeptide chain VI-A are optionally linked by or without a linker, adjacent domains of polypeptide chain VI-B are optionally linked by or without a linker, adjacent domains of polypeptide chain VI-C are optionally linked by or without a linker, and / or adjacent domains of polypeptide chain VI-D are optionally linked by or without a linker.

[0064] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

[0065] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54.

[0066] In another embodiment of the antibody-drug conjugate, both the first antigen-binding domain and the second antigen-binding domain are scFv.

[0067] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein polypeptide chain VII-A comprises the first VL, the first VH and the first Fc domain monomer (or the second Fc domain monomer), and polypeptide chain VII-B comprises the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer).

[0068] In another embodiment of the antibody-drug conjugate, the polypeptide chain VII-A comprises, from the N-terminus to the C-terminus, (i) the first VL, the first VH, and the first Fc domain monomer (or the second Fc domain monomer), or (ii) the first VH, the first VL, and the first Fc domain monomer (or the second Fc domain monomer); and / or the polypeptide chain VII-B comprises, from the N-terminus to the C-terminus, (i) the second VL, the second VH, and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the second VH, the second VL, and the second Fc domain monomer (or the first Fc domain monomer).

[0069] In another embodiment of the antibody-drug conjugate, adjacent domains of polypeptide chain VII-A are optionally linked by or without a linker, and / or adjacent domains of polypeptide chain VII-B are optionally linked by or without a linker.

[0070] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

[0071] In another embodiment of the antibody-drug conjugate, each peptide linker independently comprises the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

[0072] In another embodiment of the antibody-drug conjugate, the polypeptide chain VII-A comprises an amino acid sequence as shown in SEQ ID NO:14, and / or the polypeptide chain VII-B comprises an amino acid sequence as shown in SEQ ID NO:8.

[0073] In another embodiment of the antibody-drug conjugate, the bispecific antibody or its antigen-binding fragment comprises: (1) A polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 3. (2) A polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 9, and a polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 10. (3) A polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5, and a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6; (4) A polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8; (5) A polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11. (6) A polypeptide chain VA comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain VB comprising the amino acid sequence shown in SEQ ID NO: 9, a polypeptide chain VC comprising the amino acid sequence shown in SEQ ID NO: 13, and a polypeptide chain VD comprising the amino acid sequence shown in SEQ ID NO: 12; or, (7) A polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and a polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.

[0074] In another embodiment of the antibody-drug conjugate, the antibody-drug conjugate has enhanced tumor suppressive activity relative to a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody; wherein the CDR amino acid sequence of the monospecific anti-c-MET antibody is identical to the CDR amino acid sequence of the first antigen-binding domain, and the CDR amino acid sequence of the monospecific anti-EGFR antibody is identical to the CDR amino acid sequence of the second antigen-binding domain.

[0075] In another embodiment of the antibody-drug conjugate, the tumor-suppressive effect includes: inhibition of EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0076] In a further embodiment of the antibody-drug conjugate, M comprises , Wherein, ring A is a 5-6 membered aliphatic heterocycle or a 5-20 membered aromatic ring system, wherein the aliphatic heterocycle and aromatic ring system are optionally selected independently by one or more groups selected from oxygen (=O), halogen, cyano, amino, carboxyl, mercapto, and C. 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

[0077] In a further embodiment of the antibody-drug conjugate, M comprises Wherein ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by one or more 6-membered heteroaromatic rings connected to a benzene ring via single bonds, or a polycyclic ring formed by multiple 6-membered heteroaromatic rings connected via single bonds, wherein the aliphatic heterocycle is optionally surrounded by one or more elements selected from oxygen (=O), halogens, and C. 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

[0078] In a further embodiment of the antibody-drug conjugate, M is... Ring A is selected from , , , , , and M1 is selected from single bonds, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl or amino group.

[0079] In a further embodiment of the antibody-drug conjugate, M is selected from... , and .

[0080] In a further embodiment of the antibody-drug conjugate, M is... .

[0081] In a further embodiment of the antibody-drug conjugate, M is selected from... , , and .

[0082] In a further embodiment of the antibody-drug conjugate, M is selected from... , and .

[0083] In a further embodiment of the antibody-drug conjugate, L is selected from one or more of the following structures: C 1-6 Alkyl group, -N(R')-, carbonyl group, -O-, selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) s Natural or non-natural amino acids and their analogues (OCH3), as well as short peptides containing 1, 2, 3 or 4 amino acids. , , , , , , , , and ;

[0084] Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20.

[0085] In a further embodiment of the antibody-drug conjugate, the short peptide is selected from Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG (SEQ ID NO: 71)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 72)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 73)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 74)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 75)).

[0086] In a further embodiment of the antibody-drug conjugate, L is selected from structures comprising one or more of the following: C 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Ar g, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG (SEQ ID NO: 71)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 72)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 73)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 74)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 75)), , , , and ; where s is an integer selected from 1 to 20.

[0087] In a further embodiment of the antibody-drug conjugate, L is selected from structures comprising one or more of the following: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0088] In a further embodiment of the antibody-drug conjugate, L is selected from the following structures: , , , , , , , , , and .

[0089] In a further embodiment of the antibody-drug conjugate, L is selected from the following structures: , and .

[0090] In a further embodiment of the antibody-drug conjugate, L is selected from the following structures: and .

[0091] In a further embodiment of the antibody-drug conjugate, L is selected from the following structures: and .

[0092] In a further embodiment of the antibody-drug conjugate, E is a single bond or selected from the following structures: -NHCH2-、 , , and .

[0093] In a further embodiment of the antibody-drug conjugate, E is a single bond, -NHCH2-, or .

[0094] In a further embodiment of the antibody-drug conjugate, E is -NHCH2- or .

[0095] In a further embodiment of the antibody-drug conjugate, E is -NHCH2- or a single bond.

[0096] In a further embodiment of the antibody-drug conjugate, E is -NHCH2-.

[0097] In a further embodiment of the antibody-drug conjugate, E is a single bond.

[0098] In a further embodiment of the antibody-drug conjugate, E is... .

[0099] In a further embodiment of the antibody-drug conjugate, Selected from the following structures: .

[0100] In a further embodiment of the antibody-drug conjugate, Selected from the following structures: and .

[0101] In a further embodiment of the antibody-drug conjugate, the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

[0102] In a further embodiment of the antibody-drug conjugate, the microtubule inhibitor is an olistatin or maytansine compound.

[0103] In a further embodiment of the antibody-drug conjugate, the DNA intercalating agent is pyrrolobenzodiazepine (PBD).

[0104] In a further embodiment of the antibody-drug conjugate, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0105] In a further embodiment of the antibody-drug conjugate, the topoisomerase I inhibitor is selected from camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, rubotecan, and pharmaceutically acceptable salts, esters, or analogs thereof; the topoisomerase II inhibitor is selected from doxorubicin, PNU-159682, docalimcin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, and pharmaceutically acceptable salts, esters, or analogs thereof.

[0106] In a further embodiment of the antibody-drug conjugate, the RNA polymerase inhibitor is α-amanitin and its pharmaceutically acceptable salts, esters, or analogs.

[0107] In a further embodiment of the antibody-drug conjugate, the cytotoxic drug is selected from compounds of Formula I and II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of Formula I and II:

[0108] Among them, R1 and R2 are each independently selected from C. 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; R4 and R5 are each independently selected from H, halogens, and hydroxyl groups; or R4 and R5 are linked to form a 5-6 membered oxygen-containing heterocycle; R6 is selected from hydrogen or -C. 1-4 Alkylene-NR a R b ;and R7 is selected from C 1-6 Alkyl, -C 1-4 Alkylene-NR a R b -C 1-4 Alkylene-SiR a R b R c -SiR a R b R c -C 1-4 Alkylene = N-OR a ;where R a R b and R c Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, -SO2-C 1-6 Alkyl and -CO-C 1-6 Alkyl; wherein R is optionally present a and R b Together with the connected atoms, they form 5-6 member nitrogen-containing heterocycles.

[0109] In a further embodiment of the antibody-drug conjugate, the cytotoxic drug is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: and ; In this context, the fragment corresponding to the cytotoxic drug obtained after the cytotoxic drug is connected to the adapter is D in the above formula.

[0110] In a further embodiment of the antibody-drug conjugate, the cytotoxic drug is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: , In this context, the fragment corresponding to the cytotoxic drug obtained after the cytotoxic drug is connected to the adapter is D in the above formula.

[0111] In a further embodiment of the antibody-drug conjugate, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amine group on the cytotoxic drug.

[0112] In a further embodiment of the antibody-drug conjugate, the antibody-drug conjugate is selected from: ADC A-01

[0113] ADC A-02

[0114] ADC A-05

[0115] ADC A-06

[0116] ADC A-07

[0117] ADC A-08

[0118] ADC A-09

[0119] ADC A-10

[0120] ADC A-11

[0121] ADC A-14

[0122] ADC A-15

[0123] ADC A-16

[0124] ADC A-17

[0125] ADC A-18

[0126] ADC A-20

[0127] ADC A-21

[0128] ADC A-22

[0129] ADC A-24

[0130] ADC A-25

[0131] ADC A-26

[0132] ADC A-28

[0133] ADC A-29

[0134] ADC A-30

[0135] ADC A-32

[0136] ADC A-33

[0137] ADC A-34

[0138] ADC B-01

[0139] ADC B-03

[0140] ADC B-04

[0141] ADC B-06

[0142] ADC B-07

[0143] ADC C-02

[0144] ADC C-03

[0145] ADC C-04

[0146] ADC C-05

[0147] ADC C-06

[0148] ADC C-08

[0149] ADC C-09

[0150] ADC C-11

[0151] ADC C-12

[0152] ADC C-14

[0153] ADC C-15

[0154] ADC C-17

[0155] ADC C-18

[0156] ADC C-19

[0157] ADC C-20

[0158] ADC C-21

[0159] ADC C-22

[0160] ADC C-23

[0161] ADC C-24

[0162] ADC C-25

[0163] ADC C-26

[0164] ADC C-27

[0165] ADC C-28 ; Wherein, Ab is a bispecific antibody or its antigen-binding fragment as defined above; and This indicates the specific linkage between the thiol group of the cysteine ​​residue in the bispecific antibody or its antigen-binding fragment and M in the antibody-drug conjugate; and x represents the amount of drug payload.

[0166] In a further embodiment of the antibody-drug conjugate, the thiol group in the bispecific antibody or its antigen-binding fragment forms a thioether bond with M in the antibody-drug conjugate through an addition reaction or a substitution reaction to obtain the antibody-drug conjugate.

[0167] In a further embodiment of the antibody-drug conjugate, Ab is a bispecific antibody selected from the following: BsAb07B, BsAb10B, BsAb38B, BsAb41B, BsAb49B, BsAb55B, and BsAb56B.

[0168] The present invention also provides antibody-drug conjugates selected from the following: ADC 07B-A-05, ADC 07B-A-14, ADC 38B-A-14, ADC 49B-A-14, ADC 49B-B-01, ADC 49B-A-05, ADC 41B-A-05, ADC55B-A-14 and ADC 56B-A-14.

[0169] The present invention also provides compositions comprising one or more antibody-drug conjugates as described herein. In a further embodiment of the composition, the DAR value (drug-antibody conjugate ratio) of the composition is 1-10. In a further embodiment of the composition, the DAR value of the composition is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, ​​3~7, 3~8, 3~9, 3~10, 4~5, 4~6, 4~7, 4~8, 4~9, 4~10, 5~6, 5~7, 5~8, 5~9, 5~10, 6~7, 6~8, 6~9, 6~10, 7~8, 7~9, 7~10, 8~9, 8~10, or 9~10. In a further embodiment of the composition, the DAR value of the composition is 3-9.

[0170] In a further embodiment of the composition, the DAR value of the composition is 4 to 8. In a further embodiment of the composition, the DAR value of the composition is 3.0 to 3.5, 3.0 to 4.0, 3.0 to 4.5, 3.0 to 5.0, 3.0 to 5.5, 3.0 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 5.0, 3.5 to 5.5, 3.5 to 6.0, 3.5 to 4.0, 3.5 to 4.5, 3.5 to 7.0, 3.5 to 7.5, 3.5 to 8.0, 4.0 to 4.5, 4.0. ~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0~5.5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0171] The present invention further provides pharmaceutical compositions comprising one or more antibody-drug conjugates or compositions disclosed herein, and one or more pharmaceutically acceptable carriers and / or excipients. In another embodiment of the pharmaceutical composition, the pharmaceutical composition further comprises one or more additional pharmaceutically active agents selected from the group consisting of: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, and any combination thereof.

[0172] The present invention further provides a drug combination comprising a first therapeutic agent and a second therapeutic agent, wherein the first therapeutic agent and the second therapeutic agent are administered simultaneously or sequentially, the first therapeutic agent being selected from one or more antibody-drug conjugates or compositions described in any one of the preceding claims; and the second therapeutic agent being selected from EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0173] The present invention further provides the use of the antibody-drug conjugates, compositions, pharmaceutical compositions or pharmaceutical combinations described above in the preparation of a medicament, wherein the medicament is used in subjects for the prevention, treatment and / or as adjunctive therapy for diseases associated with c-MET and / or EGFR, and / or for the inhibition of c-MET and / or EGFR activity in vitro or in subjects; wherein the diseases associated with c-MET and / or EGFR are associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations and / or c-MET gene amplification, and optionally, the diseases associated with c-MET and / or EGFR include cancer.

[0174] In a further embodiment of the use, the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0175] In further embodiments of use, the antibody-drug conjugate or pharmaceutical composition is administered in combination with an additional pharmaceutically active agent, for example, simultaneously, separately, or sequentially; the additional pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0176] The present invention further provides a method for inhibiting the activity of c-MET and / or EGFR in cells, the method comprising contacting the cells with any of the antibody-drug conjugates, compositions, pharmaceutical compositions or drug combinations described above; wherein the cells are cells expressing c-MET and / or EGFR.

[0177] The present invention further provides a method for preventing, treating, and / or adjunctive treating diseases associated with c-MET and / or EGFR in a subject, the method comprising administering to a subject in need an effective amount of any of the preceding antibody-drug conjugates, compositions, pharmaceutical compositions, or drug combinations; wherein the c-MET and / or EGFR-associated diseases include EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification; optionally, the c-MET and / or EGFR-associated diseases include cancer.

[0178] In a further implementation of the method, the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0179] In a further embodiment of the method, the method further includes administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy may be administered simultaneously, separately, or sequentially.

[0180] The present invention further provides antibody-drug conjugates, compositions, pharmaceutical compositions, or drug combinations as described in any of the preceding claims for the prevention, treatment, and / or adjuvant therapy of c-MET and / or EGFR-related diseases in subjects, wherein the c-MET and / or EGFR-related diseases include EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification. Optionally, the c-MET and / or EGFR-related diseases are cancers. In a further embodiment, the cancers are selected from epithelial carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer. In a further embodiment, the method further includes administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy is administered simultaneously, alone, or sequentially. Attached Figure Description

[0181] Figure 1A Schematic diagram of the structure of Fab-scFv-Fc (KIH) bispecific antibody.

[0182] Figure 1B Schematic diagram of the structure of Fab-scFab-Fc (KIH) bispecific antibody.

[0183] Figure 1C Crossmab VH-VL Schematic diagram of the structure of a bispecific antibody.

[0184] Figure 1D Schematic diagram of the structure of scFv-Fc(KIH) bispecific antibody.

[0185] Figure 2 Efficacy testing of different antibody-drug conjugates on the MKN45 model.

[0186] Figure 3 Efficacy testing of different antibody-drug conjugates on the MKN45-EGFR model.

[0187] Figure 4 Efficacy testing of different antibody-drug conjugates on the MKN45 model.

[0188] Figure 5 Efficacy testing of different antibody-drug conjugates on the HCC827 model.

[0189] Figure 6 Efficacy testing of different antibody-drug conjugates on the EBC-1-EGFR model.

[0190] Figure 7 Efficacy testing of different antibody-drug conjugates on the HCC827 model.

[0191] Figure 8 : Detection of the efficacy of different antibody-drug conjugates on the PC-9 model.

[0192] Figure 9 Bystander effect detection for different antibody-drug conjugates. Invention Details

[0194] Terminology Definition

[0195] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used in this disclosure are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0196] As used herein, the term "antibody" is used in the broadest sense to encompass a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can consist of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. While not directly involved in antibody-antigen binding, the constant domain exhibits various effector functions, such as mediating interactions between immunoglobulins and host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0197] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0198] The term "antibody" also includes embodiments in which the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into a pyroglutamate salt. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or contain N-terminal glutamine or glutamate, or have an N-terminal amino acid cyclized into pyroglutamate.

[0199] As used in this article, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the antibody variable region responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modellingantibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA86:9268–9272), the MacCallum numbering system (MacCallum et al., (1996) J Mol Biol 262: 732-745, and see also Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)), and the AHo numbering system (Honegger and Plückthun, A., J. Mol. Biol. 309:657-670). The definitions in the (2001) or IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003) are readily apparent to those skilled in the art. Furthermore, the correspondences between different numbering systems are well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0200] In this document, the CDR contained in the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or antigen-binding fragment is determined by the Chothia numbering system.

[0201] The following general rules (published at www.bioinf.org.uk: Professor Andrew CR Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with the amino acids contained in the antigenic epitope that binds to the antibody. In rare cases, these generally constant features may not appear; however, Cys residues are the most conserved feature.

[0202]

[0203] V H The complete amino acid sequence is typically numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the aforementioned numbering systems. In some embodiments, V H The amino acid sites in the sequence can be numbered sequentially starting from amino acid site 1 until the end of the sequence, or they can be numbered according to Kabat. Unless otherwise stated, the V mentioned herein... H and V L The amino acid sites in the sequence are defined according to their sequential numbering.

[0204] The amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.

[0205] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0206] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0207] As used herein, the term "bispecific antibody" or "BsAb" refers to an antibody that has binding specificity to two different antigens (or epitopes) and comprises antigen-binding domains that are specific to the binding of different antigens (or epitopes), such as two antigen-binding domains that are specific to the binding of different antigens (or epitopes), thereby enabling binding to two different binding sites and / or target molecules. The individual antigen-binding domains of a bispecific antibody can be independently selected from full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., Fv, Fab, scFab, or scFv). In some cases, the individual antigen-binding domains are linked by peptide linkers.

[0208] As used herein, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to antigens, although its affinity may be lower than that of a complete binding site.

[0209] As used herein, the term "Fc fragment" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0210] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art peptide linkers consist of a repeating GGGGS amino acid sequence (SEQ ID NO: 57) or a variant thereof, such as the amino acid sequence (GGGGS)4 (SEQ ID NO: 58), but variants thereof may also be used. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0211] As used herein, the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains, typically consisting of one peptide chain containing VL and CL and another peptide chain containing VH and CH1. However, those skilled in the art will understand that the Fab domains may be arranged according to the natural orientation described above, but may also contain domain substitutions or exchanges that facilitate proper VH and VL pairing (e.g., domain exchanges in the form of Crossmab). The term "scFab" refers to a single polypeptide chain containing VL, VH, CL, and CH1 domains, wherein adjacent domains are optionally linked by a linker. In a typical structure, the single polypeptide chain contained in scFab comprises from the N-terminus to the C-terminus: (1) VL, CL, VH, and CH1, wherein CL and VH are typically linked by a peptide linker (e.g., a flexible peptide linker), or (1) VH, CH1, VL, and CL, wherein CH1 and VL are typically linked by a peptide linker (e.g., a flexible peptide linker).

[0212] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or fragment thereof derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, generally refer to antibodies or fragments thereof derived from a group of antibodies comprising at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any specific method.

[0213] As used herein, the term "CrossMab" refers to a method for constructing bispecific antibodies by exchanging the heavy and light chain domains within the antigen-binding fragment (Fab) of half of the bispecific antibody, allowing the light chain to correctly bind to its homologous heavy chain. This "exchange" preserves the antigen-binding affinity but makes the two arms so different that light chain mismatches no longer occur. Three possible forms of "CrossMab" are: CrossMab Fab CrossMab refers to the crossover or exchange of all VH-CH1 and VL-CL domain positions in half of a bispecific antibody. VH-VL This refers to the crossover or exchange of only the VH and VL domain positions in half of a bispecific antibody; and CrossMab CH1-CLThis refers to the crossover or exchange of the CH1 and CL domain positions within half of the Fab region of a bispecific antibody. CorssMab antibodies have been described or claimed in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010136172, WO2010145792 and WO2013026831. The term “CrossMab” antibody is recognized in the art; see, for example, Brinkmann and Kontennann, MAbs 9(2):182-212 (2017); Kontermann and Brinkmann, Drug Discovery Today 20(7): 838-846 (2015); Schaefer et al., PNAS, 108 11187-1191 (2011); Kleinet et al., MAbs 8(6): 1010-1020 (2016); and Klein et al., MAbs 4(6): 653-663 (2012).

[0214] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (KD) or half-maximal effective concentration (EC50) of the interaction. 50 )express.

[0215] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. The "binding rate constant" (k...) a or k on ) and "dissociation rate constant" (k dis or k off Both can be calculated from concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). dis / k on The ratio is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). KD and k can be measured by any effective method. on and k dis The dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method) in some implementations. Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used.

[0216] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and polyomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0217] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is the one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. As used herein, the term "expression vector" refers to a vector containing recombinant polynucleotides that include expression regulatory sequences effectively linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as entrapments, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0218] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0219] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48:443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the Needleman algorithm in the GAP program integrated into the GCG software package (available at www.gcg.com) can be used, employing a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0220] The twenty common amino acids involved in this disclosure are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this disclosure, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine may be represented by A or Ala.

[0221] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Stabilizers have the meaning commonly understood by those skilled in the art; stabilizers are those that can stabilize the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate).

[0222] As used herein, the terms “DAR” or “drug-antibody ratio” or “drug-antibody conjugate ratio” (as used interchangeably herein) refer to the average number of linker / payload portions present in the composition that are linked to each antibody. For a composition containing an ADC of the present disclosure, the DAR of the composition is the average of all DARs (linker-payload portions of all ADC molecules present in the composition), which may be expressed as a decimal or an integer. Thus, in some embodiments of the compositions comprising ADC disclosed herein, the DAR of the composition is a decimal or integer among 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, ​​3~7, 3~8, 3~9, 3~10, 4~5, 4~6, 4~7, 4~8, 4~9, 4~10, 5~6, 5~7, 5~8, 5~9, 5~10, 6~7, 6~8, 6~9, 6~10, 7~8, 7~9, 7~10, 8~9, 8~10, or 9~10. In additional embodiments, for compositions of the present disclosure containing an ADC, the DAR of the composition is 3.0~3.5, 3.0~4.0, 3.0~4.5, 3.0~5.0, 3.0~5.5, 3.0~6.0, 3.5~4.0, 3.5~4.5, 3.5~5.0, 3.5~5.5, 3.5~6.0, 3.5~6.5, 3.5~7.0, 3.5~7.5, 3.5~8.0, 4.0~4.5, 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5. Integers or decimals from 0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0~5.5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0, or 7.5~9.0. As used above, the term "composition" is understood to include pharmaceutical compositions. Mean DAR can be determined by a variety of conventional methods, such as ultraviolet spectroscopy, mass spectrometry, ELISA, radiometrics, hydrophobic interaction chromatography (HIC), electrophoresis, and HPLC.

[0223] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also mean prolonged survival compared to expected survival (if no treatment was received).

[0224] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) has a tumor, or is at risk of having the disease described herein.

[0225] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, a preventive effective amount is an amount sufficient to prevent, stop, or delay the onset of a disease (e.g., a tumor); a therapeutic effective amount is an amount sufficient to cure or at least partially stop an existing disease and its complications. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0226] As used herein, the term "effector function" refers to the biological activities attributable to the antibody's Fc region (either the native sequence Fc region or the Fc region of an amino acid sequence variant), and which vary with the antibody's Fc region. Examples of antibody effector functions include, but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADCP), binding to cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, and the half-life / clearance of antibodies and antigen-antibody complexes. Methods for altering antibody effector functions are known in the art, for example, by introducing mutations into the Fc region.

[0227] As used herein, the term “antibody-dependent cell-mediated cytotoxicity (ADCC)” refers to a form of cytotoxicity in which Ig interacts with Fc receptors (FcRs) present on cytotoxic cells (such as natural killer (NK) cells, neutrophils, or macrophages) to specifically bind to antigen-attached target cells and then kill the target cells by secreting cytotoxins.

[0228] In this document, combination therapy includes the use of the bispecific antibody or pharmaceutical composition disclosed herein in combination with one or more other active therapeutic agents of a second therapy (e.g., chemotherapeutic agents) or other preventive or therapeutic modalities (e.g., radiotherapy).

[0229] In such combination therapies, the various active agents often have different complementary mechanisms of action, and the combination therapy may lead to a synergistic effect. Combination therapies include therapeutic agents that affect the immune response (e.g., enhance or activate the response) and therapeutic agents that affect (e.g., inhibit or kill) tumor / cancer cells. Combination therapies can reduce the likelihood of drug-resistant cancer cells developing. Combination therapies may allow for a reduction in the dosage of one or more agents in the regimen to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies may have a synergistic therapeutic or preventative effect on underlying diseases, conditions, or symptoms.

[0230] As used herein, "combination" includes therapies that can be administered separately, such as those formulated separately for individual administration (e.g., those provided in a kit), and therapies that can be administered together as a single formulation (i.e., a "co-formulation"). In some embodiments, the bispecific antibody of this disclosure may be administered sequentially. In other embodiments, the bispecific antibody may be administered simultaneously. The bispecific antibody of this disclosure may be used in combination with at least one other (active) pharmaceutical agent in any manner.

[0231] The terms "cancer" and "tumor" are used interchangeably to refer to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0232] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, such as "C". 1-20 Alkyl", C 1-10 Alkyl", C 1-6 Alkyl", C 1-4 Alkyl", C 1-3Alkyl groups, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0233] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight-chain or branched hydrocarbon group, such as "C". 1-20 "alkylene", "C" 1-10 Alkylene, C 3-10 "alkylene", "C" 5-8 Alkylene, C 1-6 Alkylene, C 1-4 Alkylene, C 1-3 "alkylene", etc., specific examples include but are not limited to: methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene, etc.

[0234] The term "alkenyl" refers to a divalent group formed by the loss of two hydrogen atoms from a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C". 2-20 "Ideinyl", "C" 3-10 "Ideinyl", "C" 5-8 Examples of these include, but are not limited to: vinylidene, 1-propenyne, 2-propenyne, 1-butenyne, 2-butenyne, 1,3-butadiene, 1-pentenyne, 2-pentenyne, 3-pentenyne, 1,3-pentadiene, 1,4-pentadiene, 1-hexenyne, 2-hexenyne, 3-hexenyne, 1,4-hexadiene, etc.

[0235] The term "acetylenic" refers to a divalent group formed by the loss of two hydrogen atoms from a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. This includes, for example, "C..." 2-20 "ethynyl", "C" 3-10 "Immyne", "C" 5-8 Examples of "ethynyl" include, but are not limited to: ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentynyl, 1,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,4-hexynyl, etc.

[0236] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one ring member selected from N, O, and S. Specific examples include, but are not limited to, 5-6 membered aliphatic heterocycles, 5-6 membered nitrogen-containing aliphatic heterocycles, and 5-6 membered oxygen-containing aliphatic heterocycles, such as tetrahydrofuran, pyrrolidine, piperidine, and tetrahydropyran.

[0237] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O, and S. Specific examples include, but are not limited to, 5-6 membered aromatic heterocycles, 5-6 membered nitrogen-containing aromatic heterocycles, and 5-6 membered oxygen-containing aromatic heterocycles, such as furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, 1,2,4,5-tetraazine, etc.

[0238] The term "aromatic ring system" refers to a monocyclic or polycyclic system containing at least one aromatic ring (e.g., a benzene ring) or a heteroaromatic ring (e.g., a pyrimidine ring). Two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be linked by a single bond (e.g., a diamyrimidinylphenyl ring). The aromatic ring system may be divalent or higher valence (e.g., trivalent or tetravalent), such as 5-20 member aromatic ring systems.

[0239] abbreviation

[0240] The abbreviations used in this article have the following meanings:

[0241] Antibody-drug conjugates

[0242] In one aspect, this application provides a solution having the formula Ab-[MLED] x The antibody-drug conjugate shown has the following structure: Ab is a bispecific antibody or its antigen-binding fragment containing a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR. M is the linker site that is linked to the bispecific antibody or its antigen-binding fragment; L is a structural segment connecting the joint portions M and E; E is a structural segment connecting L and D; D is a cytotoxic drug or a fragment thereof; and x is any integer from 1 to 10.

[0243] Antibodies for ADC

[0244] In a specific embodiment, the bispecific antibody of the ADC is a bispecific antibody with high affinity for both EGFR and c-MET. It can specifically recognize / bind to EGFR and c-MET, and can block the binding of EGF to EGFR and / or HGF to c-MET, thereby simultaneously blocking the downstream signaling pathways of EGFR and / or c-MET, causing cell proliferation inhibition.

[0245] In some embodiments, the first antigen-binding domain includes a first light chain variable region (VL) and a first heavy chain variable region (VH), wherein the first VL and the first VH together form a domain capable of specifically binding c-MET; the second antigen-binding domain includes a second VL and a second VH, wherein the second VL and the second VH together form a domain capable of specifically binding EGFR.

[0246] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are each independently selected from scFv, Fab, and scFab.

[0247] Unless otherwise explicitly stated, Fab domains may be arranged according to their natural orientation or may contain domain substitutions or exchanges that facilitate proper VH and VL pairing (e.g., domain exchanges of the form CrossMab).

[0248] CrossMab is a method for preventing light chain and heavy chain mismatch, see US Patent US9266967B2, which is incorporated herein by reference in its entirety. This method primarily utilizes the principle of mutual repulsion among the same parts of an antibody, i.e., VH vs. VH, CH1 vs. CH1, CL vs. CL, and VL vs. VL. It prevents light chain mismatch by exchanging regions of the heavy and light chains of one of the Fab components in a bispecific antibody (e.g., exchanging light chain CL-VL with heavy chain CH1-VH, VL and VH, or CH and CL).

[0249] In some implementations, the CrossMab-style domain swaps are selected from: CrossMab Fab In the Fab, the antibody light chain CL-VL and the antibody heavy chain CH1-VH were interchanged; CrossMab VH-VL In Fab, the VL and VH of the antibody were interchanged; and CrossMab CH1-CLIn Fab, the CH and CL of the antibody are interchanged.

[0250] In some embodiments, the bispecific antibody further comprises an Fc domain. In some embodiments, the Fc domain comprises a first domain monomer and a second Fc domain monomer. In some embodiments, the first and second Fc domain monomers each independently comprise one or more amino acid modifications that, relative to homodimerization, promote heterodimerization of the first and second Fc domain monomers.

[0251] In some embodiments, the Fc domain comprises a first Fc domain monomer containing amino acid modifications capable of forming a knot structure and a second Fc domain monomer containing amino acid modifications capable of forming a hole structure, wherein the hole structure can pair with the knot structure to form a heterodimerized Fc domain (Fc heterodimer).

[0252] Those skilled in the art will readily understand that the hole structure can pair with the knot structure to form a "knobin hole structure (KIH)". The "knob in hole structure" is a method for reducing heavy chain mismatches in bispecific antibodies by introducing amino acid modifications that form a "knob" and a "hole" structure at corresponding positions in the CH3 domains of two Fc domain monomers, respectively, to form a specific interaction interface between the two Fc domain monomers (see Ridgway et al., Protein Eng., 9: 617-621 (1996); WO 2006 / 028936; the entire text of which is incorporated herein by reference). Due to the mutual attraction between the "knob" and "hole" structures and the mutual repulsion between the "knob" and "knob" structures, heavy chain mismatches in bispecific antibodies can be effectively reduced.

[0253] WO9850431 discloses amino acid modifications that promote heterodimer formation, specifically a KIH method for generating Fc domain monomer heterodimers. In this KIH method, one Fc domain monomer in the heterodimer pair contains an amino acid substitution that produces a protrusion (knob) extending outward from the surface of the Fc domain monomer. This protrusion is adapted to a depression (hole) created by an appropriate amino acid substitution in the other Fc domain monomer in the heterodimer pair. This promotes heterodimer formation more than homodimer formation. Examples of amino acid substitutions include S354C:T366W amino acid substitution of the first Fc domain monomer to form a knob, and Y349C:T366S:L368A:Y407V amino acid substitution of the second Fc domain monomer to form a hole (amino acids according to EU index numbers), wherein the first and second Fc domain monomers form a heterodimer pair. WO2014084607 discloses a KIH, wherein the Fc heterodimer comprises a first Fc domain monomer and a second Fc domain monomer, the first Fc domain monomer comprising a K409W amino acid substitution to form a knot, and the second Fc domain monomer comprising D399V and F405T amino acid substitutions to form a hole (amino acid numbers according to the EU index), wherein the first and second Fc domain monomers form a heterodimer pair. WO2013063702 discloses a KIH, wherein the Fc heterodimer comprises a first Fc domain monomer comprising amino acid modifications at positions T350, L351, F405, and Y407, and a second Fc domain monomer comprising amino acid modifications at positions T350, T366, K392, and T394 (amino acid numbers according to the EU index), wherein the first and second Fc domain monomers form a heterodimer pair.

[0254] In some embodiments, the Fc domain monomer is derived from the Fc domain of a human immunoglobulin and modified based on the Fc domain to obtain an Fc domain monomer capable of forming a knock or hole structure. For example, the amino acid sequence of a first Fc domain monomer is modified in a specific region to provide a knock structure, and the amino acid sequence of a second Fc domain monomer is modified in a corresponding region to provide a hole structure capable of accepting a knock structure on the first Fc domain monomer, thereby allowing the first and second Fc domain monomers to form heterodimer pairs while preventing them from forming homodimer pairs. In some embodiments, the human immunoglobulin is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain monomer contains one or more modifications that form a knock structure, wherein the one or more modifications contain S354C and / or T366W amino acid substitutions, numbered according to the Eu numbering scheme. In some embodiments, the Fc domain monomer contains S354 and T366W amino acid substitutions, numbered according to the Eu numbering scheme. In some embodiments, the Fc domain monomer comprises one or more modifications forming a hole structure, wherein said one or more modifications comprise amino acid substitutions of Y349C, T366S, L368A, and / or Y407V, wherein the substitutions are numbered according to the Eu numbering scheme.

[0255] In some embodiments, the Fc domain further possesses altered effector functions (e.g., enhanced ADCC activity) compared to the wild-type Fc region. These altered effector functions can be introduced, for example, by mutation or chemical modification of the Fc domain monomer in the Fc heterodimer.

[0256] In a further embodiment, the first and second Fc domain monomers further comprise one or more amino acid substitutions that reduce or eliminate the function of the Fc region effectors. In certain specific embodiments, the first and second Fc domain monomers further comprise: (i) E233A and L235A (EALA) amino acid substitutions, wherein the amino acids are numbered according to the Eu numbering scheme; (ii) L234A and L235A (LALA) amino acid substitutions, wherein the amino acids are numbered according to the Eu numbering scheme; (iii) L234A L235A D265S (LALADS) amino acid substitutions, wherein the amino acids are numbered according to the Eu numbering scheme; (iv) L234A L235A P329G (LALAPG) amino acid substitutions, wherein the amino acids are numbered according to the Eu numbering scheme; (v) L235E (LE) amino acid substitutions, wherein the amino acids are numbered according to the Eu numbering scheme; (vi) D265A (DS) amino acid substitutions, wherein the amino acids are numbered according to the Eu numbering scheme; (vii) D265A N297G (DANG) amino acid substitutions, wherein the amino acids are numbered according to the Eu numbering scheme; (viii) N297X amino acid substitution, where X is any amino acid other than N, numbered according to the Eu numbering scheme; or (ix) N297A / D356E / L358M (NADELM) amino acid substitution, numbered according to the Eu numbering scheme; or (x) D356E L358M (DELM) amino acid substitution, numbered according to the Eu numbering scheme.

[0257] In some embodiments, the first Fc domain monomer contains an amino acid sequence as shown in SEQ ID NO: 49 or 51, and the second Fc domain monomer contains an amino acid sequence as shown in SEQ ID NO: 50 or 52.

[0258] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are each linked to one of the first and second Fc domain monomers of the Fc heterodimer.

[0259] In some embodiments, the first antigen-binding domain is linked to the first Fc domain monomer, and the second antigen-binding domain is linked to the second Fc domain monomer; or, the first antigen-binding domain is linked to the second Fc domain monomer, and the second antigen-binding domain is linked to the first Fc domain monomer.

[0260] In some embodiments, the first antigen-binding domain is linked to a first Fc domain monomer, and the second antigen-binding domain is linked to a second Fc domain monomer; or the first antigen-binding domain is linked to a second Fc domain monomer, and the second antigen-binding domain is linked to a first Fc domain monomer.

[0261] The individual CDRs of the antigen-binding domains of the bispecific antibodies of the ADCs provided in this article can be determined according to any CDR numbering scheme known in the art.

[0262] In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises light chain CDR 1 (CDR-L1), CDR-L2, and / or CDR-L3 of the VL amino acid sequence as determined by the Kabat numbering scheme. In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises heavy chain CDR 1 (CDR-H1), CDR-H2, and / or CDR-H3 of the VH amino acid sequence as determined by the Kabat numbering scheme.

[0263] In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-L1, CDR-L2, and CDR-L3 of the VL amino acid sequence as defined by SEQ ID NOs: 15, 17, and 59, as determined by the Chothia numbering scheme. In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-H1, CDR-H2, and / or CDR-H3 of the VH amino acid sequence as defined by SEQ ID NOs: 16, 18, and 60, as determined by the Chothia numbering scheme.

[0264] In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-L1, CDR-L2, and / or CDR-L3 of the VL amino acid sequence as shown in any one of SEQ ID NOs: 15, 17, and 59 as determined by the MacCallum numbering scheme. In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-H1, CDR-H2, and / or CDR-H3 of the VH amino acid sequence as shown in any one of SEQ ID NOs: 16, 18, and 60 as determined by the MacCallum numbering scheme.

[0265] In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-L1, CDR-L2, and / or CDR-L3 of the VL amino acid sequence as shown in any one of SEQ ID NOs: 15, 17, and 59 as determined by the IMGT numbering scheme. In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-H1, CDR-H2, and / or CDR-H3 of the VH amino acid sequence as shown in any one of SEQ ID NOs: 16, 18, and 60 as determined by the IMGT numbering scheme.

[0266] In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-L1, CDR-L2, and / or CDR-L3 of the VL amino acid sequence as shown in any one of SEQ ID NOs: 15, 17, and 59 as determined by the AbM numbering scheme. In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-H1, CDR-H2, and / or CDR-H3 of the VH amino acid sequence as shown in any one of SEQ ID NOs: 16, 18, and 60 as determined by the AbM numbering scheme.

[0267] In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-L1, CDR-L2, and / or CDR-L3 of the VL amino acid sequence as shown in any one of SEQ ID NOs: 15, 17, and 59 as determined by the AHo numbering scheme. In some embodiments, the antigen-binding domain of the bispecific antibody of the ADC provided herein comprises CDR-H1, CDR-H2, and / or CDR-H3 of the VH amino acid sequence as shown in any one of SEQ ID NOs: 16, 18, and 60 as determined by the AHo numbering scheme.

[0268] In some embodiments, each CDR of the antigen-binding domain of the bispecific antibody of the ADC provided herein is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the antigen-binding domain, wherein the structural analysis identifies residues in the variable region that are expected to contact the epitope region of c-MET or EGFR.

[0269] In some embodiments, this disclosure provides an ADC comprising a bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises a VL comprising the amino acid sequences CDR-L1, CDR-L2, and CDR-L3 of the VL amino acid sequence shown in SEQ ID NO: 17 or 59, and a VH comprising the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 of the VH amino acid sequence shown in SEQ ID NO: 18 or 60, and / or wherein the second antigen-binding domain comprises a VL comprising the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 of the VL amino acid sequence shown in SEQ ID NO: 15, and a VH comprising the amino acid sequences CDR-H1, CDR-H2, and CDR-H3 of the VH amino acid sequence shown in SEQ ID NO: 16. Each CDR is independently determined according to one of the numbering schemes of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM, or by structural analysis of the antigen-binding domain, wherein the structural analysis identifies residues in the variable region that are expected to contact the epitope region of c-MET or EGFR.

[0270] In some embodiments, this disclosure provides an ADC comprising a bispecific antibody, the bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises VL of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17, and VH of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 18, and / or the second antigen-binding domain comprises VL of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 15, and VH of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16. Each CDR is independently determined according to one of the numbering schemes of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM, or by structural analysis of the antigen-binding domain, wherein the structural analysis identifies residues in the variable region that are expected to contact the epitope region of c-MET or EGFR.

[0271] In some embodiments, this disclosure provides an ADC comprising a bispecific antibody, the bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises VL of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 59, and VH of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 60, and / or the second antigen-binding domain comprises VL of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 15, and VH of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16. Each CDR is independently determined according to one of the numbering schemes of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM, or by structural analysis of the antigen-binding domain, wherein the structural analysis identifies residues in the variable region that are expected to contact the epitope region of c-MET or EGFR.

[0272] In some embodiments, this disclosure provides an ADC comprising a bispecific antibody, the bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises VL of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17, and VH of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 60, and / or the second antigen-binding domain comprises VL of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 15, and VH of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16. Each CDR is independently determined according to one of the numbering schemes of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM, or by structural analysis of the antigen-binding domain, wherein the structural analysis identifies residues in the variable region that are expected to contact the epitope region of c-MET or EGFR.

[0273] In some embodiments, this disclosure provides an ADC comprising a bispecific antibody, the bispecific antibody comprising a first antigen-binding domain specifically binding to c-MET and a second antigen-binding domain specifically binding to EGFR, wherein the first antigen-binding domain comprises VL of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 59, and VH of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 18, and / or the second antigen-binding domain comprises VL of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 15, and VH of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16. Each CDR is independently determined according to one of the numbering schemes of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM, or by structural analysis of the antigen-binding domain, wherein the structural analysis identifies residues in the variable region that are expected to contact the epitope region of c-MET or EGFR.

[0274] In some embodiments of the ADC provided herein, the first VL of the bispecific antibody of the ADC comprises: CDR-L1, which contains the amino acid sequence shown in SEQ ID NO: 34; CDR-L2, which contains the amino acid sequence shown in SEQ ID NO: 36; and CDR-L3, which contains the amino acid sequence shown in SEQ ID NO: 38; wherein the CDRs are defined by the Kabat numbering system. CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Chothia numbering system; CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the Abm numbering system; or CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 35, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 37, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38; wherein the CDR is defined by the IMGT numbering system; and / or The first VH includes: CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 39; CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 43; and CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 47; wherein the CDR is defined by the Kabat numbering system. CDR-H1, comprising the amino acid sequence shown in SEQ ID NO: 40; CDR-H2, comprising the amino acid sequence shown in SEQ ID NO: 44; and CDR-H3, comprising the amino acid sequence shown in SEQ ID NO: 47; wherein the CDR is defined by the Chothia numbering system. CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 42, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47; wherein the CDR is defined by the Abm numbering system; or, CDR-H1, which contains the amino acid sequence shown in SEQ ID NO: 41; CDR-H2, which contains the amino acid sequence shown in SEQ ID NO: 45; and CDR-H3, which contains the amino acid sequence shown in SEQ ID NO: 48; wherein the CDR is defined by the IMGT numbering system.

[0275] In some embodiments, the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 17 or 59. In some embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO: 17 or 59.

[0276] In some embodiments, the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO: 18 or 60. In some embodiments, the first VH consists of the amino acid sequence shown in SEQ ID NO: 18 or 60.

[0277] In some embodiments, the first VL comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 17 or 59, and the first VH comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 18 or 60. In some embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO: 17 or 59, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 18 or 60.

[0278] In some embodiments, the first VL comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 17, and the first VH comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, and the first VH comprises the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO: 17, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 18.

[0279] In some embodiments, the first VL comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 59, and the first VH comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO: 59, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 60.

[0280] In some embodiments, the first VL comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 60, and the first VH comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO: 17, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 60.

[0281] In some embodiments, the first VL comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 59, and the first VH comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the first VL consists of the amino acid sequence shown in SEQ ID NO: 59, and the first VH consists of the amino acid sequence shown in SEQ ID NO: 18.

[0282] In some implementations of the ADC provided herein, the second VL includes: (i) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Kabat numbering system; (ii) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Chothia numbering system; (iii) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the Abm numbering system; or (iv) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 20, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 22, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23; wherein the CDR is defined by the IMGT numbering system; and / or The second VH of the bispecific antibody of the ADC comprises: (i) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 24, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 28, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32; wherein the CDR is defined by the Kabat numbering system; (ii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 25, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 29, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32; wherein the CDR is defined by the Chothia numbering system; (iii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 27, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32; wherein the CDR is defined by the Abm numbering system; or (iv) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 26, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 30, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 33; wherein the CDR is defined by the IMGT numbering system.

[0283] In some embodiments, the second VL comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the second VL consists of the amino acid sequence shown in SEQ ID NO: 15.

[0284] In some embodiments, the second VH comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the second VH consists of the amino acid sequence shown in SEQ ID NO: 16.

[0285] In some embodiments, the second VL comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 15, and the second VH comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the second VL consists of the amino acid sequence shown in SEQ ID NO: 15, and the second VH consists of the amino acid sequence shown in SEQ ID NO: 16.

[0286] In some embodiments, this disclosure provides an ADC comprising a bispecific antibody that cross-competes with any antibody described herein for binding to c-MET and / or EGFR. In some embodiments, this disclosure provides an ADC comprising a bispecific antibody that binds to the same or overlapping epitopes of c-MET and / or EGFR with the antibody described herein.

[0287] In some implementations, the epitopes of the antibody can be determined by, for example, NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange and mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any method known in the art (e.g., Gigé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are incorporated herein by reference in their entirety). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) volumes 114 & 115, edited by Eds. Wyckoff HW et al.; US Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr. D. Biocrystals 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323, all of which are incorporated herein by reference in their entirety). Mutagenic mapping studies can be performed using any method known to those skilled in the art. Descriptions of mutagenic techniques, including alanine scan mutagenesis, can be found, for example, in Champe M et al. (1995) and Cunningham BC and Wells JA (1989). In one specific embodiment, alanine scan mutagenesis is used to identify the epitopes of the antibody. Additionally, bispecific antibodies that recognize and bind to the same or overlapping epitopes of c-MET and / or EGFR can be identified using conventional techniques such as immunoassays, for example by demonstrating the ability of one antibody to block the binding of another antibody to the target antigen, i.e., a competitive binding assay. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity to the epitopes.Competitive binding can be determined in assays that specifically bind to a reference antibody against a common antigen (such as c-MET or EGFR). Several types of competitive binding assays are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli C et al., (1983) Method Enzyme Mole 9: 242-253); solid-phase direct biotinylate EIA (see Kirkland TN et al., (1986) J immunl 137: 3614-9); solid-phase direct labeling, solid-phase direct labeling sandwich assay (see Harlow E & Lane D, (1988) Antibodies: Laboratory Handbook, Cold Spring Harbor); solid-phase direct labeling RIA using I-125 labeling (see Morel GA et al., (1988) Mol Immunol 25(1): 7-15); solid-phase direct biotin-avidinase immunoassay (see Zhang RC et al., (1990) Virology 176:546-52); and directly labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32: 77-82), all of which are incorporated herein by reference in their entirety. Typically, such assays involve the use of purified antigens (e.g., c-MET or EGFR) or cells containing any of the above, bound to a solid surface, unlabeled target immunoglobulin, and labeled reference immunoglobulin. Competitive inhibition can be determined by measuring the amount of labeling bound to the solid surface or cells in the presence of the target immunoglobulin. Typically, an excess of the target immunoglobulin is present. Typically, when an excess of the competitive antibody is present, it will inhibit the specific binding of the reference or antibody to the same antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured in a wide variety of formats using labeled antigens or labeled antibodies. In a common version of this assay, the antigen is immobilized on a 96-well plate. Radioactive or enzyme labeling is then used to determine the ability of the unlabeled antibody to block the binding of the labeled antibody to the antigen.For further details, see, for example, Wagener C et al., (1983) J immunl 130: 2308-2315; Weiner et al., (1984) Immunomethods 68: 269-274; Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D eds., pp. 386-389, all of which are incorporated herein by reference in their entirety.

[0288] In some embodiments of the ADC provided herein, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1, according to the EU numbering system) and / or the hinge region (residues 216-230, according to the EU numbering system) of the bispecific antibody of the ADC to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding and / or antigen-dependent cytotoxicity.

[0289] In some implementations, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region to, for example, reduce or eliminate the effector function of the Fc region. In some embodiments, the antibody comprises an Fc region containing: (i) amino acid substitutions for E233A and L235A (EALA), wherein the amino acid substitutions are numbered according to the Eu numbering scheme; (ii) amino acid substitutions for L234A and L235A (LALA), wherein the amino acid substitutions are numbered according to the Eu numbering scheme; (iii) amino acid substitutions for L234A, L235A, and D265S (LALADS), wherein the amino acid substitutions are numbered according to the Eu numbering scheme; (iv) amino acid substitutions for L234A, L235A, and P329G (LALAPG), wherein the amino acid substitutions are numbered according to the Eu numbering scheme; (v) amino acid substitutions for L234A, L235A, and P329A (LALAPA), wherein the amino acid substitutions are numbered according to the Eu numbering scheme; (vi) amino acid substitutions for L235E (LE), wherein the amino acid substitutions are numbered according to the Eu numbering scheme; (vii) amino acid substitutions for D265A (DS), wherein the amino acid substitutions are numbered according to the Eu numbering scheme; (viii) amino acid substitutions for D265A... N297G(DANG) amino acid substitution, wherein the numbering follows the Eu numbering scheme; (ix) N297X amino acid subunit substitution, wherein X is any amino acid other than N, wherein the numbering follows the Eu numbering scheme; (x) N297A / D356E / L358M(NADELM) amino acid substitution, wherein the numbering follows the Eu numbering scheme; or (xi) D356E L358M(DELM) amino acid substitution, wherein the numbering follows the Eu numbering scheme.

[0290] In some implementations, any constant region mutation or modification described herein may be introduced into one or both heavy chain constant regions of the antibody described herein that has two heavy chain constant regions.

[0291] Fab-scFv structure

[0292] In some implementations, the first antigen-binding domain is Fab, and the second antigen-binding domain is scFv.

[0293] In some embodiments, the bispecific antibody comprises polypeptide chain IA, polypeptide chain IB, and polypeptide chain IC; wherein (a) the elements contained in polypeptide chain IA include a first VL and a first CL; the elements contained in polypeptide chain IB include a first VH, a first heavy chain CH1 region, and a first Fc domain monomer; the elements contained in polypeptide chain IC include a second VL, a second VH, a second heavy chain CH1 hinge region, and a second Fc domain monomer; or (b) the elements contained in polypeptide chain IA include a first VL and a first CL; the elements contained in polypeptide chain IB include a first VH, a first heavy chain CH1 region, and a second Fc domain monomer; the elements contained in polypeptide chain IC include a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc domain monomer.

[0294] In some embodiments, (a) the element contained in polypeptide chain IA comprises a first VL and a first CL from the N-terminus to the C-terminus; the element contained in polypeptide chain IB comprises a first VH, a first heavy chain CH1 region, and a first Fc domain monomer from the N-terminus to the C-terminus; and / or the element contained in polypeptide chain IC comprises: (i) a second VL, a second VH, a second heavy chain CH1 hinge region, and a second Fc domain monomer from the N-terminus to the C-terminus, or (ii) a second VH, a second VL, a second heavy chain CH1 hinge region, and a second Fc domain monomer. The elements contained in polypeptide chain IA include, from the N-terminus to the C-terminus, a first VL and a first CL; the elements contained in polypeptide chain IB include, from the N-terminus to the C-terminus, a first VH, a first heavy chain CH1, and a second Fc domain monomer; and / or the elements contained in polypeptide chain IC include, from the N-terminus to the C-terminus, (i) a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc domain monomer, or (ii) a second VH, a second VL, a second heavy chain CH1 hinge region, and a first Fc domain monomer.

[0295] In some embodiments, adjacent elements contained in polypeptide chain IA are interconnected with or without peptide connectors; adjacent elements contained in polypeptide chain IB are interconnected with or without peptide connectors; and / or adjacent elements contained in polypeptide chain IC are interconnected with or without peptide connectors.

[0296] In some embodiments, the peptide linkers are each independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers). In some embodiments, each peptide linker independently comprises 3 to 55 amino acid residues (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55). In some embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S) residues. In some embodiments, each peptide linker may independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues. In some embodiments, each peptide linker may independently comprise peptide GGGG (SEQ ID NO: 61). In some embodiments, the peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which may be composed of (GGGGS). n The expression indicates that n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 57, 65, 55, 58, 66-68, 56, 69, or 70, respectively). In some embodiments, each peptide linker independently comprises an amino acid sequence shown in any of SEQ ID NO: 55-58 or 65-70.

[0297] In some embodiments, the first CL comprises the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 63. In some embodiments, the first heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, the second heavy chain CH1 hinge region comprises the amino acid sequence shown in SEQ ID NO: 62.

[0298] In some embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 60, and the first VL comprises the amino acid sequence shown in SEQ ID NO: 59. In some embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 60, the first VL comprises the amino acid sequence shown in SEQ ID NO: 59, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15.

[0299] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52.

[0300] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51.

[0301] In some embodiments, the polypeptide chain IA contains the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB contains the amino acid sequence shown in SEQ ID NO: 2 or 9, and / or the polypeptide chain IC contains the amino acid sequence shown in SEQ ID NO: 3 or 10.

[0302] scFv-Fab structure

[0303] In some implementations, the first antigen-binding domain is scFv and the second antigen-binding domain is Fab.

[0304] In some embodiments, the bispecific antibody comprises peptide chain II-A, peptide chain II-B, and peptide chain II-C; wherein (a) the element contained in peptide chain II-A comprises a second VL and a second CL; the element contained in peptide chain II-B comprises a second VH, a second heavy chain CH1 region, and a first Fc domain monomer; the element contained in peptide chain II-C comprises a first VL, a first VH, a first heavy chain CH1 hinge region, and a second Fc domain monomer, or (b) the element contained in peptide chain II-A comprises a second VL and a second CL; the element contained in peptide chain II-B comprises a second VH, a second heavy chain CH1 region, and a second Fc domain monomer; and the element contained in peptide chain II-C comprises a first VL, a first VH, a first heavy chain CH1 hinge region, and a first Fc domain monomer.

[0305] In some embodiments, (a) the element contained in polypeptide chain II-A comprises a second VL and a second CL from the N-terminus to the C-terminus; the element contained in polypeptide chain II-B comprises a second VH, a second heavy chain CH1 region, and a first Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in polypeptide chain II-C comprises, from the N-terminus to the C-terminus: (i) a first VL, a first VH, a first heavy chain CH1 hinge region, and a second Fc domain monomer, or (ii) a first VH, a first VL, a first heavy chain CH1 hinge region, and a second Fc domain monomer, or (b) The element contained in the polypeptide chain II-A includes a second VL and a second CL from the N-terminus to the C-terminus; the element contained in the polypeptide chain II-B includes a second VH, a second heavy chain CH1 region, and a second Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in the polypeptide chain II-C includes: (i) a first VL, a first VH, a first heavy chain CH1 hinge region, and a first Fc domain monomer from the N-terminus to the C-terminus, or (ii) a first VH, a first VL, a first heavy chain CH1 hinge region, and a first Fc domain monomer.

[0306] In some embodiments, adjacent elements contained in polypeptide chain II-A are optionally connected by or without a connector, adjacent elements contained in polypeptide chain II-B are optionally connected by or without a connector, and / or adjacent elements contained in polypeptide chain II-C are optionally connected by or without a connector.

[0307] In some embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid or flexible peptide linkers). In some embodiments, each peptide linker is independently composed of 3 to 55 amino acid residues (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55). In some embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S). In some embodiments, each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which may be composed of (GGGGS). n The expression indicates that n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as shown in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, and 70, respectively). In some embodiments, each peptide linker independently comprises an amino acid sequence as shown in one of SEQ ID NOs: 55-58 or 65-70.

[0308] In some embodiments, the second CL comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54, and / or the heavy chain CH1 hinge region comprises an amino acid sequence as shown in SEQ ID NO: 62.

[0309] In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.

[0310] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52.

[0311] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51.

[0312] In some embodiments, polypeptide chain II-A comprises an amino acid sequence as shown in SEQ ID NO: 4, polypeptide chain II-B comprises an amino acid sequence as shown in SEQ ID NO: 5 or 7, and / or, polypeptide chain II-C comprises an amino acid sequence as shown in SEQ ID NO: 6 or 8.

[0313] Fab-scFab structure

[0314] In some implementations, the first antigen-binding domain is Fab, and the second antigen-binding domain is scFab (single-chain Fab).

[0315] In some embodiments, the bispecific antibody comprises polypeptide chain III-A, polypeptide chain III-B, and polypeptide chain III-C; wherein (a) the element contained in polypeptide chain III-A comprises a first VL and a first CL; the element contained in polypeptide chain III-B comprises a first VH, a first heavy chain CH1 region, and a first Fc domain monomer; and the element contained in polypeptide chain III-C comprises a second VL, a second CL, a second VH, a second heavy chain CH1 region, and a second Fc domain monomer; or, (b) the element contained in polypeptide chain III-A comprises a first VL and a first CL; the element contained in polypeptide chain III-B comprises a first VH, a first heavy chain CH1 region, and a second Fc domain monomer; and the element contained in polypeptide chain III-C comprises a second VL, a second CL, a second VH, a second heavy chain CH1 region, and a first Fc domain monomer.

[0316] In some embodiments, (a) the element contained in polypeptide chain III-A contains a first VL and a first CL from the N-terminus to the C-terminus; the element contained in polypeptide chain III-B contains a first VH, a first heavy chain CH1 region, and a first Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in polypeptide chain III-C contains (i) a second VL, a second CL, a second VH, a second heavy chain CH1 region, and a second Fc domain monomer from the N-terminus to the C-terminus, or (ii) a second VH, a second heavy chain CH1 region, a second VL, CL, and a second Fc domain monomer; or, (b) The element contained in polypeptide chain III-A contains a first VL and a first CL from the N-terminus to the C-terminus; the element contained in polypeptide chain III-B contains a first VH, a first heavy chain CH1 region, and a second Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in polypeptide chain III-C contains (i) a second VL, a second CL, a second VH, a second heavy chain CH1 region, and a first Fc domain monomer from the N-terminus to the C-terminus, or (ii) a second VH, a second heavy chain CH1 region, a second VL, a second CL, and a first Fc domain monomer.

[0317] In some embodiments, adjacent elements contained in polypeptide chain III-A are optionally connected by or without a connector, adjacent elements contained in polypeptide chain III-B are optionally connected by or without a connector, and / or adjacent elements contained in polypeptide chain III-C are optionally connected by or without a connector.

[0318] In some embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid or flexible peptide linkers). In some embodiments, each peptide linker is independently composed of 3 to 55 amino acid residues (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55). In some embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S). In some embodiments, each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which may be composed of (GGGGS). n The expression indicates that n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as shown in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, and 70, respectively). In some embodiments, each peptide linker independently comprises an amino acid sequence as shown in one of SEQ ID NOs: 55-58 or 65-70.

[0319] In some embodiments, the first CL and the second CL contain amino acid sequences as shown in SEQ ID NO: 53 or SEQ ID NO: 63, the second heavy chain CH1 region contains amino acid sequences as shown in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region contains amino acid sequences as shown in SEQ ID NO: 62.

[0320] In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.

[0321] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52.

[0322] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51.

[0323] scFab-Fab structure

[0324] In some embodiments, the first antigen-binding domain is scFab, and the second antigen-binding domain is Fab.

[0325] In some embodiments, the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C, wherein (a) the element contained in polypeptide chain IV-A comprises a second VL and a second CL; the element contained in polypeptide chain IV-B comprises a second VH, a second heavy chain CH1 region, and a first Fc domain monomer; and the element contained in polypeptide chain IV-C comprises a first VL, a first CL, a first VH, a first heavy chain CH1 hinge region, and a second Fc domain monomer; or, (b) the element contained in polypeptide chain IV-A comprises a second VL and a second CL; the element contained in polypeptide chain IV-B comprises a second VH, a second heavy chain CH1 region, and a second Fc domain monomer; and the element contained in polypeptide chain IV-C comprises a first VL, a first CL, a first VH, a first heavy chain CH1 hinge region, and a first Fc domain monomer.

[0326] In some embodiments, (a) the element contained in the polypeptide chain IV-A comprises a second VL and a second CL from the N-terminus to the C-terminus; the element contained in the polypeptide chain IV-B comprises a second VH, a second heavy chain CH1 region, and a first Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in the polypeptide chain IV-C comprises a first VL, a first CL, a first VH, a first heavy chain CH1 hinge region, and a second Fc domain monomer from the N-terminus to the C-terminus; or, (b) the element contained in the polypeptide chain IV-A comprises a second VL and a second CL from the N-terminus to the C-terminus; the element contained in the polypeptide chain IV-B comprises a second VL and a second CL, a second VH, a second heavy chain CH1 region, and a second Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in the polypeptide chain IV-C comprises (i) a first VL, a first CL, a first VH, a first heavy chain CH1 hinge region, and a first Fc domain monomer from the N-terminus to the C-terminus.

[0327] In some embodiments, adjacent elements contained in polypeptide chain IV-A are optionally connected by or without a connector, adjacent elements contained in polypeptide chain IV-B are optionally connected by or without a connector, and / or adjacent elements contained in polypeptide chain IV-C are optionally connected by or without a connector.

[0328] In some embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid or flexible peptide linkers). In some embodiments, each peptide linker is independently composed of 3 to 55 amino acid residues (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55). In some embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S). In some embodiments, each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which may be composed of (GGGGS). n The expression indicates that n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as shown in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, and 70, respectively). In some embodiments, each peptide linker independently comprises an amino acid sequence as shown in one of SEQ ID NOs: 55-58 or 65-70.

[0329] In some embodiments, the first and second CLs contain amino acid sequences as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region contains an amino acid sequence as shown in SEQ ID NO: 54, and / or the first heavy chain CH1 hinge region contains an amino acid sequence as shown in SEQ ID NO: 62.

[0330] In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.

[0331] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52.

[0332] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51.

[0333] In some embodiments, polypeptide chain IV-A comprises the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain IV-B comprises the amino acid sequence shown in SEQ ID NO: 7, and / or polypeptide chain IV-C comprises the amino acid sequence shown in SEQ ID NO: 11.

[0334] Fab-Fab (including crossMab-style domain exchange) structure

[0335] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are Fab, and the Fab of the second antigen-binding domain contains domain exchanges of the form of CrossMab.

[0336] In some embodiments, the Fab of the second antigen-binding domain comprises CrossMab VH-VL The domains of VH and VL in the form of swapping.

[0337] In some embodiments, the Fab of the second antigen-binding domain contains CrossMab. CH1-CL The CH1 and CL structure domains are swapped.

[0338] In some embodiments, the Fab of the second antigen-binding domain contains CrossMab. Fab Domain swapping in the form of structure.

[0339] In some implementations, the CrossMab CH1-CL A bispecific antibody comprises polypeptide chains VA, VB, VC, and VD; wherein (a) the elements contained in the polypeptide chain VA include a first VL and a first CL; the elements contained in the polypeptide chain VB include a first VH, a first heavy chain CH1 region, and a first Fc domain monomer; the elements contained in the polypeptide chain VC include a second VH, a second CL, a second heavy chain CH1 hinge region, and a second Fc domain monomer; and the elements contained in the polypeptide chain VD include a second VL and a second heavy chain CH1 region; or, (b) the elements contained in the polypeptide chain VA include a first VL and a first CL; the elements contained in the polypeptide chain VB include a first VH, a first heavy chain CH1 region, and a second Fc domain monomer; the elements contained in the polypeptide chain VC include a second VH, a second CL, a second heavy chain CH1 hinge region, and a first Fc domain monomer; and the elements contained in the polypeptide chain VD include a second VL and a second heavy chain CH1 region.

[0340] In some embodiments, (a) the element contained in the polypeptide chain VA comprises a first VL and a first CL from the N-terminus to the C-terminus; the element contained in the polypeptide chain VB comprises a first VH, a first heavy chain CH1 region, and a first Fc domain monomer from the N-terminus to the C-terminus; the element contained in the polypeptide chain VC comprises a second VH, a second CL, a second heavy chain CH1 hinge region, and a second Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in the polypeptide chain VD comprises a second VL and a second heavy chain CH1 region from the N-terminus to the C-terminus; or, (b) The elements contained in the polypeptide chain VA include a first VL and a first CL from the N-terminus to the C-terminus; the elements contained in the polypeptide chain VB include a first VH, a first heavy chain CH1 region, and a second Fc domain monomer from the N-terminus to the C-terminus; the elements contained in the polypeptide chain VC include a second VH, a second CL, a second heavy chain CH1 hinge region, and a first Fc domain monomer from the N-terminus to the C-terminus; and / or, the elements contained in the polypeptide chain VD include a second VL and a second heavy chain CH1 region from the N-terminus to the C-terminus.

[0341] In some embodiments, adjacent elements contained in the polypeptide chain VA are optionally connected by or without a connector, adjacent elements contained in the polypeptide chain VB are optionally connected by or without a connector, adjacent elements contained in the polypeptide chain VC are optionally connected by or without a connector, and / or adjacent elements contained in the polypeptide chain VD are optionally connected by or without a connector.

[0342] In some embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid or flexible peptide linkers). In some embodiments, each peptide linker is independently composed of 3 to 55 amino acid residues (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55). In some embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S). In some embodiments, each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which may be composed of (GGGGS). n The expression indicates that n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as shown in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, and 70, respectively). In some embodiments, each peptide linker independently comprises an amino acid sequence as shown in one of SEQ ID NOs: 55-58 or 65-70.

[0343] In some implementations, the first and second CLs

[0344] It contains an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, the heavy chain CH1 region contains an amino acid sequence as shown in SEQ ID NO: 54, and / or the heavy chain CH1 hinge region contains an amino acid sequence as shown in SEQ ID NO: 62.

[0345] In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.

[0346] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52.

[0347] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51.

[0348] In some embodiments, the polypeptide chain VA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain VB comprises the amino acid sequence shown in SEQ ID NO: 9, the polypeptide chain VC comprises the amino acid sequence shown in SEQ ID NO: 13, and / or, the polypeptide chain VD comprises the amino acid sequence shown in SEQ ID NO: 12.

[0349] Fab (including crossMab-style domain exchanges) - Fab structure

[0350] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are Fabs, and the Fab of the first antigen-binding domain contains domain exchanges of the form of CrossMab.

[0351] In some embodiments, the Fab of the first antigen-binding domain contains the CrossMab. CH1-CL The CH1 and CL structure domains are swapped.

[0352] In some embodiments, the Fab of the first antigen-binding domain contains CrossMab. VH-VL The VH and VL domains of the form are swapped.

[0353] In some embodiments, the Fab of the first antigen-binding domain contains CrossMab. Fab Domain swapping in the form of structure.

[0354] In some embodiments, the bispecific antibody comprises polypeptide chain VI-A, polypeptide chain VI-B, polypeptide chain VI-C, and polypeptide chain VI-D; wherein, (a) the element contained in polypeptide chain VI-A comprises the second light chain variable region and the second CL, the element contained in polypeptide chain VI-B comprises: the second heavy chain variable region, the second heavy chain CH1 region, and the first Fc domain monomer, the element contained in polypeptide chain VI-C comprises: the first heavy chain variable region, the first CL, and the second Fc domain monomer, and the element contained in polypeptide chain VI-D comprises: the first light chain variable region and the first heavy chain CH1 region; or, (b) the element contained in polypeptide chain VI-A comprises the second light chain variable region and the second CL, the element contained in polypeptide chain VI-B comprises: the second heavy chain variable region, the second heavy chain CH1 region, and the second Fc domain monomer, the element contained in polypeptide chain VI-C comprises: the first heavy chain variable region, the first CL, and the first Fc domain monomer, and the element contained in polypeptide chain VI-D comprises: the first light chain variable region and the first heavy chain CH1 region.

[0355] In some embodiments, polypeptide chain VI-A includes the second light chain variable region and the second CL from the N-terminus to the C-terminus; polypeptide chain VI-B includes the second heavy chain variable region, the second heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus; polypeptide chain VI-C includes the first heavy chain variable region, the first CL, and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus; and / or, polypeptide chain VI-D includes the first light chain variable region and the first heavy chain CH1 region from the N-terminus to the C-terminus.

[0356] In some embodiments, (a) the element included in polypeptide chain VI-A comprises a second VL and a second CL from the N-terminus to the C-terminus; the element included in polypeptide chain VI-B comprises a second VL and a second CL, a second VH, a heavy chain CH1 region, and a first Fc domain monomer from the N-terminus to the C-terminus; the element included in polypeptide chain VI-C comprises a first VH, a first CL, a first heavy chain CH1 hinge region, and a second Fc domain monomer from the N-terminus to the C-terminus; and / or, the element included in polypeptide chain VI-D comprises a first VL and a heavy chain CH1 region from the N-terminus to the C-terminus; or, (b) The element contained in polypeptide chain VI-A includes a second VL and a second CL from the N-terminus to the C-terminus; the element contained in polypeptide chain VI-B includes a second VH, a heavy chain CH1 region, and a second Fc domain monomer from the N-terminus to the C-terminus; the element contained in polypeptide chain VI-C includes a first VH, a first CL, a first heavy chain CH1 hinge region, and a first Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in polypeptide chain VI-D includes a first VL and a heavy chain CH1 region from the N-terminus to the C-terminus.

[0357] In some embodiments, adjacent elements contained in polypeptide chains VI-A are optionally connected by or without a connector, adjacent elements contained in polypeptide chains VI-B are optionally connected by or without a connector, adjacent elements contained in polypeptide chains VI-C are optionally connected by or without a connector, and / or adjacent elements contained in polypeptide chains VI-D are optionally connected by or without a connector.

[0358] In some embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid or flexible peptide linkers). In some embodiments, each peptide linker is independently composed of 3 to 55 amino acid residues (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55). In some embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S). In some embodiments, each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which may be composed of (GGGGS). nThe expression indicates that n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as shown in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, and 70, respectively). In some embodiments, each peptide linker independently comprises an amino acid sequence as shown in one of SEQ ID NOs: 55-58 or 65-70.

[0359] In some embodiments, the first CL and the second CL contain amino acid sequences as shown in SEQ ID NO: 53 or SEQ ID NO: 63, the heavy chain CH1 region contains amino acid sequences as shown in SEQ ID NO: 54, and / or the first heavy chain CH1 hinge region includes the amino acid sequence shown in SEQ ID NO: 62.

[0360] In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.

[0361] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52.

[0362] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51.

[0363] In some implementations, the CrossMab VH-VL A bispecific antibody comprises polypeptide chains VA, VB, VC, and VD; wherein, (a) the elements contained in the polypeptide chain VA include a first light chain variable region and a first CL; the elements contained in the polypeptide chain VB include a first heavy chain variable region, a first heavy chain CH1 region, and a first Fc domain monomer; the elements contained in the polypeptide chain VC include a second light chain variable region, a second heavy chain CH1 hinge region, and a second Fc domain monomer; and the elements contained in the polypeptide chain VD include a second heavy chain variable region and a second CL; or, (b) the elements contained in the polypeptide chain VA include a first light chain variable region and a first CL; the elements contained in the polypeptide chain VB include a first heavy chain variable region, a first heavy chain CH1 region, and a second Fc domain monomer; the elements contained in the polypeptide chain VC include a second light chain variable region, a second heavy chain CH1 hinge region, and a first Fc domain monomer; and the elements contained in the polypeptide chain VD include a second heavy chain variable region and a second CL.

[0364] In some embodiments, (a) the element contained in the polypeptide chain VA comprises a first VL and a first CL from the N-terminus to the C-terminus; the element contained in the polypeptide chain VB comprises a first VH, a first heavy chain CH1 region, and a first Fc domain monomer from the N-terminus to the C-terminus; the element contained in the polypeptide chain VC comprises a second VL, a second heavy chain CH1 hinge region, and a second Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in the polypeptide chain VD comprises a second VH and a second CL from the N-terminus to the C-terminus; or, (b) the element contained in the polypeptide chain VA comprises a first VL and a first CL from the N-terminus to the C-terminus; the element contained in the polypeptide chain VB comprises a first VH, a first heavy chain CH1 region, and a second Fc domain monomer from the N-terminus to the C-terminus; the element contained in the polypeptide chain VC comprises a second VL, a second heavy chain CH1 hinge region, and a first Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in the polypeptide chain VD comprises a second VH and a second CL from the N-terminus to the C-terminus.

[0365] In some embodiments, adjacent elements contained in the polypeptide chain VA are optionally connected by or without a connector, adjacent elements contained in the polypeptide chain VB are optionally connected by or without a connector, adjacent elements contained in the polypeptide chain VC are optionally connected by or without a connector, and / or adjacent elements contained in the polypeptide chain VD are optionally connected by or without a connector.

[0366] In some embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid or flexible peptide linkers). In some embodiments, each peptide linker is independently composed of 3 to 55 amino acid residues (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55). In some embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S). In some embodiments, each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which may be composed of (GGGGS). n The expression indicates that n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as shown in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, and 70, respectively). In some embodiments, each peptide linker independently comprises an amino acid sequence as shown in one of SEQ ID NOs: 55-58 or 65-70.

[0367] In some embodiments, the first and second CLs contain amino acid sequences as shown in SEQ ID NO: 53 or SEQ ID NO: 63, the first and second heavy chain CH1 regions contain amino acid sequences as shown in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region contains amino acid sequences as shown in SEQ ID NO: 62.

[0368] In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.

[0369] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52.

[0370] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51.

[0371] In some implementations, the CrossMab Fab A bispecific antibody comprises polypeptide chains VA, VB, VC, and VD; wherein (a) the elements contained in the polypeptide chain VA include a first light chain variable region and a first CL; the elements contained in the polypeptide chain VB include a first heavy chain variable region, a first heavy chain CH1 region, and a first Fc domain monomer; the elements contained in the polypeptide chain VC include a second light chain variable region, a second CL region, a second heavy chain CH1 hinge region, and a second Fc domain monomer; and the elements contained in the polypeptide chain VD include a second heavy chain variable region and a second heavy chain CH1 region; or (b) the elements contained in the polypeptide chain VA include a first light chain variable region and a first CL; the elements contained in the polypeptide chain VB include a first heavy chain variable region, a first light chain CH1 region, and a second Fc domain monomer; the elements contained in the polypeptide chain VC include a second light chain variable region, a second CL, a second heavy chain CH1 hinge region, and a first Fc domain monomer; and the elements contained in the polypeptide chain VD include a second heavy chain variable region and a second heavy chain CH1 region.

[0372] In some embodiments, (a) the element contained in the polypeptide chain VA comprises a first VL and a first CL from the N-terminus to the C-terminus; the element contained in the polypeptide chain VB comprises a first VH, a first heavy chain CH1 region, and a first Fc domain monomer from the N-terminus to the C-terminus; the element contained in the polypeptide chain VC comprises a second VL, a second heavy chain CH1 hinge region, and a second Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in the polypeptide chain VD comprises a second VH and a second heavy chain CH1 region from the N-terminus to the C-terminus; or, (b) the element contained in the polypeptide chain VA comprises a first VL and a second CL from the N-terminus to the C-terminus; the element contained in the polypeptide chain VB comprises a first VH, a first heavy chain CH1 region, and a second Fc domain monomer from the N-terminus to the C-terminus; the element contained in the polypeptide chain VC comprises a second VL, a second heavy chain CH1 hinge region, and a first Fc domain monomer from the N-terminus to the C-terminus; and / or, the element contained in the polypeptide chain VD comprises a second VH and a second heavy chain CH1 region from the N-terminus to the C-terminus.

[0373] In some embodiments, adjacent elements contained in the polypeptide chain VA are optionally connected by or without a connector, adjacent elements contained in the polypeptide chain VB are optionally connected by or without a connector, adjacent elements contained in the polypeptide chain VC are optionally connected by or without a connector, and / or adjacent elements contained in the polypeptide chain VD are optionally connected by or without a connector.

[0374] In some embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid or flexible peptide linkers). In some embodiments, each peptide linker is independently composed of 3 to 55 amino acid residues (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55). In some embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S). In some embodiments, each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which may be composed of (GGGGS). n The expression indicates that n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as shown in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, and 70, respectively). In some embodiments, each peptide linker independently comprises an amino acid sequence as shown in one of SEQ ID NOs: 55-58 or 65-70.

[0375] In some embodiments, the first and second CLs contain amino acid sequences as shown in SEQ ID NO: 53 or SEQ ID NO: 63, the first and second heavy chain CH1 regions contain amino acid sequences as shown in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region contains amino acid sequences as shown in SEQ ID NO: 62.

[0376] In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.

[0377] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52.

[0378] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51.

[0379] scFv-scFv structure

[0380] In some implementations, both the first antigen-binding domain and the second antigen-binding domain are scFv.

[0381] In some embodiments, the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein, (a) the elements contained in polypeptide chain VII-A include the first VL, the first VH, the first heavy chain CH1 hinge region, and the first Fc domain monomer; the elements contained in polypeptide chain VII-B include the second VL, the second VH, the second heavy chain CH1 hinge region, and the second Fc domain monomer; or (b) the elements contained in polypeptide chain VII-A include the first VL, the first VH, the first heavy chain CH1 hinge region, and the second Fc domain monomer; and the elements contained in polypeptide chain VII-B include the second VL, the second VH, the second heavy chain CH1 hinge region, and the first Fc domain monomer.

[0382] In some embodiments, (a) the element contained in polypeptide chain VII-A from the N-terminus to the C-terminus includes (i) a first VL, a first VH, a first heavy chain CH1 hinge region and a first Fc domain monomer, or (ii) a first VH, a first VL, a first heavy chain CH1 hinge region and a first Fc domain monomer; and / or the element contained in polypeptide chain VII-B from the N-terminus to the C-terminus includes (i) a second VL, a second VH, a second heavy chain CH1 hinge region and a second Fc domain monomer, or (ii) a second VH, a second VL, a second heavy chain CH1 hinge region and a second Fc domain monomer; Or (b) the element contained in polypeptide chain VII-A from the N-terminus to the C-terminus includes (i) a first VL, a first VH, a first heavy chain CH1 hinge region, and a second Fc domain monomer, or (ii) a first VH, a first VL, a first heavy chain CH1 hinge region, and a second Fc domain monomer; and / or the element contained in polypeptide chain VII-B from the N-terminus to the C-terminus includes (i) a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc domain monomer, or (ii) a second VH, a second VL, a second heavy chain CH1 hinge region, and a first Fc domain monomer.

[0383] In some embodiments, adjacent elements contained in the polypeptide chain VII-A are optionally connected by or without a connector, and / or adjacent elements contained in the polypeptide chain VII-B are optionally connected by or without a connector.

[0384] In some embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid or flexible peptide linkers). In some embodiments, each peptide linker is independently composed of 3 to 55 amino acid residues (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55). In some embodiments, each peptide linker is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S). In some embodiments, each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which may be composed of (GGGGS). n The expression indicates that n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as shown in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, and 70, respectively). In some embodiments, each peptide linker independently comprises an amino acid sequence as shown in one of SEQ ID NOs: 55-58 or 65-70.

[0385] In some embodiments, the first and second heavy chain CH1 hinge regions contain the amino acid sequence shown in SEQ ID NO: 62.

[0386] In some embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the first VL comprises the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first VH comprises the amino acid sequence shown in SEQ ID NO: 18, the first VL comprises the amino acid sequence shown in SEQ ID NO: 17, the second VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the second VL comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15.

[0387] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52.

[0388] In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the first Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO: 51.

[0389] In some embodiments, the polypeptide chain VII-A comprises an amino acid sequence as shown in SEQ ID NO: 14, and / or the polypeptide chain VII-B comprises an amino acid sequence as shown in SEQ ID NO: 8.

[0390] In some exemplary embodiments, the bispecific antibody in the ADC provided in this disclosure comprises: The polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 2, and the polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 3. The polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 9, and the polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 10. Polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5, and polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6; Polypeptide chain II-A containing the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain II-B containing the amino acid sequence shown in SEQ ID NO: 7, and polypeptide chain II-C containing the amino acid sequence shown in SEQ ID NO: 8; The polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, the polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and the polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11. The polypeptide chain VA comprising the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain VB comprising the amino acid sequence shown in SEQ ID NO: 9, the polypeptide chain VC comprising the amino acid sequence shown in SEQ ID NO: 13, and the polypeptide chain VD comprising the amino acid sequence shown in SEQ ID NO: 12; or, The polypeptide chain VII-A contains the amino acid sequence shown in SEQ ID NO: 14, and the polypeptide chain VII-B contains the amino acid sequence shown in SEQ ID NO: 8.

[0391] In some embodiments, the bispecific antibody has enhanced tumor-suppressive activity relative to the monospecific anti-c-MET antibody and / or the monospecific anti-EGFR antibody; wherein the CDR amino acid sequence of the monospecific anti-c-MET antibody is identical to the CDR amino acid sequence of the first antigen-binding domain, and the CDR amino acid sequence of the monospecific anti-EGFR antibody is identical to the CDR amino acid sequence of the second antigen-binding domain.

[0392] In some embodiments, the tumor suppression effect includes: inhibition of EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0393] Drug-connector for ADCs

[0394] The ADC provided herein comprises a bispecific antibody as described herein, and further comprises a cytotoxic drug fragment D, which is linked to the antibody via a linker-MLE-.

[0395] In some embodiments, the linker described in European Patent Publication EP 4349372 is incorporated herein by reference. In some embodiments, the linker (MLE) and the individual components M, L, and E as described in European Patent Publication EP 4349372 are incorporated herein by reference. In some embodiments, the cytotoxic drug described in European Patent Publication EP 4349372 is incorporated herein by reference. In some embodiments, the linker / cytotoxic drug described in European Patent Publication EP 4349372 is incorporated herein by reference.

[0396] In some implementations, M in the ADC includes In this embodiment, ring A is a 5-6 membered alicyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic ring and aromatic ring system are optionally selected independently by one or more groups selected from oxygen (=O), halogen, cyano, amino, carboxyl, mercapto, and C. 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

[0397] In some implementations, M includes Wherein ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by one or more 6-membered heteroaromatic rings connected to a benzene ring via single bonds, or a polycyclic ring formed by multiple 6-membered heteroaromatic rings connected via single bonds, wherein the aliphatic heterocycle is optionally surrounded by one or more elements selected from oxygen (=O), halogens, and C. 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

[0398] In some implementations, M is Ring A is selected from , , , , , and M1 is selected from single bonds and C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl and amino groups.

[0399] In some implementations, M is selected from , and .

[0400] In some implementations, M is .

[0401] In some implementations, M is selected from , , and .

[0402] In a further embodiment of the antibody-drug conjugate, M is selected from... , and .

[0403] In some embodiments, L in the antibody-drug conjugate is selected from one or more of the following structures: C 1-6 Alkyl groups, -N(R')-, carbonyl groups, -O-, natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) sOCH3), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly(GGFG (SEQ ID NO: 71)), Gly-Gly-Val-Ala). (GGVA (SEQ ID NO: 72)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 73)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 74)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 75)), , , , , , , , , and ;

[0404] Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20.

[0405] In some implementations, the L in the ADC is selected from one or more of the following structures: C 1-6Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Al a. Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val- Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG (SEQ ID NO: 71)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 72)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 73)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 74)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 75)), , , , and ; where s is an integer selected from 1 to 20.

[0406] In some implementations, L is selected from one or more of the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0407] In some implementations, L is selected from the following structures: , , , , , , , , , and .

[0408] In some implementations, L is selected from the following structures: , and .

[0409] In some implementations, L is selected from the following structures: and .

[0410] In some implementations, L is selected from the following structures: and .

[0411] In some embodiments, E in the antibody-drug conjugate is a single bond or selected from the following structures: -NHCH2-、 , , and .

[0412] In some implementations, E is a single bond, -NHCH2-, or .

[0413] In some implementations, E is -NHCH2- or .

[0414] In some implementations, E is -NHCH2-.

[0415] In some implementations, E is a single bond.

[0416] In some implementations, E is .

[0417] In some implementations, M is selected from , , , and ; L is selected from , , , , and ; E is selected from -NHCH2- and .

[0418] In some embodiments, the antibody-drug conjugate contains Selected from the following structures: .

[0419] In some implementation schemes, Selected from the following structures: and .

[0420] In some embodiments, the cytotoxic agent is selected from tubulin inhibitors, DNA intercalating agents, DNA topoisomerase inhibitors, and RNA polymerase inhibitors. In some embodiments, the tubulin inhibitor is an olistatin or maytansine compound. In some embodiments, the DNA intercalating agent is pyrrolobenzodiazepine (PBD). In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, or rubotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, docalmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester, or analogue thereof.

[0421] In some embodiments, the cytotoxic drug is selected from compounds of Formula I or II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of Formula I or II.

[0422] Among them, R1 and R2 are each independently selected from C. 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; R4 and R5 are each independently selected from H, halogens, and hydroxyl groups; or R4 and R5 form a 5-6 membered oxygen-containing heterocycle with the attached carbon atom. R6 is selected from hydrogen or -C. 1-4 Alkylene-NR a R b -C 1-4 Alkylene-SiR a R b R c -SiR a R b R c -C 1-4 Alkylene = N-OR a ;and R7 is selected from C 1-6 Alkyl, -C 1-4 Alkylene-NR a R b -C 1-4 Alkylene-SiR a R b R c-SiR a R b R c -C 1-4 Alkylene = N-OR a ;where R a R b and R c Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, -SO2-C 1-6 Alkyl and -CO-C 1-6 Alkyl; wherein R is optionally present a R b The atoms it is connected to form 5-6 member nitrogen-containing heterocycles.

[0423] In a further embodiment of the antibody-drug conjugate, the cytotoxic drug is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: and .

[0424] The cytotoxic drugs disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), mercapto (-SH), primary amine (-NH2), and secondary amine (-NR). A H) or tertiary amine group (-NR) B R C ), while R in this article A R B and R C These represent only non-hydrogen substituents on N, and the cytotoxic drug can be linked to the linker in the conjugate via these functional groups.

[0425] In some embodiments, the cytotoxic agent is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: and .

[0426] Connection between connector and drug (load)

[0427] In some embodiments, the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, -SH, -NH2, secondary amine group, or tertiary amine group.

[0428] In some embodiments, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amine group on the cytotoxic drug.

[0429] ADC Examples

[0430] In some embodiments, the antibody-drug conjugate is selected from ADC A-01 to ADC A-34, ADC B-01 to ADC B-07, or ADC C-01 to ADC C-28, wherein the structure of the ADC is as follows: ADC A-01

[0431] ADC A-02

[0432] ADC A-04

[0433] ADC A-05

[0434] ADC A-06

[0435] ADC A-07

[0436] ADC A-08

[0437] ADC A-09

[0438] ADC A-10

[0439] ADC A-11

[0440] ADC A-13

[0441] ADC A-14

[0442] ADC A-15

[0443] ADC A-16

[0444] ADC A-17

[0445] ADC A-18

[0446] ADC A-20

[0447] ADC A-21

[0448] ADC A-22

[0449] ADC A-24

[0450] ADC A-25

[0451] ADC A-26

[0452] ADC A-28

[0453] ADC A-29

[0454] ADC A-30

[0455] ADC A-32

[0456] ADC A-33

[0457] ADC A-34

[0458] ADC B-01

[0459] ADC B-02

[0460] ADC B-03

[0461] ADC B-04

[0462] ADC B-06

[0463] ADC B-07

[0464] ADC C-01

[0465] ADC C-02

[0466] ADC C-03

[0467] ADC C-04

[0468] ADC C-05

[0469] ADC C-06

[0470] ADC C-07

[0471] ADC C-08

[0472] ADC C-09

[0473] ADC C-10

[0474] ADC C-11

[0475] ADC C-12

[0476] ADC C-13

[0477] ADC C-14

[0478] ADC C-15

[0479] ADC C-16

[0480] ADC C-17

[0481] ADC C-18

[0482] ADC C-19

[0483] ADC C-20

[0484] ADC C-21

[0485] ADC C-22

[0486] ADC C-23

[0487] ADC C-24

[0488] ADC C-25

[0489] ADC C-26

[0490] ADC C-27 or ADC C-28 Wherein, Ab is a bispecific antibody or its antigen-binding fragment as defined above; and This indicates the specific connection method between the thiol group in the bispecific antibody or its antigen-binding fragment and other parts of the antibody-drug conjugate; and x represents the amount of drug load.

[0491] In some embodiments, the thiol group in the bispecific antibody or its antigen-binding fragment forms a thioether bond with the precursor of other parts of the antibody-drug conjugate through an addition reaction or substitution reaction to obtain the antibody-drug conjugate.

[0492] In some implementations, the Ab includes: (1) A polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 2 or 9, and / or a polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 3 or 10. (2) A polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5 or 7, and / or a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6 or 8; (3) A polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and / or a polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11; (4) A polypeptide chain VA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain VB containing the amino acid sequence shown in SEQ ID NO: 9, a polypeptide chain VC containing the amino acid sequence shown in SEQ ID NO: 13, and / or, a polypeptide chain VD containing the amino acid sequence shown in SEQ ID NO: 12; or (5) A polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and / or a polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.

[0493] In some implementations, the Ab includes: (1) A polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 3. (2) A polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 9, and a polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 10. (3) A polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5, and a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6; (4) A polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8; (5) A polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11. (6) A polypeptide chain VA comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain VB comprising the amino acid sequence shown in SEQ ID NO: 9, a polypeptide chain VC comprising the amino acid sequence shown in SEQ ID NO: 13, and a polypeptide chain VD comprising the amino acid sequence shown in SEQ ID NO: 12; or, (7) A polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and a polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.

[0494] In the specific implementation plan, Ab is selected from bispecific antibodies 07B, 10B, 38B, 41B, 49B, 55B and 56B.

[0495] In specific embodiments of the antibody-drug conjugates disclosed herein, the heavy chain constant region of the bispecific antibody of the antibody-drug conjugate may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments, the N-terminal amino acid of the variable region of the bispecific antibody of the antibody-drug conjugate may contain a glutamic acid or glutamine residue, or the glutamic acid or glutamine residue may be cyclized to pyroglutamic acid.

[0496] In some embodiments, the N-terminal amino acid of the bispecific antibody or its antigen-binding fragment variable region of the antibody-drug conjugate may be cyclized to pyroglutamic acid. Therefore, in the antibody-drug conjugate compositions provided in this disclosure, each antibody-drug conjugate may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or contain an N-terminal glutamine or glutamate residue, or have an N-terminal glutamine or glutamate cyclized to a pyroglutamate salt.

[0497] Therefore, in some embodiments, this application further provides compositions comprising the antibody-drug conjugates described herein, wherein the major antibody-drug conjugate in the composition comprises (i) an antibody lacking a lysine residue at the C-terminus of the heavy chain; (ii) an antibody containing a glutamine, glutamic acid, or pyroglutamic acid residue at the N-terminus of the heavy chain; (iii) an antibody lacking a lysine residue at the C-terminus of the heavy chain and having a glutamine, glutamic acid, or pyroglutamic acid residue at the N-terminus of the heavy chain; (iv) an antibody lacking a lysine residue at the C-terminus of the heavy chain and having a pyroglutamic acid residue at the N-terminus of the heavy chain; or, (v) an antibody lacking a lysine residue at the C-terminus of the heavy chain and having a glutamine or glutamic acid residue at the N-terminus of the heavy chain.

[0498] As those skilled in the art know, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution. Therefore, this application also provides a composition comprising one or more of the antibody-drug conjugates described in any of the preceding claims. For example, the present invention provides an ADC embodiment comprising the amino acid sequence described in SEQ ID NO: 2, 9, 18 or 60, wherein the N-terminal glutamic acid has been converted to pyroglutamate, and / or provides an ADC embodiment comprising the amino acid sequence described in SEQ ID NO: 5, 7, 13 or 16, wherein the N-terminal glutamine has been converted to pyroglutamate.

[0499] In some embodiments, the DAR value (drug-antibody conjugate ratio) of the composition is 1-10, for example: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, ​​3~7, 3~8, 3~9, 3~10, 4~5, 4~6, 4~7, 4~8, 4~9, 4~10, 5~6, 5~7, 5~8, 5~9, 5~10, 6~7, 6~8, 6~9, 6~10, 7~8, 7~9, 7~10, 8~9, 8~10, or 9~10.

[0500] In some embodiments, the DAR value of the composition is 3-9 or 4-8, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5-6.0, 3.5-6.5, 3.5-7.0, 3.5-7.5, 3.5-8.0, 4.0-4.5. 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0~5.5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0501] Couplet

[0502] In another aspect, this application provides a method for preparing the antibody-drug conjugate (ADC) described herein by combining the adapter-load described herein with the antibody described herein.

[0503] In some implementations, the antibody described herein binds to the adapter-load described herein by binding to lysine residues in the antibody.

[0504] In some embodiments, the antibody described herein binds to the linker-loador described herein by binding to cysteine ​​residues in the antibody. In some embodiments, the cysteine ​​residues are derived from reduced intrachain disulfide bonds in the antibody. In some embodiments, the cysteine ​​residues are derived from reduced interchain disulfide bonds in the antibody.

[0505] In some embodiments, the antibody is conjugated to the linker-payload by conjugation to reduced interchain disulfide bonds in the antibody. For example, an IgG1 antibody consists of four polypeptide chains, two heavy chains containing VH, CH1, and Fc (e.g., hinge region, CH2, and CH3) domains, and two light chains containing VL and CL domains, linked by interchain cysteine ​​disulfide bonds (-SS-) (e.g., two heavy-to-light chain interchain disulfide bonds and two hinge-to-heavy chain interchain disulfide bonds). In some embodiments, when these disulfide bonds break under reducing conditions, eight (8) reactive cysteine ​​thiol moieties are generated. In some embodiments, each of the eight reactive cysteine ​​thiol moieties is a linker-drug conjugate site, thus allowing up to eight (x = 8) linker-payloads to be conjugated to the reduced antibody. In some embodiments, any one of the four disulfide bonds breaks under reducing conditions, generating two (2) reactive cysteine ​​thiol moieties. In a further embodiment, each of the two reactive cysteine ​​thiol moieties is a linker-drug junction, thus allowing two (x = 2) linker-drugs to the reducing antibody. In some embodiments, any two of the four disulfide bonds break under reducing conditions, resulting in four (4) reactive cysteine ​​thiol moieties. In a further embodiment, each of the four reactive cysteine ​​thiol moieties is a linker-drug junction, thus allowing four (x = 4) linker-drugs to the reducing antibody. In some embodiments, any three of the four disulfide bonds break under reducing conditions, resulting in six (6) reactive cysteine ​​thiol moieties. In a further embodiment, each of the six reactive cysteine ​​thiol moieties is a linker-drug junction, thus allowing six (x = 6) linker-drugs to the reducing antibody.

[0506] In some embodiments, the interchain disulfide bond is located between two cysteine ​​residues that cleave under reducing conditions to produce two reactive cysteine ​​thiol moieties. In further embodiments, the interchain disulfide bond in the antibody is located between a heavy chain and a light chain, for example, between C220 of the heavy chain according to EU numbering and C214 of the kappa light chain according to EU and / or Kabat numbering, or between C220 of the heavy chain according to EU numbering and C214 of the lambda light chain according to EU and / or Kabat numbering. And / or, the interchain disulfide bond in the antibody is located between two heavy chains, for example, between C226 and / or C229 of the first heavy chain according to EU numbering and C226 and / or C229 of the second heavy chain. In some embodiments, the cysteine ​​residues are located in the hinge region of the antibody. In some embodiments, the cysteine ​​residue is located at one or more of positions 220, 226, or 229 in the heavy chain according to EU numbering (also referred to herein as C220, C226, or C229, respectively). In some embodiments, the cysteine ​​residue is located at position 214 in the light chain according to EU and / or Kabat numbering (also referred to herein as C214, e.g., position 214 in the kappa light chain according to EU and Kabat numbering or position 214 in the lambda light chain according to EU and Kabat numbering). In one embodiment, the cysteine ​​residue is located at each of positions 220, 226, and 229 in the heavy chain according to EU numbering and position 214 in the light chain according to EU or Kabat numbering. In one embodiment, the cysteine ​​residue is located at each of positions 220, 226, and 229 in the heavy chain according to EU numbering and position 214 in the kappa light chain according to EU and Kabat numbering. In one embodiment, the cysteine ​​residue is located at each of positions 220, 226, and 229 in the heavy chain according to EU numbers, and at position 214 in the lambda light chain according to EU and Kabat numbers. In one embodiment, the cysteine ​​residue is located at any one or more of the following positions: (i) Based on any one, two, or three of the 220, 226, and 229 bits in the first chain of the EU number; (ii) Based on any one, two, or three of the 220, 226, and 229 bits in the second chain of the EU number; (iii) Bit 214 of the first light chain according to the Kabat number and / or EU number; and / or (iv) 214th bit in the second light chain according to the Kabat number and / or EU number.

[0507] As used herein, C220, C226, and C229 refer to amino acid residues (cysteine, Cys, C) of immunoglobulins identified according to EU numbers. Those skilled in the art will understand that such numbers correspondingly represent amino acid residues of peptides aligned with amino acid residues identified in immunoglobulins, such as those shown at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0508] As used herein, the cysteine ​​residue at position 214 of the κ light chain refers to the amino acid residue (cysteine, Cys, C) of the immunoglobulin identified by Eu or Kabat numbering. Those skilled in the art will understand that such numbering correspondingly represents amino acid residues of a polypeptide aligned with amino acid residues identified in the immunoglobulin, such as those shown at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html.

[0509] As used herein, the cysteine ​​residue at position 214 of the λ light chain refers to the amino acid residue (cysteine, Cys, C) of the immunoglobulin identified by Eu or Kabat numbering. Those skilled in the art will understand that such numbering correspondingly indicates amino acid residues of polypeptides aligned with the amino acid residues identified in the immunoglobulin, such as the amino acid residues shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGLCnber.html.

[0510] In some embodiments, the antibody described herein contains four interchain disulfide bonds in the hinge region that can be reduced to break the bonds and expose a reactive thiol moiety that can bind to the maleimide portion on the linker-payload (e.g., the maleimide portion on the linker-payload described herein).

[0511] In some embodiments, the antibodies described herein comprise lysine residues, wherein the reactive amine side chains of the lysine residues can bind to the adapter-load, such as the maleimide portion of the adapter-load described herein.

[0512] In one embodiment, this disclosure provides a method for preparing the ADC described herein, comprising the steps of: a) providing a solution containing an antibody; b) contacting the solution of a) with a reducing agent; and c) contacting the solution of b) with a solution containing a linker-payload or a salt thereof, as described herein, to prepare the ADC.

[0513] In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0514] Pharmaceutical compositions or combinations

[0515] In another aspect, this application provides a pharmaceutical composition comprising one or more antibody-drug conjugates or compositions as described in any one of the preceding claims, and a pharmaceutically acceptable carrier and / or excipient.

[0516] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In some embodiments, the additional pharmaceutically active agent is a drug with antitumor activity. In some embodiments, the additional pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof. In some embodiments, the ADC or composition as described herein and the additional pharmaceutically active agent are provided as separate components or as a mixture of components.

[0517] In some embodiments, the ADC or composition in the pharmaceutical composition of the present invention is sufficient (e.g., in a subject) to exert a tumor-suppressive effect (e.g., superior to the tumor-suppressive effect of monospecific anti-c-MET antibody and / or monospecific anti-EGFR antibody, wherein the amino acid sequence of the CDR of the monospecific anti-c-MET antibody is identical to the amino acid sequence of the CDR of the first antigen-binding domain, and the amino acid sequence of the CDR of the monospecific anti-EGFR antibody is identical to the amino acid sequence of the CDR of the second antigen-binding domain).

[0518] In some implementations, tumor suppression includes: inhibition of EGFR and c-MET signaling, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0519] In some embodiments, the antibody-drug conjugates described herein are typically formulated into unit injection forms, such as intravenous boluses, intravenous injections, intratumoral injections, etc., together with a pharmaceutically acceptable parenteral carrier for parenteral use. Optionally, antibody-drug conjugates of desired purity are mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers to form lyophilized or solution forms (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.). The antibody-drug conjugates described herein, or pharmaceutical compositions comprising said antibody-drug conjugates, may be administered to subjects via any suitable route.

[0520] Therapeutic applications of ADC

[0521] On the other hand, this disclosure provides the use of the antibody-drug conjugates, compositions, pharmaceutical compositions, or combinations thereof in the preparation of medicaments for the prevention and / or treatment and / or as adjunctive therapy for diseases associated with c-MET and / or EGFR in subjects, and / or for the inhibition of c-MET and / or EGFR activity in vitro or in subjects; wherein the diseases associated with c-MET and / or EGFR include, but are not limited to, cancers associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification.

[0522] In some implementations, the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0523] In some embodiments, the antibody-drug conjugate or pharmaceutical composition is administered in combination with an additional pharmaceutically active agent, for example, simultaneously, separately, or sequentially. In some embodiments, the additional pharmaceutically active agent is a drug with antitumor activity. In some embodiments, the additional pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0524] In another aspect, the present invention provides a method for inhibiting the activity of c-MET and / or EGFR in cells, comprising contacting the cells with the antibody-drug conjugate or pharmaceutical composition described herein. The cells are cells expressing c-MET and / or EGFR, such as tumor cells.

[0525] In another aspect, the present invention provides a method for preventing, treating and / or acting as adjunctive medicine for diseases associated with c-MET and / or EGFR in a subject, the method comprising administering to a subject in need an effective amount of any of the preceding antibody-drug conjugates, compositions and / or pharmaceutical compositions.

[0526] In some embodiments, the disease associated with c-MET and / or EGFR is cancer. In some embodiments, the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification.

[0527] In some embodiments, the cancer is epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell carcinoma, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer. In some embodiments, the method further includes administering a second therapy to the subject selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0528] In some implementations, the second therapy may be administered simultaneously, alone, or sequentially with the methods described above.

[0529] In certain embodiments, the present invention provides an ADC, composition, or pharmaceutical composition as described herein for use in subjects to prevent, treat, and / or as adjunctive therapy for diseases associated with c-MET and / or EGFR. In some embodiments, the disease associated with c-MET and / or EGFR is cancer. In some embodiments, the cancer is associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification.

[0530] In some implementations, the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0531] In some embodiments, the method further includes administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof. In some embodiments, the second therapy may be administered simultaneously, separately, or sequentially.

[0532] The ADC and pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including solutions for injection, sterile powders for injection, and concentrates for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by adding the desired dose of the ADC or pharmaceutical composition of the present invention to a suitable solvent, and optionally simultaneously adding other desired ingredients (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration for sterilization. Furthermore, for ease of storage and use, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying). This sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0533] Furthermore, the ADC of the present invention can be present in the pharmaceutical composition in unit dose form for convenient administration.

[0534] The ADC and pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, groin, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some preferred embodiments, the ADC and pharmaceutical composition of the present invention are administered by intravenous or injectable injection.

[0535] The pharmaceutical compositions of the present invention may comprise a "therapeutic effective amount" or a "preventive effective amount" of the bispecific antibody of the present invention. A "preventive effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A "therapeutic effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutic effective amount of the bispecific antibody of the present invention may vary depending on factors such as the severity of the disease to be treated, the general state of the patient's immune system, the patient's general condition, such as age, weight, and sex, the route of administration of the drug, and other concurrent treatments.

[0536] In this invention, the dosing regimen can be adjusted to obtain the optimal desired response (e.g., therapeutic or preventative response). For example, a single dose can be administered, multiple doses can be administered over time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0537] In this invention, the subject can be a mammal, such as a human.

[0538] Reagent test kit

[0539] Kits comprising one or more ADCs or pharmaceutical compositions thereof described herein are also provided. In certain embodiments, a pharmaceutical package or kit is provided herein comprising one or more containers containing one or more components of the pharmaceutical compositions described herein, such as one or more ADCs provided herein. In some embodiments, the kit comprises the pharmaceutical compositions described herein and any preventative or therapeutic agent, such as those described herein. In some embodiments, the kit may include T-cell mitogens, such as phytohemagglutinin (PHA) and / or phorbol myristate (PMA), or TCR complex stimulating antibodies, such as anti-CD3 antibodies and anti-CD28 antibodies. Optionally, associated with such containers may be a notification in the form of a government agency directing the manufacture, use, or sale of a pharmaceutical or biological product, reflecting the agency's approval for the manufacture, use, or sale for human administration.

[0540] Kits for use with the methods described herein are also provided. In some embodiments, the kit includes the ADC described herein, preferably purified ADC, housed in one or more containers. In a particular embodiment, the kit described herein includes substantially isolated c-MET and / or EGFR antigens as controls. In another particular embodiment, the kit described herein also includes a control antibody that does not react with the c-MET or EGFR antigen. In yet another particular embodiment, the kit described herein includes one or more elements for detecting the binding of an antibody to the c-MET and / or EGFR antigen (e.g., the antibody may be conjugated to a detectable substrate (e.g., a fluorescent compound, enzyme substrate, radioactive compound, or luminescent compound), or a second antibody recognizing a first antibody may be conjugated to a detectable substrate). In a particular embodiment, the kits provided herein may include recombinantly generated or chemically synthesized c-MET and / or EGFR antigens. The antigens provided in the kit may also be attached to a solid support. In more specific embodiments, the detection means of the above-described kits includes a solid-phase carrier with c-MET and / or EGFR antigens attached. Such kits may also include anti-human antibodies or anti-mouse / rat antibodies without attached reporter labels. In this implementation, the binding of the bispecific antibody to the antigen can be detected by the binding of the reporter label antibody.

[0541] The specific implementation scheme of this invention includes: Implementation Scheme 1. An antibody-drug conjugate containing the structure shown below: Ab-[MLED] x in: Ab It is a bispecific antibody or its antigen-binding fragment containing a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR; M is a linker site connected to the bispecific antibody or its antigen-binding fragment; L is a structural fragment connecting the linker site M and E; E is a structural fragment connecting L and D; D is a cytotoxic drug or its fragment; x is selected from any integer from 1 to 10.

[0542] Implementation Scheme 2. The antibody-drug conjugate of Implementation Scheme 1, wherein the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), the first VL and the first VH together forming a domain capable of specifically binding c-MET; the second antigen-binding domain comprises a second VL and a second VH, the second VL and the second VH together forming a domain capable of specifically binding EGFR. Wherein, the first VL includes: (i) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38, wherein the CDR is defined by the Kabat numbering system; (ii) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDR is defined by the Chothia numbering system; (iii) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDRs are defined by the Abm numbering system; or, (iv) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 35, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 37, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38, wherein the CDR is defined by the IMGT numbering system; And / or, the first heavy chain (HC) variable region (VH) includes: (i) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 47, wherein the CDR is defined by the Kabat numbering system; (ii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 40, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47, wherein the CDR is defined by the Chothia numbering system; (iii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 42, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47, wherein the CDR is defined by the Abm numbering system; or, (iv) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 41, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 45, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 48, wherein the CDR is defined by the IMGT numbering system.

[0543] Implementation Scheme 3. The antibody-drug conjugate of Implementation Scheme 2, wherein the second VL comprises: (i) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 23, wherein the CDR is defined by the Kabat numbering system; (ii) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDR is defined by the Chothia numbering system; (iii) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDRs are defined by the Abm numbering system; or, (iv) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 20, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 22, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23, wherein the CDR is defined by the IMGT numbering system; And / or, the second VH includes: (i) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 24, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 28, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 32, wherein the CDR is defined by the Kabat numbering system; (ii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 25, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 29, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32, wherein the CDR is defined by the Chothia numbering system; (iii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 27, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32, wherein the CDR is defined by the Abm numbering system; or, (iv) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 26, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 30, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 33, wherein the CDR is defined by the IMGT numbering system.

[0544] Implementation Scheme 4. The antibody-drug conjugate according to any one of Implementation Schemes 2-3, wherein (i) the first VL contains the amino acid sequence shown in SEQ ID NO: 17 and the first VH contains the amino acid sequence shown in SEQ ID NO: 18; or (ii) the first VL contains the amino acid sequence shown in SEQ ID NO: 59 and the first VH contains the amino acid sequence shown in SEQ ID NO: 60.

[0545] Implementation Scheme 5. The antibody-drug conjugate according to any one of Implementation Schemes 2-4, wherein the second VL comprises the amino acid sequence shown in SEQ ID NO: 15, and / or the second VH comprises the amino acid sequence shown in SEQ ID NO: 16.

[0546] Implementation Scheme 6. The antibody-drug conjugate according to any one of Implementation Schemes 1-5, wherein the bispecific antibody or its antigen-binding fragment further comprises an Fc domain, the Fc domain comprising first and second Fc domain monomers, and each of the first and second Fc domain monomers independently comprises a modification of one or more amino acids, the modification being capable of promoting heterodimerization of the first and second Fc domain monomers.

[0547] Implementation Scheme 7. The antibody-drug conjugate of Implementation Scheme 6, wherein the Fc domain comprises a first Fc domain monomer containing amino acid modifications capable of forming a knob structure and a second Fc domain monomer containing amino acid modifications capable of forming a hole structure, wherein the hole structure can pair with the knob structure to form a heterodimerized Fc domain dimer.

[0548] Implementation Scheme 8. The antibody-drug conjugate of Implementation Scheme 6 or 7, wherein (a) the first Fc domain monomer contains the amino acid sequence shown in SEQ ID NO: 49, and the second Fc domain monomer contains the amino acid sequence shown in SEQ ID NO: 50; or (b) the first Fc domain monomer contains the amino acid sequence shown in SEQ ID NO: 51, and the second Fc domain monomer contains the amino acid sequence shown in SEQ ID NO: 52.

[0549] Implementation Scheme 9. The antibody-drug conjugate according to any one of Implementation Schemes 6-8, wherein the first antigen-binding domain is linked to the first Fc domain monomer, and the second antigen-binding domain is linked to the second Fc domain monomer; or, the first antigen-binding domain is linked to the second Fc domain monomer, and the second antigen-binding domain is linked to the first Fc domain monomer.

[0550] Implementation Scheme 10. The antibody-drug conjugate according to any one of Implementation Schemes 1-9, wherein the first antigen-binding domain is Fab and the second antigen-binding domain is scFv.

[0551] Implementation Scheme 11. The antibody-drug conjugate of Implementation Scheme 10, wherein the bispecific antibody comprises polypeptide chain IA, polypeptide chain IB, and polypeptide chain IC; wherein, (a) The element contained in the polypeptide chain IA comprises, from the N-terminus to the C-terminus, the first VL and a light chain constant region; the element contained in the polypeptide chain IB comprises, from the N-terminus to the C-terminus, the first VH, a heavy chain CH1 region, and a first Fc domain monomer; and / or, the element contained in the polypeptide chain IC comprises, from the N-terminus to the C-terminus, (i) the second VL, the second VH, and the second Fc domain monomer; or, (ii) the second VH, the second VL, and the second Fc domain monomer; or... (b) The element contained in the polypeptide chain IA includes the first VL and a light chain constant region from the N-terminus to the C-terminus, and the element contained in the polypeptide chain IB includes the first VH, a heavy chain CH1 region and a second Fc domain monomer from the N-terminus to the C-terminus, and / or the element contained in the polypeptide chain IC includes (i) the second VL, the second VH and the first Fc domain monomer from the N-terminus to the C-terminus, or (ii) the second VH, the second VL and the first Fc domain monomer.

[0552] Implementation Scheme 12. The antibody-drug conjugate of Implementation Scheme 10 or 11, wherein (a) the polypeptide chain IA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB comprises the amino acid sequence shown in SEQ ID NO: 2, and / or the polypeptide chain IC comprises the amino acid sequence shown in SEQ ID NO: 3; or (b) the polypeptide chain IA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB comprises the amino acid sequence shown in SEQ ID NO: 9, and / or the polypeptide chain IC comprises the amino acid sequence shown in SEQ ID NO: 10.

[0553] Implementation Scheme 13. The antibody-drug conjugate according to any one of Implementation Schemes 1-9, wherein the first antigen-binding domain is scFv and the second antigen-binding domain is Fab.

[0554] Implementation Scheme 14. The antibody-drug conjugate of Implementation Scheme 13, wherein (a) the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein the element contained in polypeptide chain II-A comprises, from the N-terminus to the C-terminus, the second VL and a light chain constant region, the element contained in polypeptide chain II-B comprises, from the N-terminus to the C-terminus, the second VH, a heavy chain CH1 region, and a monomer of the first Fc domain, and / or, the element contained in polypeptide chain II-C comprises, from the N-terminus to the C-terminus, (i) the first VL, the first VH, and a monomer of the second Fc domain, or (ii) the first VH, the first VL, and a monomer of the second Fc domain; or

[0555] (b) The bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B and polypeptide chain II-C; wherein the element contained in polypeptide chain II-A comprises the second VL and a light chain constant region from the N-terminus to the C-terminus, the element contained in polypeptide chain II-B comprises the second VH, a heavy chain CH1 region and a second Fc domain monomer from the N-terminus to the C-terminus, and / or the element contained in polypeptide chain II-C comprises (i) the first VL, the first VH and the first Fc domain monomer from the N-terminus to the C-terminus, or (ii) the first VH, the first VL and the first Fc domain monomer.

[0556] Implementation Scheme 15. The antibody-drug conjugate of Implementation Scheme 13 or 14, wherein (a) polypeptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 5, and / or, polypeptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 6; or (b) polypeptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4, polypeptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 7, and / or, polypeptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 8.

[0557] Implementation Scheme 16. The antibody-drug conjugate according to any one of Implementation Schemes 1-9, wherein the first antigen-binding domain is scFab and the second antigen-binding domain is Fab.

[0558] Implementation Scheme 17. The antibody-drug conjugate of Implementation Scheme 16, wherein (a) the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C; wherein the element contained in polypeptide chain IV-A comprises, from the N-terminus to the C-terminus, the second VL and a light chain constant region, the element contained in polypeptide chain IV-B comprises, from the N-terminus to the C-terminus, the second VH, a heavy chain CH1 region, and a monomer of the first Fc domain, and / or, the element contained in polypeptide chain IV-C comprises, from the N-terminus to the C-terminus, (i) the first VL, the light chain constant region, the first VH, the heavy chain CH1 region, and a monomer of the second Fc domain, or (ii) the first VH, the heavy chain CH1 region, the first VL, the light chain constant region, and a monomer of the second Fc domain; or, (b) The bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B and polypeptide chain IV-C; wherein, polypeptide chain IV-A comprises, from the N-terminus to the C-terminus, the second VL and the light chain constant region, polypeptide chain IV-B comprises, from the N-terminus to the C-terminus, the second VH, the heavy chain CH1 region and the second Fc domain monomer, and / or, polypeptide chain IV-C comprises, from the N-terminus to the C-terminus, (i) the first VL, the light chain constant region, the first VH, the heavy chain CH1 region and the first Fc domain monomer, or (ii) the first VH, the heavy chain CH1 region, the first VL, the light chain constant region and the first Fc domain monomer.

[0559] Implementation Scheme 18. The antibody-drug conjugate of Implementation Scheme 16 or 17, wherein the polypeptide chain IV-A comprises the amino acid sequence shown in SEQ ID NO: 4, the polypeptide chain IV-B comprises the amino acid sequence shown in SEQ ID NO: 7, and / or the polypeptide chain IV-C comprises the amino acid sequence shown in SEQ ID NO: 11.

[0560] Implementation Scheme 19. The antibody-drug conjugate according to any one of Implementation Schemes 1-9, wherein the first antigen-binding domain and the second antigen-binding domain are Fab, and the Fab of the second antigen-binding domain contains domain exchanges of the form of CrossMab.

[0561] Implementation Scheme 20. The antibody-drug conjugate of Implementation Scheme 19, wherein (a) the bispecific antibody comprises polypeptide chain VA, polypeptide chain VB, polypeptide chain VC, and polypeptide chain VD; wherein the element contained in polypeptide chain VA comprises, from the N-terminus to the C-terminus, the first VL and a light chain constant region; the element contained in polypeptide chain VB comprises, from the N-terminus to the C-terminus, the first VH, a heavy chain CH1 region, and a first Fc domain monomer; the element contained in polypeptide chain VC comprises, from the N-terminus to the C-terminus, the second VH, a light chain constant region, and a second Fc domain monomer; and / or, the element contained in polypeptide chain VD comprises, from the N-terminus to the C-terminus, the second VL and a heavy chain CH1 region; or

[0562] (b) The bispecific antibody comprises polypeptide chain VA, polypeptide chain VB, polypeptide chain VC and polypeptide chain VD; wherein, the element contained in polypeptide chain VA comprises, from the N-terminus to the C-terminus, the first VL and the light chain constant region; the element contained in polypeptide chain VB comprises, from the N-terminus to the C-terminus, the first VH, the heavy chain CH1 region and the second Fc domain monomer; the element contained in polypeptide chain VC comprises, from the N-terminus to the C-terminus, the second VH, the light chain constant region and the first Fc domain monomer; and / or, the element contained in polypeptide chain VD comprises, from the N-terminus to the C-terminus, the second VL and the heavy chain CH1 region.

[0563] Implementation Scheme 21. The antibody-drug conjugate of Implementation Scheme 19 or 20, wherein the polypeptide chain VA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain VB comprises the amino acid sequence shown in SEQ ID NO: 9, the polypeptide chain VC comprises the amino acid sequence shown in SEQ ID NO: 13, and / or, the polypeptide chain VD comprises the amino acid sequence shown in SEQ ID NO: 12.

[0564] Implementation Scheme 22. The antibody-drug conjugate according to any one of Implementation Schemes 1-9, wherein the first antigen-binding domain and the second antigen-binding domain are both scFv.

[0565] Implementation Scheme 23. The antibody-drug conjugate of Implementation Scheme 22, wherein (a) the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein the element contained in polypeptide chain VII-A comprises, from the N-terminus to the C-terminus: (i) the first VL, the first VH, and the first Fc structural domain monomer, or, (ii) the first VH, the first VL, and the first Fc domain monomers; and / or, The elements contained in the polypeptide chain VII-B from the N-terminus to the C-terminus include: (x) The second VL, the second VH, and the second Fc structural domain monomers, or, (y) The second VH, the second VL, and the second Fc structural domain monomers; or (b) The bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein the elements contained in polypeptide chain VII-A, from the N-terminus to the C-terminus, comprise: (i) the first VL, the first VH, and the second Fc structural domain monomers, or, (ii) the first VH, the first VL, and the second Fc domain monomers; and / or, the elements contained in the polypeptide chain VII-B from the N-terminus to the C-terminus comprise: (x) The second VL, the second VH, and the first Fc structural domain monomer, or, (y) The second VH, the second VL, and the first Fc domain monomer.

[0566] Implementation Scheme 24. The antibody-drug conjugate of Implementation Scheme 22 or 23, wherein the polypeptide chain VII-A comprises the amino acid sequence shown in SEQ ID NO: 14, and / or the polypeptide chain VII-B comprises the amino acid sequence shown in SEQ ID NO: 8.

[0567] Implementation Scheme 25. The antibody-drug conjugate of Implementation Schemes 11, 14, 17, 20 or 23, wherein adjacent elements contained in each of the polypeptide chains are optionally linked by or without a linker.

[0568] Implementation Scheme 26. The antibody-drug conjugate of Implementation Scheme 25, wherein each of the linkers is independently the same or different peptide linkers, wherein each peptide linker is selected from rigid peptide linkers or flexible peptide linkers.

[0569] Implementation Scheme 27. The antibody-drug conjugate of Implementation Scheme 26, wherein each of the peptide linkers is independently selected from peptide linkers comprising one or more glycine (G) and / or serine (S).

[0570] Implementation Scheme 28. The antibody-drug conjugate of Implementation Scheme 27, wherein each peptide linker independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 tandem copies of a peptide linker subunit, and the peptide linker subunit comprises the amino acid sequence GGGGS (SEQ ID NO: 57).

[0571] Implementation Scheme 29. The antibody-drug conjugate of Implementation Scheme 28, wherein each of the peptide linkers independently comprises an amino acid sequence as shown in SEQ ID NO: 55 or 56.

[0572] Implementation Scheme 30. The antibody-drug conjugate of Implementation Scheme 11, 14, 17, 20 or 23, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO: 54.

[0573] Implementation Scheme 31. The antibody-drug conjugate according to any one of Implementation Schemes 1-30, wherein the bispecific antibody or its antigen-binding fragment comprises: (a) A polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 2 or 9, and / or a polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 3 or 10. (b) A polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5 or 7, and / or a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6 or 8; (c) A polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and / or a polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11; (d) A polypeptide chain VA comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain VB comprising the amino acid sequence shown in SEQ ID NO: 9, a polypeptide chain VC comprising the amino acid sequence shown in SEQ ID NO: 13, and / or, a polypeptide chain VD comprising the amino acid sequence shown in SEQ ID NO: 12; or (e) a polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and / or a polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.

[0574] Implementation Scheme 32. The antibody-drug conjugate of Implementation Scheme 28, wherein the bispecific antibody or its antigen-binding fragment comprises: (a) a polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO: 3. (b) a polypeptide chain IA comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB comprising the amino acid sequence shown in SEQ ID NO: 9, and a polypeptide chain IC comprising the amino acid sequence shown in SEQ ID NO: 10. (c) A polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 5, and a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 6; (d) A polypeptide chain II-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide chain II-C comprising the amino acid sequence shown in SEQ ID NO: 8; (e) a polypeptide chain IV-A comprising the amino acid sequence shown in SEQ ID NO: 4, a polypeptide chain IV-B comprising the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide chain IV-C comprising the amino acid sequence shown in SEQ ID NO: 11. (f) A polypeptide chain VA comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain VB comprising the amino acid sequence shown in SEQ ID NO: 9, a polypeptide chain VC comprising the amino acid sequence shown in SEQ ID NO: 13, and a polypeptide chain VD comprising the amino acid sequence shown in SEQ ID NO: 12; or, (g) a polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and a polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO: 8.

[0575] Implementation Scheme 33. The antibody-drug conjugate according to any one of Implementation Schemes 1-30, wherein the bispecific antibody or its antigen-binding fragment has enhanced tumor-suppressive activity relative to the monospecific anti-c-MET antibody and / or the monospecific anti-EGFR antibody; wherein, (a) The CDR amino acid sequence of the monospecific anti-c-MET antibody is identical to the CDR amino acid sequence of the first antigen-binding domain, and the CDR amino acid sequence of the monospecific anti-EGFR antibody is identical to the CDR amino acid sequence of the second antigen-binding domain; or (b) The CDR amino acid sequence of the monospecific anti-c-MET antibody is the same as the CDR amino acid sequence of the second antigen-binding domain, and the CDR amino acid sequence of the monospecific anti-EGFR antibody is the same as the CDR amino acid sequence of the first antigen-binding domain.

[0576] Implementation Scheme 34. The antibody-drug conjugate of Implementation Scheme 33, wherein the tumor inhibitory effect includes: inhibition of EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity.

[0577] Implementation Scheme 35. The antibody-drug conjugate according to any one of Implementation Schemes 1-34, wherein M comprises , Wherein, ring A is a 5-6 membered aliphatic heterocycle or a 5-20 membered aromatic ring system, wherein the aliphatic heterocycle and aromatic ring system are optionally selected independently by one or more groups selected from oxygen (=O), halogen, cyano, amino, carboxyl, mercapto, and C. 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

[0578] Implementation Scheme 36. The antibody-drug conjugate according to any one of Implementation Schemes 1-34, wherein M comprises Wherein ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by one or more 6-membered heteroaromatic rings connected to a benzene ring via single bonds, or a polycyclic ring formed by multiple 6-membered heteroaromatic rings connected via single bonds, wherein the aliphatic heterocycle is optionally surrounded by one or more elements selected from oxygen (=O), halogens, and C. 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

[0579] Implementation Scheme 37. The antibody-drug conjugate according to any one of Implementation Schemes 1-34, wherein M is Ring A is selected from , , , , , and M1 is selected from single bonds and C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl or amino group.

[0580] Implementation Scheme 38. The antibody-drug conjugate according to any one of Implementation Schemes 1-34, wherein M is selected from... , and .

[0581] Implementation Scheme 39. The antibody-drug conjugate according to any one of Implementation Schemes 1-34, wherein M is .

[0582] Implementation Scheme 40. The antibody-drug conjugate according to any one of Implementation Schemes 1-34, wherein M is selected from... , , and .

[0583] Implementation Scheme 41. The antibody-drug conjugate according to any one of Implementation Schemes 1-34, wherein M is selected from... , and .

[0584] Implementation Scheme 42. The antibody-drug conjugate according to any one of Implementation Schemes 1-41, wherein L is selected from one or more of the following structures: C 1-6 Alkyl group, -N(R')-, carbonyl group, -O-, selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) s Natural or non-natural amino acids and their analogues (OCH3), as well as short peptides containing 1, 2, 3 or 4 amino acids. , , , , , , , , and ;

[0585] Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20.

[0586] Implementation Scheme 43. The antibody-drug conjugate of Implementation Scheme 42, wherein the short peptide is selected from Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG (SEQ ID NO: 71)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 72)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 73)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 74)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 75)).

[0587] Implementation Scheme 44. The antibody-drug conjugate according to any one of Implementation Schemes 1-41, wherein L is selected from structures comprising one or more of the following: C 1-6Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Al a. Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val- Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG (SEQ ID NO: 71)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 72)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 73)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 74)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 75)), , , , and ; where s is an integer selected from 1 to 20.

[0588] Implementation Scheme 45. The antibody-drug conjugate according to any one of Implementation Schemes 1-41, wherein L is selected from one or more of the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0589] Implementation Scheme 46. The antibody-drug conjugate according to any one of Implementation Schemes 1-41, wherein the L is selected from the following structures: , , , , , , , , , and .

[0590] Implementation Scheme 47. The antibody-drug conjugate according to any one of Implementation Schemes 1-41, wherein the L is selected from the following structures: , and ; Implementation Scheme 48. The antibody-drug conjugate according to any one of Implementation Schemes 1-41, wherein the L is selected from the following structures: and .

[0591] Implementation Scheme 49. The antibody-drug conjugate according to any one of Implementation Schemes 1-41, wherein the L is selected from the following structures: .

[0592] Implementation Scheme 50. The antibody-drug conjugate according to any one of Implementation Schemes 1-49, wherein E is a single bond or selected from the following structures: -NHCH2-、 , , and .

[0593] Implementation Scheme 51. The antibody-drug conjugate according to any one of Implementation Schemes 1-49, wherein E is a single bond, -NHCH2-, or .

[0594] Implementation Scheme 52. The antibody-drug conjugate according to any one of Implementation Schemes 1-49, wherein E is -NHCH2- or ; Implementation Scheme 53. The antibody-drug conjugate according to any one of Implementation Schemes 1-49, wherein E is -NHCH2-; preferably, E is a single bond.

[0595] Implementation Scheme 54. The antibody-drug conjugate according to any one of Implementation Schemes 1-49, wherein E is... .

[0596] Implementation Scheme 55. The antibody-drug conjugate described in any one of Implementation Schemes 1-55, Selected from the following structures:

[0597] and .

[0598] Implementation Scheme 56. The antibody-drug conjugate according to any one of Implementation Schemes 1-54, wherein, Selected from the following structures:

[0599] and .

[0600] Implementation Scheme 57. The antibody-drug conjugate according to any one of Implementation Schemes 1-56, wherein the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

[0601] Implementation Scheme 58. The antibody-drug conjugate described in Implementation Scheme 57, wherein the microtubule inhibitor is an olistatin compound or a maytansine compound.

[0602] Implementation Scheme 59. The antibody-drug conjugate of Implementation Scheme 57, wherein the DNA intercalating agent is pyrrolobenzodiazepine (PBD).

[0603] Implementation Scheme 60. The antibody-drug conjugate of Implementation Scheme 57, wherein the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0604] Implementation Scheme 61. The antibody-drug conjugate of Implementation Scheme 60, wherein the topoisomerase I inhibitor is selected from camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, rubotecan, and pharmaceutically acceptable salts, esters, or analogs thereof; and the topoisomerase II inhibitor is selected from doxorubicin, PNU-159682, docalimcin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, and pharmaceutically acceptable salts, esters, or analogs thereof.

[0605] Implementation Scheme 62. The antibody-drug conjugate of Implementation Scheme 57, wherein the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester or analogue thereof; Implementation Scheme 63. The antibody-drug conjugate according to any one of Implementation Schemes 1-56, wherein the cytotoxic drug is selected from compounds of Formula I or II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of Formula I or II:

[0606] Among them, R1 and R2 are each independently selected from C. 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; R4 and R5 are each independently selected from H, halogens, and hydroxyl groups; or R4 and R5 are connected to the adjacent carbon atom to form a 5-6 membered oxygen-containing heterocycle; R6 is selected from hydrogen or -C. 1-4 Alkylene-NR a R b ; R7 is selected from C 1-6 Alkyl, -C 1-4 Alkylene-NR a R b -C 1-4 Alkylene-SiR a R b R c -SiR a R b R c -C 1-4 Alkylene = N-OR a ; Where R a R b Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, -SO2-C 1-6 Alkyl and -CO-C 1-6 Alkyl group; wherein Ra and Rb optionally form a 5-6 member nitrogen-containing heterocycle with the atoms to which they are attached.

[0607] Implementation Scheme 64. The antibody-drug conjugate according to any one of Implementation Schemes 1-56, wherein the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: ; The residues of the cytotoxic drug obtained after linking the cytotoxic drug to the linker are D in the general formula of embodiment 1.

[0608] Implementation Scheme 65. The antibody-drug conjugate according to any one of Implementation Schemes 1-56, wherein the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: , The fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula of embodiment 1.

[0609] Implementation Scheme 66. The antibody-drug conjugate of Implementation Scheme 64 or 65, wherein D is a monovalent structure obtained by losing an H from the -OH, -NH2 or secondary amino group on the cytotoxic drug.

[0610] Implementation Scheme 67. The antibody-drug conjugate described in any one of Implementation Schemes 1-66, selected from: ADC A-01

[0611] ADC A-02

[0612] ADC A-04

[0613] ADC A-05

[0614] ADC A-06

[0615] ADC A-07

[0616] ADC A-08

[0617] ADC A-09

[0618] ADC A-10

[0619] ADC A-11

[0620] ADC A-13

[0621] ADC A-14

[0622] ADC A-15

[0623] ADC A-16

[0624] ADC A-17

[0625] ADC A-18

[0626] ADC A-20

[0627] ADC A-21

[0628] ADC A-22

[0629] ADC A-24

[0630] ADC A-25

[0631] ADC A-26

[0632] ADC A-28

[0633] ADC A-29

[0634] ADC A-30

[0635] ADC A-32

[0636] ADC A-33

[0637] ADC A-34

[0638] ADC B-01

[0639] ADC B-02 , ADC B-03

[0640] ADC B-04

[0641] ADC B-06

[0642] ADC B-07

[0643] ADC C-01

[0644] ADC C-02

[0645] ADC C-03

[0646] ADC C-04 , ADC C-05

[0647] ADC C-06

[0648] ADC C-07

[0649] ADC C-08

[0650] ADC C-09

[0651] ADC C-10

[0652] ADC C-11

[0653] ADC C-12

[0654] ADC C-13

[0655] ADC C-14

[0656] ADC C-15

[0657] ADC C-16

[0658] ADC C-17

[0659] ADC C-18

[0660] ADC C-19

[0661] ADC C-20

[0662] ADC C-21

[0663] ADC C-22

[0664] ADC C-23

[0665] ADC C-24

[0666] ADC C-25

[0667] ADC C-26

[0668] ADC C-27

[0669] and

[0670] ADC C-28 Wherein, Ab is a bispecific antibody or its antigen-binding fragment as defined in any one of embodiments 1-34; and This indicates the specific linkage between the thiol group of the cysteine ​​residue in the bispecific antibody or its antigen-binding fragment and other parts of the antibody-drug conjugate; and x represents the amount of drug payload.

[0671] Implementation Scheme 68. Implementation Scheme 67, wherein the antibody-drug conjugate is obtained by forming a thioether bond with M in the bispecific antibody or its antigen-binding fragment through an addition reaction or a substitution reaction.

[0672] Implementation Scheme 69. The antibody-drug conjugate described in Implementation Schemes 67-68, wherein the Ab is a bispecific antibody selected from BsAb07B, BsAb10B, BsAb38B, BsAb41B, BsAb49B, BsAb55B and BsAb56B.

[0673] Implementation Scheme 70. An antibody-drug conjugate selected from ADC 07B-A-05, ADC 07B-A-14, ADC 38B-A-14, ADC 49B-A-14, ADC 49B-B-01, ADC 49B-A-05, ADC 41B-A-05, ADC 5B-A-14 and ADC56B-A-14.

[0674] Implementation Scheme 71. A composition comprising one or more antibody-drug conjugates as described in any one of Implementation Schemes 1-70.

[0675] Implementation Scheme 72. The composition of Implementation Scheme 71, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 1-10.

[0676] Implementation Scheme 73. The composition of Implementation Scheme 71, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, ​​3~7, 3~8, 3~9, 3~10, 4~5, 4~6, 4~7, 4~8, 4~9, 4~10, 5~6, 5~7, 5~8, 5~9, 5~10, 6~7, 6~8, 6~9, 6~10, 7~8, 7~9, 7~10, 8~9, 8~10, or 9~10.

[0677] Implementation Scheme 74. The composition of Implementation Scheme 71, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 3 to 9.

[0678] Implementation Scheme 75. The composition of Implementation Scheme 71, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 4 to 8.

[0679] Implementation Scheme 76. The composition of Implementation Scheme 71, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 3.0~3.5, 3.0~4.0, 3.0~4.5, 3.0~5.0, 3.0~5.5, 3.0~6.0, 3.5~4.0, 3.5~4.5, 3.5~5.0, 3.5~5.5, 3.5~6.0, 3.5~6.5, 3.5~7.0, 3.5~7.5, 3.5~8.0, 4.0~4.5 4.0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0~5.5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0680] Implementation Scheme 77. A pharmaceutical composition comprising one or more antibody-drug conjugates as described in any one of embodiments 1-70 or the composition described in embodiment 71, and a pharmaceutically acceptable carrier and / or excipient.

[0681] Implementation Scheme 78. The pharmaceutical composition of Implementation Scheme 77, wherein the pharmaceutical composition further comprises one or more additional pharmaceutically active agents selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0682] Implementation Scheme 79. A drug combination comprising a first therapeutic agent and a second therapeutic agent, wherein the first therapeutic agent and the second therapeutic agent are administered simultaneously or sequentially, wherein the first therapeutic agent is selected from one or more antibody-drug conjugates according to any one of Implementation Schemes 1-70 or compositions according to any one of Implementation Schemes 71-76; and the second therapeutic agent is selected from EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

[0683] Implementation Scheme 80. Use of the antibody-drug conjugate of any one of Implementation Schemes 1-70, the composition of any one of Implementation Schemes 71-76, the pharmaceutical composition of Implementation Scheme 77 or 78, or the combination drug of Implementation Scheme 80 in the preparation of a medicament for the prevention and / or treatment and / or adjunctive treatment of diseases associated with c-MET and / or EGFR in a subject, and / or for the inhibition of cellular c-MET and / or EGFR activity in vitro or in a subject; wherein the diseases associated with c-MET and / or EGFR are diseases associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification.

[0684] Implementation Scheme 81. The use described in Implementation Scheme 81, wherein the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0685] Implementation Scheme 82. Use of Implementation Scheme 80 or 81, wherein the antibody-drug conjugate or pharmaceutical composition is administered simultaneously, alone or sequentially in combination with another drug; wherein the other pharmaceutical active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents or any combination thereof.

[0686] Implementation Scheme 83. A method for inhibiting the activity of c-MET and / or EGFR in cells, comprising contacting the cells with an antibody-drug conjugate of any one of embodiments 1-70, a composition of any one of embodiments 71-76, a pharmaceutical composition of embodiment 77 or 78, or a pharmaceutical combination of embodiment 79; wherein the cells are cells expressing c-MET and / or EGFR.

[0687] Implementation Scheme 84. A method for preventing and / or treating and / or adjuvant treating a c-MET and / or EGFR-related disease in a subject, the method comprising administering to a subject in need an effective amount of the antibody-drug conjugate of any one of Implementation Schemes 1-70, the composition of any one of Implementation Schemes 71-76, the pharmaceutical composition of Implementation Scheme 77 or 78, or the pharmaceutical combination of Implementation Scheme 79; wherein the c-MET and / or EGFR-related disease is a cancer associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification.

[0688] Implementation Scheme 85. The method described in Implementation Scheme 84, wherein the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0689] Implementation Scheme 86. The method of Implementation Scheme 84 or 85, wherein the method further comprises administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy may be administered simultaneously, separately, or sequentially.

[0690] Implementation Scheme 87. The antibody-drug conjugate of any one of Implementation Schemes 1-70, the composition of any one of Implementation Schemes 71-76, the pharmaceutical composition of Implementation Scheme 77 or 78, or the pharmaceutical combination of Implementation Scheme 79, for the prevention and / or treatment and / or adjuvant treatment of a subject with c-MET and / or EGFR-related diseases, wherein the c-MET and / or EGFR-related diseases are cancers comprising EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification.

[0691] Implementation Scheme 88. The antibody-drug conjugate according to Implementation Scheme 87, wherein the cancer is selected from the group consisting of epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0692] Implementation Scheme 89. The antibody-drug conjugate according to Implementation Scheme 87 or 88, wherein the prevention and / or treatment and / or adjuvant treatment further comprises administering a second therapy to the subject, the second therapy being selected from the group consisting of: surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; the second therapy may be administered simultaneously, alone, or sequentially as needed.

[0693] Implementation Scheme 90. A bispecific antibody (BsAb) conjugate comprising a bispecific antibody selected from the group consisting of: BsAb 07B, BsAb 10B, BsAb 38B, BsAb 41B, BsAb 49B, BsAb 55B, and BsAb 56B; and conjugated to one or more drug-linkers selected from M-01, A-05, A-07, A-14, B-01, B-02, B-03, C-10, C-17, C-19, C-21, and C-23.

[0694] Implementation Scheme 91. A bispecific antibody (BsAb) conjugate comprising bispecific antibodies selected from the group consisting of BsAb 07B, BsAb 10B, BsAb 38B, BsAb 41B, BsAb 49B, BsAb 55B, and BsAb 56B; and a drug-linker conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 molecules, wherein the drug-linker is selected from the group consisting of M-01, A-05, A-07, A-14, B-01, B-02, B-03, C-10, C-17, C-19, C-21, and C-23. Implementation Scheme 92. A method for generating bispecific antibody (BsAb) conjugates, comprising: providing BsAb selected from the group consisting of BsAb07B, BsAb10B, BsAb38B, BsAb41B, BsAb49B, BsAb55B, BsAb56B, and a drug-linker selected from the group consisting of M-01, A-05, A-07, A-14, B-01, B-02, B-03, C-10, C-17, C-19, C-21, and C-23; mixing the BsAb with the drug-linker to provide a reaction mixture; incubating the reaction mixture under conditions suitable for generating BsAb conjugates; and separating the BsAb conjugate from the reaction mixture to generate the BsAb conjugate.

[0695] Implementation Scheme 93. A method for generating a bispecific antibody (BsAb) conjugate having approximate DARs of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, comprising: providing BsAbs selected from the group consisting of BsAb 07B, BsAb 10B, BsAb 38B, BsAb 41B, BsAb 49B, BsAb 55B, and BsAb 56B; and drug-linkers selected from the group consisting of M-01, A-05, A-07, A-14, B-01, B-02, B-03, C-10, C-17, C-19, and C-21. C-23; mixing a certain amount of BsAb with a certain amount of connector-effective load to provide a reaction mixture capable of producing BsAb conjugates with a DAR approximation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; incubating the reaction mixture under conditions suitable for producing BsAb conjugates with a DAR approximation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; separating the BsAb conjugates from the reaction mixture to produce BsAb conjugates with a DAR approximation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0696] Sequence information

[0697] The descriptions of the sequences involved in this application are provided in the table below.

[0698]

[0699] Example

[0700] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the teachings of the present invention without departing from the basic ideas and scope of the invention.

[0701] The structures of the compounds described in the following examples were determined by NMR ( 1It can be determined by 1H NMR or mass spectrometry (MS).

[0702] Nuclear magnetic resonance (NMR) 1 The H NMR measurements were performed using a Bruker 400 MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0703] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0704] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d6: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0705] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0706] Example 1: Preparation of the compound

[0707] 1.1 Preparation of N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide (M-01)

[0708] Compound IM-1 (0.40 g, 640.59 μmol, its synthesis is described in patent application CN 111936169A), eczema mesylate (0.37 g, 704.65 μmol), and DMF (8 mL) were dissolved in DMF. HATU (0.32 g, 832.77 μmol) and DIPEA (0.25 g, 1.92 mmol) were added, and the mixture was reacted at 25 °C for 4 hours. DIPEA was removed under reduced pressure, and water was added and freeze-dried to remove most of the DMF to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography (under the following conditions) to obtain compound M-01 273 mg.

[0709] Column: Waters XBridge Prep C18 OBD 45 mm × 450 mm × 8.0 μm

[0710] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0711] The structural characterization data of M-01 are as follows: ESI-MS (m / z): 1034.4 [M+H] + . 1.2 Preparation of N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxy-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide (A-05)

[0712] Under nitrogen protection, 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetic acid ester (IM-2, 0.66 g, 1.80 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid (IM-3, 0.75 g, 1.77 mmol) were added to DMF (19 In a solution of 1,2-methyl-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-10,13-dione (1-4, 1.00 g, 1.77 mmol), the solution was cooled to 5-15°C with ice water, DMTMM (0.98 g, 3.53 mmol) was added, followed by the addition of DIPEA (1.14 g, 8.84 mmol), and the solution was reacted at 25°C for 16 hours. The reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes. The DCM phase was separated, washed with brine (100 mL), and concentrated to obtain the crude product. After purification by preparative high performance liquid chromatography, the product was freeze-dried to obtain 0.98 g of compound A-05.

[0713] The separation and purification method for A-05 is as follows: Column: Waters SunFire Prep C18 OBD (5 μm) 19 mm 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0714] The structural characterization data for A-05 are as follows: MS m / z (ESI): 1107.3 [M+H]+ 1 H NMR (400 MHz, DMSO) δ 9.10 (s, 2H), 8.66 - 8.63 (m, 1H), 8.51 (d,J = 8.8 Hz, 1H), 8.34 - 8.31 (m, 1H), 8.21 - 8.19 (m, 1H), 8.17 – 8.09 (m,2H), 8.08 - 8.04 (m, 1H), 7.30 (s, 1H), 7.26 – 7.15 (m, 5H), 6.55 (s, 1H),5.56 - 5.55 (m, 1H), 5.48 – 5.35 (m, 2H), 5.25 – 5.10 (m, 2H), 4.64 (d, J =6.4 Hz, 2H), 4.45 - 4.44 (m, 1H), 4.06 - 3.98 (m, 2H), 3.77 - 3.52 (m, 6H), 3.41 (s, 3H), 3.25 - 3.12 (m, 2H), 3.03 - 3.00 (m, 1H), 2.83 – 2.72 (m, 1H),2.58 – 2.56 (m, 2H), 2.48 (s, 3H), 2.33 – 2.30 (m, 2H), 2.21 – 2.13 (m, 2H),1.91 – 1.76 (m, 4H), 0.87 (t, J = 7.2 Hz, 3H). 1.3 Preparation of N-((S)-10-benzyl-1-(((1S,9S)-5-fluoro-9-ethyl-9-hydroxy-4-chloro-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxy-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide (A-07)

[0715] Under nitrogen protection, 2,5-dioxopyrrolidone-1-yl-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylacetic acid ester (IM-2, 21.6 mg, 0.059 mmol) and (R)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanoic acid (IM-3, 24.5 mg, 0.058 mmol) were added to DMF (1 mL), and the mixture was heated to 35 °C and reacted for 16 hours. Then, (1S, The reaction mixture consisted of 9S)-1-amino-5-fluoro-9-ethyl-9-hydroxy-4-chloro-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-10,13-dione trifluoroacetate (30.0 mg, 0.053 mmol), HATU (30 mg, 0.079 mmol), and DIPEA (27.2 mg, 0.21 mmol). The reaction mixture was reacted at 25 °C for 16 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 26.4 mg of compound A-07.

[0716] The separation and purification method for A-07 is as follows: Column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0717] The structural characterization data for A-07 are as follows: ESI-MS (m / z): 1111.3 [M+H] + . 1.4 Preparation of N-((7S,10S,13S)-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinolin-1-yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecane-13-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-acetylamide (A-14)

[0718] Step 1: Compound IM-4 (657 mg, 1.22 mmol) and compounds 1-4 (500 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (10 mL), followed by the addition of HATU (630.67 mg, 1.66 mmol) and N,N-diisopropylethylamine (428 mg, 3.32 mmol), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was purified directly by preparative high-performance liquid chromatography and then freeze-dried to obtain 700 mg of compound IM-5.

[0719] The preparation method for high performance liquid chromatography purification is as follows: Column: Waters SunFire Prep C18 OBD (5 μm) 19 mm 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0720] Step Two: Compound IM-5 (500 mg, 0.513 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (75.05 mg, 1.03 mmol) was added. The reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly purified by preparative high performance liquid chromatography and then freeze-dried to obtain 307 mg of compound IM-6.

[0721] The preparation method for high performance liquid chromatography purification is as follows: Column: Waters SunFire Prep C18 OBD (5 μm) 19 mm 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0722] Step 3: IM-6 (170 mg, 0.226 mmol) and compound IM-2 (90.83 mg, 0.249 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (29.21 mg, 0.226 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified by preparative high-performance liquid chromatography and then freeze-dried to obtain 50.56 mg of compound A-14.

[0723] Its structural characterization data are as follows: MS m / z (ESI): 1002.4 [M+H]+ The preparation method for high performance liquid chromatography purification is as follows: Column: Waters SunFire Prep C18 OBD (5 μm) 19 mm 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0724] 1 H NMR (400 MHz, DMSO) δ 9.11 (s, 2H), 8.68 (t, J = 6.4 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.16 (s, 1H), 8.10 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 7.2Hz, 1H), 7.91 (d, J = 6.8 Hz, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65-5.55 (m,1H), 5.43 (s, 2H), 5.21 (s, 2H), 4.67-4.55 (m, 2H), 4.29-4.15 (m, 3H), 3.98(s, 2H), 3.41 (s, 3H), 3.25-3.15 (m, 2H), 2.57-2.56 (m, 2H), 2.35-2.27 (m,2H), 2.22-2.12 (m, 2H), 1.91-1.75 (m, 4H), 1.23-1.09 (m, 9H), 0.87 (t, J =7.2 Hz, 3H).

[0725] 1.5 Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((S)-4-ethyl-11-(2-(N-(isopropyl)methylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl) carbonate (B-01)

[0726] Step 1: At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis is referenced in patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (72.95 mg, 0.503 mmol) and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynylamide (95.10 mg, 0.302 mmol) were added. After stirring for 1 h, the mixture was filtered, and the filtrate was purified by preparative high performance liquid chromatography (under the following conditions) to obtain 30.00 mg of compound B-01-2.

[0727] Column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0728] Mobile phase A: Acetonitrile; Mobile phase B: Water

[0729] Step Two: Compound B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL) and trifluoroacetic acid (0.2 mL) was added. The reaction was carried out at room temperature for 30 min. The reaction solution was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (under the following conditions) to obtain 20.00 mg of trifluoroacetate of compound B-01.

[0730] Column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0731] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0732] The structural characterization data are as follows: ESI-MS (m / z): 1631.7 [M+H] + 816.0 [M / 2+H] + .

[0733] 1.6 Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-acetylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxy-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentadecanoamide)benzyl((1S,9R)-9-ethyl-5-fluoro-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-9-yl) carbonate (B-02)

[0734] Step 1:

[0735] At 25°C, 1-1 methanesulfonate (30.00 mg, 56.44 μmol) was dissolved in N,N-dimethylformamide (1 mL), followed by the sequential addition of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (58.74 mg, 112.88 μmol), N,N-diisopropylethylamine (43.76 mg, 338.63 μmol), and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (26.62 mg, 84.66 μmol). The reaction was maintained at 25°C for 1 hour, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was separated by thin-layer chromatography (dichloromethane:methanol = 15:1) to obtain 27.00 mg of compound B-02-1.

[0736] Step Two:

[0737] At 0°C, B-02-1 (20 mg, 27.33 μmol) was dissolved in dichloromethane (2 mL), followed by the addition of a dichloromethane solution (0.5 mL) of 4-dimethylaminopyridine (26.71 mg, 218.61 μmol) and triphosgene (8.11 mg, 27.33 μmol), and the reaction was maintained at 0°C for 0.5 hours. After replacing the residual triphosgene with nitrogen, a dichloromethane solution (1 mL) of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonazo-6-azatridodecanoamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexamamide (43.46 mg, 40.99 μmol) was added dropwise. The reaction was carried out at 0℃ for 0.5 hours. The reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was concentrated and the crude product was purified by thin-layer chromatography (dichloromethane:methanol=15:1) to obtain 30.00 mg of compound B-02-2.

[0738] Step 3:

[0739] At 25°C, B-02-2 (250.00 mg, 137.51 μmol) was dissolved in a mixed solvent of DMSO (2 mL) and water (0.4 mL), and 6-(2-(methanesulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynylamide (62.98 mg, 206.26 μmol) and cuprous bromide (39.45 mg, 275.01 μmol) were added. The reaction was maintained at 25°C for 1 hour. The reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was lyophilized to obtain 150.00 mg of compound B-02-3.

[0740] Column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0741] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0742] Step Four:

[0743] At 25°C, B-02-3 (150 mg, 49.45 μmol) was dissolved in tetrahydrofuran (1 mL), and a mixture of tetrabutylammonium fluoride (1M tetrahydrofuran solution) / glacial acetic acid (v / v=13 / 1) (50 uL) was added dropwise. The reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography (under the following conditions), and the preparative solution was lyophilized to obtain 50.00 mg of compound B-02-4.

[0744] Column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0745] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0746] Step 5:

[0747] B-02-4 (50 mg, 26.52 μmol) was dissolved in dichloromethane (1 mL) at 25 °C, and trifluoroacetic acid (60.49 mg, 530.49 μmol) was added. The reaction was maintained at 25 °C for 0.5 hours. The reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by preparative high performance liquid chromatography (under the following conditions). The preparative solution was lyophilized to obtain 23.69 mg of compound B-02.

[0748] Column: SunFire Prep C18 OBD 19 mm × 150 mm × 5.0 μm

[0749] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0750] The structural characterization data of B-02 are as follows:

[0751] ESI-MS (m / z): 1613.6 [M+H] + .

[0752] 1.7 Preparation of N-((7S,10S,13S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4':6,7]indoleazine[1,2-b]quinoline-1-yl)amino)-7,10,13-trimethyl-1,6,9,12,15-pentoxo-3,17,20,23-tetraoxa-5,8,11,14-tetraazapecopentane-25-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzamide (C-07)

[0753] Step 1: The starting material C-07-1 (4.80 g, 16.33 mmol), tributyl(2-methylthiopyrimidin-4-yl)tin (16.27 g, 39.19 mmol) and bis(triphenylphosphine)palladium dichloride (2.29 g, 3.27 mmol) were dissolved in 1,4-dioxane (100 mL). The reaction system was stirred at 110 °C for 5 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The reaction system was concentrated and purified by column chromatography (EA / PE = 0-50%) to obtain 1.36 g of compound C-07-2.

[0754] Step Two: Compound C-07-2 (510 mg, 1.33 mol) and NaOH (212.24 mg, 5.31 mmol) were dissolved in THF (12.5 mL), MeOH (12.5 mL), and H2O (2.5 mL). The reaction was stirred at 25 °C for 2 hours. The reaction was monitored by LC-MS. The pH of the system was adjusted to approximately 2 with 3N HCl. A large amount of solid precipitated. The solid was filtered, the filter cake was collected, and dried to obtain 380 mg of compound C-07-3.

[0755] Step 3: Compound C-07-3 (315 mg, 850.32 μmol), 2-[2-(2-aminoethoxy)ethoxy]ethoxyacetate tert-butyl ester (246.31 mg, 935.35 μmol), HATU (484.99 mg, 1.28 mmol), and DIPEA (329.69 mg, 2.55 mmol) were added to DMF (3 mL) and reacted at 25 °C for 2 h. The reaction was monitored by LC-MS. The reaction solution was purified by preparative high performance liquid chromatography and then freeze-dried to obtain 40 mg of compound C-07-3.

[0756] Its preparation method is as follows: Column: Waters XBridge Prep C18OBD (5 μm) 19mm 150 mm) Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0757] Step Four: Compound C-07-4 (40 mg, 64.96 μmol) was dissolved in DCM (3 mL)...

Claims

1. An antibody-drug conjugate containing the structure shown below: Ab-[M-L-E-D] x in: Ab is a bispecific antibody or its antigen-binding fragment comprising a first antigen-binding domain that specifically binds to c-MET and a second antigen-binding domain that specifically binds to EGFR; wherein, the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), the first VL and the first VH together forming a domain capable of specifically binding to c-MET; the second antigen-binding domain comprises a second VL and a second VH, the second VL and the second VH together forming a domain capable of specifically binding to EGFR. M is the linker site that is linked to the bispecific antibody or its antigen-binding fragment; L is a structural segment connecting the joint portions M and E; E is a structural segment connecting L and D; D is a cytotoxic drug or a fragment thereof; and x is any integer from 1 to 10.

2. The antibody-drug conjugate of claim 1, wherein the first antigen-binding domain and the second antigen-binding domain are each independently selected from scFv, Fab, and scFab.

3. The antibody-drug conjugate according to claim 1 or 2, wherein the bispecific antibody or its bispecific antigen-binding fragment further comprises an Fc domain, the Fc domain comprising a first Fc domain monomer and a second Fc domain monomer, and wherein the first and second Fc domain monomers comprise one or more modifications that promote heterodimerization of the Fc domain monomers.

4. The antibody-drug conjugate according to claim 3, wherein the Fc domain comprises a first Fc domain monomer and a second Fc domain monomer, the first Fc domain monomer comprising a modification to form a knock structure, the second Fc domain monomer comprising a modification to form a hole structure, wherein the hole structure can pair with the knock structure to form a heterodimerized Fc domain.

5. The antibody-drug conjugate according to claim 3 or 4, wherein the first Fc domain monomer comprises an amino acid sequence as shown in SEQ ID NO: 49 or 51, and the second Fc domain monomer comprises an amino acid sequence as shown in SEQ ID NO: 50 or 52.

6. The antibody-drug conjugate according to any one of claims 3-5, wherein the first antigen-binding domain and the second antigen-binding domain are each linked to one of the first and second Fc domain monomers of the Fc domain.

7. The antibody-drug conjugate of any one of claims 3-6, wherein the first antigen-binding domain is linked to the first Fc domain monomer, and the second antigen-binding domain is linked to the second Fc domain monomer; or the first antigen-binding domain is linked to the second Fc domain monomer, and the second antigen-binding domain is linked to the first Fc domain monomer.

8. The antibody-drug conjugate of any one of claims 1-7, wherein the first VL comprises the amino acid sequences of the complementarity-determining regions (CDRs)-L1, CDR-L2, and CDR-L3 of the VL amino acid sequence described in SEQ ID NO: 17 or SEQ ID NO: 59; and / or the first VH comprises the amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 of the VH amino acid sequence described in SEQ ID NO: 18 or SEQ ID NO:

60.

9. The antibody-drug conjugate of any one of claims 1-8, wherein: The first VL includes: (i) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 36, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 38; or, (ii) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 35, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 37, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 38; And / or, The first VH includes: (i) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 39, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 43, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 47; (ii) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 40, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 47; (iii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 42, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 47; or, (iv) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 45, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO:

48.

10. The antibody-drug conjugate according to any one of claims 1-9, wherein: The first VL contains the amino acid sequence shown in SEQ ID NO: 17 or 59, and / or the first VH contains the amino acid sequence shown in SEQ ID NO: 18 or 60; or The first VL contains the amino acid sequence shown in SEQ ID NO:17, and the first VH contains the amino acid sequence shown in SEQ ID NO:18; or the first VL contains the amino acid sequence shown in SEQ ID NO:59, and the first VH contains the amino acid sequence shown in SEQ ID NO:

60.

11. The antibody-drug conjugate of any one of claims 1-10, wherein, The second VL comprises the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence described in SEQ ID NO: 15; and / or the second VH comprises the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence described in SEQ ID NO:

16.

12. The antibody-drug conjugate of any one of claims 1-11, wherein: The second VL includes: (i) CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 23; or, (ii) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 20, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 22, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 23; And / or, The second VH includes: (i) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 24, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 28, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 32; (ii) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 25, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 29, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 32; (iii) CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 27, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 31, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 32; or, (iv) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 26, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 30, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO:

33.

13. The antibody-drug conjugate of any one of claims 1-12, wherein the second VL comprises the amino acid sequence shown in SEQ ID NO:15, and / or the second VH comprises the amino acid sequence shown in SEQ ID NO:

16.

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

15. The antibody-drug conjugate of claim 14, wherein the bispecific antibody comprises polypeptide chain IA, polypeptide chain IB, and polypeptide chain IC; wherein polypeptide chain IA comprises the first VL and a light chain constant region, polypeptide chain IB comprises the first VH, a heavy chain CH1 region, and a first Fc domain monomer (or a second Fc domain monomer), and / or polypeptide chain IC comprises the second VL, the second VH, and a second Fc domain monomer (the first Fc domain monomer).

16. The antibody-drug conjugate of claim 15, wherein the polypeptide chain IA comprises the first VL and a light chain constant region from the N-terminus to the C-terminus, the polypeptide chain IB comprises the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or the polypeptide chain IC comprises (i) the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the second VH, the second VL and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus.

17. The antibody-drug conjugate of claim 15 or 16, wherein adjacent domains of the polypeptide chain IA are optionally linked by or without a linker, adjacent domains of the polypeptide chain IB are optionally linked by or without a linker, and / or adjacent domains of the polypeptide chain IC are optionally linked by or without a linker.

18. The antibody-drug conjugate of claim 17, wherein each of the linkers is independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or each of the peptide linkers is independently selected from peptide linkers comprising one or more glycine (G) and / or serine (S), for example, having a structure such as (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

19. The antibody-drug conjugate of any one of claims 15-18, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

20. The antibody-drug conjugate of any one of claims 15-19, wherein the polypeptide chain IA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain IB comprises the amino acid sequence shown in SEQ ID NO: 2 or 9, and / or the polypeptide chain IC comprises the amino acid sequence shown in SEQ ID NO: 3 or 10.

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

22. The antibody-drug conjugate of claim 21, wherein the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein polypeptide chain II-A comprises the second VL and a light chain constant region, polypeptide chain II-B comprises the second VH, a heavy chain CH1 region, and a first Fc domain monomer (or a second Fc domain monomer), and / or polypeptide chain II-C comprises the first VL, the first VH, and a second Fc domain monomer (or a first Fc domain monomer).

23. The antibody-drug conjugate of claim 22, wherein the polypeptide chain II-A comprises the second VL and a light chain constant region from the N-terminus to the C-terminus, the polypeptide chain II-B comprises the second VH, a heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or the polypeptide chain II-C comprises (i) the first VL, the first VH and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the first VH, the first VL and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus.

24. The antibody-drug conjugate of claim 22 or 23, wherein adjacent domains of polypeptide chain II-A are optionally linked by or without a linker, adjacent domains of polypeptide chain II-B are optionally linked by or without a linker, and / or adjacent domains of polypeptide chain II-C are optionally linked by or without a linker.

25. The antibody-drug conjugate of claim 24, wherein each of the linkers is independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or each of the peptide linkers is independently selected from peptide linkers comprising one or more glycine (G) and / or serine (S), for example, having a structure such as (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

26. The antibody-drug conjugate of any one of claims 22-25, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

27. The antibody-drug conjugate of any one of claims 22-26, wherein the polypeptide chain II-A comprises the amino acid sequence shown in SEQ ID NO: 4, the polypeptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 5 or 7, and / or the polypeptide chain II-C comprises the amino acid sequence shown in SEQ ID NO: 6 or 8.

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

29. The antibody-drug conjugate of claim 28, wherein the bispecific antibody comprises polypeptide chain III-A, polypeptide chain III-B, and polypeptide chain III-C; wherein polypeptide chain III-A comprises a first VL and a light chain constant region, polypeptide chain III-B comprises a first VH, a heavy chain CH1 region, and a first Fc domain monomer (or a second Fc domain monomer), and / or polypeptide chain III-C comprises a second VL, a light chain constant region, a second VH, a heavy chain CH1 region, and a second Fc domain monomer (or a first Fc domain monomer).

30. The antibody-drug conjugate of claim 29, wherein polypeptide chain III-A comprises a first VL and a light chain constant region from the N-terminus to the C-terminus, polypeptide chain III-B comprises a first VH, the heavy chain CH1 region and a first Fc domain monomer (or a second Fc domain monomer) from the N-terminus to the C-terminus, and / or polypeptide chain III-C comprises (i) the second VL, the light chain constant region, the second VH, the heavy chain CH1 region and a second Fc domain monomer (or a first Fc domain monomer), or (ii) the second VH, the heavy chain CH1 region, the second VL, the light chain constant region and a second Fc domain monomer (or a first Fc domain monomer).

31. The antibody-drug conjugate of claim 29 or 30, wherein adjacent domains of polypeptide chain III-A are optionally linked by or without a linker, adjacent domains of polypeptide chain III-B are optionally linked by or without a linker, and / or adjacent domains of polypeptide chain III-C are optionally linked by or without a linker.

32. The antibody-drug conjugate of claim 31, wherein each of the linkers is independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or each of the peptide linkers is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

33. The antibody-drug conjugate of any one of claims 29-32, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

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

35. The antibody-drug conjugate of claim 34, wherein the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C; wherein polypeptide chain IV-A comprises the second VL and a light chain constant region, polypeptide chain IV-B comprises the second VH, a heavy chain CH1 region, and a first Fc domain monomer (or the second Fc domain monomer), and / or polypeptide chain IV-C comprises the first VL, a light chain constant region, the first VH, a heavy chain CH1 region, and a second Fc domain monomer (or the first Fc domain monomer).

36. The antibody-drug conjugate of claim 35, wherein polypeptide chain IV-A comprises the second VL and a light chain constant region from the N-terminus to the C-terminus, polypeptide chain IV-B comprises the second VH, a heavy chain CH1 region and a first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, and / or polypeptide chain IV-C comprises (i) the first VL, the light chain constant region, the first VH, the heavy chain CH1 region and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the first VH, the heavy chain CH1 region, the first VL, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer).

37. The antibody-drug conjugate of claim 35 or 36, wherein adjacent domains of polypeptide chain IV-A are optionally linked by or without a linker, adjacent domains of polypeptide chain IV-B are optionally linked by or without a linker, and / or adjacent domains of polypeptide chain IV-C are optionally linked by or without a linker.

38. The antibody-drug conjugate of claim 37, wherein each of the linkers is independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or each of the peptide linkers is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having a structure such as (GGGGS). )n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

39. The antibody-drug conjugate of any one of claims 35-38, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

40. The antibody-drug conjugate of any one of claims 35-39, wherein the polypeptide chain IV-A comprises the amino acid sequence shown in SEQ ID NO: 4, the polypeptide chain IV-B comprises the amino acid sequence shown in SEQ ID NO: 7, and / or the polypeptide chain IV-C comprises the amino acid sequence shown in SEQ ID NO:

11.

41. The antibody-drug conjugate according to any one of claims 1-13, wherein the first antigen-binding domain and the second antigen-binding domain are Fab, and the Fab of the second antigen-binding domain comprises domain exchanges of the form of CrossMab.

42. The antibody-drug conjugate of claim 41, wherein the bispecific antibody comprises polypeptide chain VA, polypeptide chain VB, polypeptide chain VC, and polypeptide chain VD; wherein polypeptide chain VA comprises the first VL and a light chain constant region, polypeptide chain VB comprises the first VH, a heavy chain CH1 region, and a monomer of the first Fc domain (or a monomer of the second Fc domain), polypeptide chain VC comprises the second VH, a light chain constant region, and a monomer of the second Fc domain (or a monomer of the first Fc domain), and / or polypeptide chain VD comprises the second VL and a heavy chain CH1 region from the N-terminus to the C-terminus.

43. The antibody-drug conjugate of claim 42, wherein the polypeptide chain VA comprises the first VL and the light chain constant region from the N-terminus to the C-terminus, the polypeptide chain VB comprises the first VH, the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer) from the N-terminus to the C-terminus, the polypeptide chain VC comprises the second VH, the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer) from the N-terminus to the C-terminus, and / or the polypeptide chain VD comprises the second VL and the heavy chain CH1 region from the N-terminus to the C-terminus.

44. The antibody-drug conjugate of claim 42 or 43, wherein adjacent domains of the polypeptide chain VA are optionally linked by or without a linker, adjacent domains of the polypeptide chain VB are optionally linked by or without a linker, adjacent domains of the polypeptide chain VC are optionally linked by or without a linker, and / or adjacent domains of the polypeptide chain VD are optionally linked by or without a linker.

45. The antibody-drug conjugate of claim 44, wherein each of the linkers is independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or each of the peptide linkers is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

46. ​​The antibody-drug conjugate according to any one of claims 42-45, wherein the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as shown in SEQ ID NO:

54.

47. The antibody-drug conjugate of any one of claims 42-46, wherein the polypeptide chain VA comprises the amino acid sequence shown in SEQ ID NO: 1, the polypeptide chain VB comprises the amino acid sequence shown in SEQ ID NO: 9, the polypeptide chain VC comprises the amino acid sequence shown in SEQ ID NO: 13, and / or the polypeptide chain VD comprises the amino acid sequence shown in SEQ ID NO:

12.

48. The antibody-drug conjugate of any one of claims 1-13, wherein the first antigen-binding domain and the second antigen-binding domain are Fab, and the Fab of the first antigen-binding domain comprises domain exchanges of the form of CrossMab.

49. The antibody-drug conjugate of claim 48, wherein the bispecific antibody comprises polypeptide chain VI-A, polypeptide chain VI-B, polypeptide chain VI-C, and polypeptide chain VI-D; wherein polypeptide chain VI-A comprises the second VL and a light chain constant region, polypeptide chain VI-B comprises the second VH, a heavy chain CH1 region, and a monomer of the first Fc domain (or a monomer of the second Fc domain), polypeptide chain VI-C comprises the first VH, a light chain constant region, and a monomer of the second Fc domain (or a monomer of the first Fc domain); and polypeptide chain VI-D comprises the first VL and a heavy chain CH1 region.

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

51. The antibody-drug conjugate of claim 49 or 50, wherein adjacent domains of polypeptide chain VI-A are optionally linked by or without a linker, adjacent domains of polypeptide chain VI-B are optionally linked by or without a linker, adjacent domains of polypeptide chain VI-C are optionally linked by or without a linker, and / or adjacent domains of polypeptide chain VI-D are optionally linked by or without a linker.

52. The antibody-drug conjugate of claim 51, wherein each of the linkers is independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or each of the peptide linkers is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

53. The antibody-drug conjugate of any one of claims 49-52, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises the amino acid sequence shown in SEQ ID NO:

54.

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

55. The antibody-drug conjugate of claim 54, wherein the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein polypeptide chain VII-A comprises the first VL, the first VH and the first Fc domain monomer (or the second Fc domain monomer), and polypeptide chain VII-B comprises the second VL, the second VH and the second Fc domain monomer (or the first Fc domain monomer).

56. The antibody-drug conjugate of claim 55, wherein the polypeptide chain VII-A comprises, from the N-terminus to the C-terminus, (i) the first VL, the first VH, and the first Fc domain monomer (or the second Fc domain monomer), or (ii) the first VH, the first VL, and the first Fc domain monomer (or the second Fc domain monomer); and / or the polypeptide chain VII-B comprises, from the N-terminus to the C-terminus, (i) the second VL, the second VH, and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the second VH, the second VL, and the second Fc domain monomer (or the first Fc domain monomer).

57. The antibody-drug conjugate of claim 55 or 56, wherein adjacent domains of said polypeptide chain VII-A are optionally linked by or without a linker, and / or adjacent domains of said polypeptide chain VII-B are optionally linked by or without a linker.

58. The antibody-drug conjugate of claim 57, wherein each of the linkers is independently the same peptide linker or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); or each of the peptide linkers is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, having (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 55-58 or 65-70); or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

59. The antibody-drug conjugate of claim 58, wherein each peptide linker independently comprises the amino acid sequence shown in SEQ ID NO: 55-58, 61 or 65-70.

60. The antibody-drug conjugate of claims 55-59, wherein the polypeptide chain VII-A comprises the amino acid sequence shown in SEQ ID NO:14, and / or the polypeptide chain VII-B comprises the amino acid sequence shown in SEQ ID NO:

8.

61. The antibody-drug conjugate according to any one of claims 1-60, wherein the bispecific antibody or its antigen-binding fragment comprises: (1) A polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 2, and a polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO:

3. (2) A polypeptide chain IA containing the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain IB containing the amino acid sequence shown in SEQ ID NO: 9, and a polypeptide chain IC containing the amino acid sequence shown in SEQ ID NO:

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

11. (6) A polypeptide chain VA comprising the amino acid sequence shown in SEQ ID NO: 1, a polypeptide chain VB comprising the amino acid sequence shown in SEQ ID NO: 9, a polypeptide chain VC comprising the amino acid sequence shown in SEQ ID NO: 13, and a polypeptide chain VD comprising the amino acid sequence shown in SEQ ID NO: 12; or, (7) A polypeptide chain VII-A comprising the amino acid sequence shown in SEQ ID NO: 14, and a polypeptide chain VII-B comprising the amino acid sequence shown in SEQ ID NO:

8.

62. The antibody-drug conjugate according to any one of claims 1-61, having enhanced tumor-suppressive activity relative to a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody; wherein the CDR amino acid sequence of the monospecific anti-c-MET antibody is identical to the CDR amino acid sequence of the first antigen-binding domain, and the CDR amino acid sequence of the monospecific anti-EGFR antibody is identical to the CDR amino acid sequence of the second antigen-binding domain.

63. The antibody-drug conjugate of claim 62, wherein the tumor-suppressive effect comprises: Inhibition of EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity.

64. The antibody-drug conjugate according to any one of claims 1-63, wherein M comprises , in, Ring A is a 5-6 membered aliphatic heterocycle or a 5-20 membered aromatic ring system, wherein the aliphatic heterocycle and aromatic ring system are optionally selected independently by one or more groups selected from oxygen (=O), halogen, cyano, amino, carboxyl, mercapto, and C. 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

65. The antibody-drug conjugate according to any one of claims 1-63, wherein M comprises Wherein ring A is a 5-membered aliphatic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by one or more 6-membered heteroaromatic rings connected to a benzene ring via single bonds, or a polycyclic ring formed by multiple 6-membered heteroaromatic rings connected via single bonds, wherein the aliphatic heterocycle is optionally surrounded by one or more elements selected from oxygen (=O), halogens, and C. 1-4 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 imidene group, C 2-20 Alynyl or amino group.

66. The antibody-drug conjugate according to any one of claims 1-63, wherein M comprises Ring A is selected from , , , , , and M1 is selected from single bonds, C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Alynyl or amino group.

67. The antibody-drug conjugate according to any one of claims 1-63, wherein M is selected from... , and .

68. The antibody-drug conjugate according to any one of claims 1-63, wherein M is .

69. The antibody-drug conjugate according to any one of claims 1-63, wherein M is selected from... , , and .

70. The antibody-drug conjugate according to any one of claims 1-63, wherein M is selected from... , and .

71. The antibody-drug conjugate according to any one of claims 1-70, wherein L is selected from one or more of the following structures: C 1-6 Alkyl group, -N(R')-, carbonyl group, -O-, selected from Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2)) s Natural or non-natural amino acids and their analogues (OCH3), as well as short peptides containing 1, 2, 3 or 4 amino acids. , , , , , , , , and ; Where R' represents hydrogen, C 1-6 Alkyl or polyethylene glycol fragment containing 1-10 EO units; s is an integer selected from 1-20.

72. The antibody-drug conjugate of claim 71, wherein the short peptide is selected from Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG (SEQ ID NO: 71)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 72)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 73)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 74)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 75)).

73. The antibody-drug conjugate according to any one of claims 1-70, wherein L is selected from structures comprising one or more of the following: C 1-6 Alkylene, Carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Ar g, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG (SEQ ID NO: 71)), Gly-Gly-Val-Ala (GGVA (SEQ ID NO: 72)), Gly-Phe-Leu-Gly (GFLG (SEQ ID NO: 73)), Glu-Ala-Ala-Ala (EAAA (SEQ ID NO: 74)), Gly-Gly-Gly-Gly-Gly (GGGGG (SEQ ID NO: 75)), , , , and ; where s is an integer selected from 1 to 20.

74. The antibody-drug conjugate according to any one of claims 1-70, wherein L is selected from one or more of the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

75. The antibody-drug conjugate according to any one of claims 1-70, wherein the L is selected from the following structures: , , , , , , , , , and .

76. The antibody-drug conjugate according to any one of claims 1-70, wherein the L is selected from the following structures: , and .

77. The antibody-drug conjugate according to any one of claims 1-70, wherein the L is selected from the following structures: and .

78. The antibody-drug conjugate according to any one of claims 1-70, wherein the L is selected from the following structures: and .

79. The antibody-drug conjugate according to any one of claims 1-78, wherein E is a single bond or selected from the following structures: -NHCH2-、 , , and .

80. The antibody-drug conjugate according to any one of claims 1-78, wherein E is a single bond, -NHCH2-, or .

81. The antibody-drug conjugate according to any one of claims 1-78, wherein E is -NHCH2- or .

82. The antibody-drug conjugate according to any one of claims 1-78, wherein E is -NHCH2- or a single bond.

83. The antibody-drug conjugate according to any one of claims 1-78, wherein E is... .

84. The antibody-drug conjugate according to any one of claims 1-83, Selected from the following structures: and 。 85. The antibody-drug conjugate according to any one of claims 1-83, wherein, Selected from the following structures: and 。 86. The antibody-drug conjugate according to any one of claims 1-85, wherein the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors, and RNA polymerase inhibitors.

87. The antibody-drug conjugate of claim 86, wherein the microtubule inhibitor is an olistatin or maytansine compound.

88. The antibody-drug conjugate of claim 86, wherein the DNA intercalating agent is pyrrolobenzodiazepine (PBD).

89. The antibody-drug conjugate of claim 86, wherein the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.

90. The antibody-drug conjugate of claim 89, wherein the topoisomerase I inhibitor is selected from camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, rubotecan, and pharmaceutically acceptable salts, esters, or analogs thereof; and the topoisomerase II inhibitor is selected from doxorubicin, PNU-159682, docalimcin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, and pharmaceutically acceptable salts, esters, or analogs thereof.

91. The antibody-drug conjugate of claim 86, wherein the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester or analogue thereof.

92. The antibody-drug conjugate according to any one of claims 1-85, wherein the cytotoxic drug is selected from compounds of formula I and II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of formula I and II: in, R1 and R2 are each independently selected from C 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; R4 and R5 are each independently selected from H, halogens, and hydroxyl groups; or R4 and R5 are linked to form a 5-6 membered oxygen-containing heterocycle; R6 is selected from hydrogen or -C. 1-4 Alkylene-NR a R b ; R7 is selected from C 1-6 Alkyl, -C 1-4 Alkylene-NR a R b -C 1-4 Alkylene-SiR a R b R c -SiR a R b R c -C 1-4 Alkylene = N-OR a ;where R a R b and R c Each time it appears, it is independently selected from H and C. 1-6 Alkyl group, -SO2-C 1-6 Alkyl and -CO-C 1-6 Alkyl; wherein R is optionally present a and R b It forms 5-6 member nitrogen-containing heterocycles with the connected atoms.

93. The antibody-drug conjugate according to any one of claims 1-85, wherein the cytotoxic drug is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: ； The fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula of claim 1.

94. The antibody-drug conjugate according to any one of claims 1-85, wherein the cytotoxic drug is selected from compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: ， The fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula of claim 1.

95. The antibody-drug conjugate of claim 93 or 94, wherein, D is a monovalent structure obtained by losing an H from the -OH, -NH2, or secondary amine group on the cytotoxic drug.

96. The antibody-drug conjugate according to any one of claims 1-95, wherein the antibody-drug conjugate is selected from: ADC A-01 ADC A-02 ADC A-05 ADC A-06 ADC A-07 ADC A-08 ADC A-09 ADC A-10 ADC A-11 ADC A-14 ADC A-15 ADC A-16 ADC A-17 ADC A-18 ADC A-20 ADC A-21 ADC A-22 ADC A-24 ADC A-25 ADC A-26 ADC A-28 ADC A-29 ADC A-30 ADC A-32 ADC A-33 ADC A-34 ADC B-01 ADC B-03 ADC B-04 ADC B-06 ADC B-07 ADC C-02 ADC C-03 ADC C-05 ADC C-06 ADC C-08 ADC C-09 ADC C-11 ADC C-12 ADC C-14 ADC C-15 ADC C-17 ADC C-18 ADC C-19 ADC C-20 ADC C-21 ADC C-22 ADC C-23 ADC C-24 ADC C-25 ADC C-26 ADC C-27 and ADC C-28 in, Ab is a bispecific antibody or its antigen-binding fragment as defined in any one of claims 1-63; and This indicates the specific linkage between the thiol group of the cysteine ​​residue in the bispecific antibody or its antigen-binding fragment and M in the antibody-drug conjugate; and x represents the amount of drug load.

97. The antibody-drug conjugate of claim 96, wherein the thiol group in the bispecific antibody or its antigen-binding fragment forms a thioether bond with M in the antibody-drug conjugate through an addition reaction or a substitution reaction to obtain the antibody-drug conjugate.

98. The antibody-drug conjugate of claim 96 or 97, wherein the Ab is a bispecific antibody selected from BsAb 07B, BsAb 10B, BsAb 38B, BsAb 41B, BsAb 49B, BsAb 55B, and BsAb 56B.

99. Antibody-drug conjugates selected from ADC 07B-A-05, ADC 07B-A-14, ADC 38B-A-14, ADC 49B-A-14, ADC 49B-B-01, ADC 49B-A-05, ADC 41B-A-05, ADC 55B-A-14 and ADC 56B-A-14.

100. A composition comprising one or more antibody-drug conjugates as described in any one of claims 1-99.

101. The composition of claim 100, wherein the DAR value (drug-antibody conjugate ratio) of the composition is 1-10.

102. The composition of claim 100, wherein the DAR value of the composition is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1~2, 1~3, 1~4, 1~5, 1~6, 1~7, 1~8, 1~9, 1~10, 2~3, 2~4, 2~5, 2~6, 2~7, 2~8, 2~9, 2~10, 3~4, 3~5, 3~6, ​​3~7, 3~8, 3~9, 3~10, 4~5, 4~6, 4~7, 4~8, 4~9, 4~10, 5~6, 5~7, 5~8, 5~9, 5~10, 6~7, 6~8, 6~9, 6~10, 7~8, 7~9, 7~10, 8~9, 8~10, or 9~10.

103. The composition of claim 100, wherein the DAR value of the composition is 3 to 9.

104. The composition of claim 100, wherein the DAR value of the composition is 4 to 8.

105. The composition of claim 100, wherein the DAR value of the composition is 3.0~3.5, 3.0~4.0, 3.0~4.5, 3.0~5.0, 3.0~5.5, 3.0~6.0, 3.5~4.0, 3.5~4.5, 3.5~5.0, 3.5~5.5, 3.5~6.0, 3.5~6.5, 3.5~7.0, 3.5~7.5, 3.5~8.0, 4.0~4.5, 4 0~5.0, 4.0~5.5, 4.0~6.0, 4.0~6.5, 4.0~7.0, 4.0~7.5, 4.0~8.0, 4.5~5.0, 4.5~5.5, 4.5~6.0, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.5~8.0, 5.0~5.5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.

0.

106. A pharmaceutical composition comprising one or more antibody-drug conjugates as described in any one of claims 1-99 or the composition as described in claim 100, and a pharmaceutically acceptable carrier and / or excipient.

107. The pharmaceutical composition of claim 106, wherein the pharmaceutical composition further comprises one or more additional pharmaceutically active agents selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

108. A pharmaceutical combination comprising a first therapeutic agent and a second therapeutic agent, wherein the first therapeutic agent and the second therapeutic agent are administered simultaneously or sequentially, wherein the first therapeutic agent is selected from one or more antibody-drug conjugates according to any one of claims 1-99 or compositions according to any one of claims 100-105; and the second therapeutic agent is selected from EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents, or any combination thereof.

109. Use in the preparation of a medicament of any one of claims 1-99, any one of claims 100-105, any one of claims 106 or 107, or any one of claims 108, of the antibody-drug conjugate of claim 1-99, the composition of claim 100-105, the pharmaceutical composition of claim 106 or 107, or the pharmaceutical combination of claim 108, wherein the medicament is used in a subject for the prevention, treatment, and / or as an adjunct to the treatment of diseases associated with c-MET and / or EGFR, and / or for the inhibition of c-MET and / or EGFR activity in vitro or in a subject; wherein the diseases associated with c-MET and / or EGFR are associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification, and optionally, the diseases associated with c-MET and / or EGFR include cancer.

110. The use according to claim 109, wherein the cancer is selected from epithelial carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

111. The use of claim 109 or 110, wherein the antibody-drug conjugate or pharmaceutical composition and the additional pharmaceutically active agent are administered simultaneously, alone or in sequence; wherein the additional pharmaceutically active agent is selected from: EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-MET or VEGF inhibitors, chemotherapeutic agents and any combination thereof.

112. A method for inhibiting the activity of c-MET and / or EGFR in cells, the method comprising contacting the cells with an antibody-drug conjugate according to any one of claims 1-99, a composition according to any one of claims 100-105, a pharmaceutical composition according to claim 106 or 107, or a pharmaceutical combination according to claim 108; wherein the cells are cells expressing c-MET and / or EGFR.

113. A method for preventing, treating, and / or acting as adjunctive therapy for c-MET and / or EGFR-related diseases in a subject, the method comprising administering to a subject in need an effective amount of an antibody-drug conjugate of any one of claims 1-99, a composition of any one of claims 100-105, a pharmaceutical composition of claim 106 or 107, or a pharmaceutical combination of claim 108; wherein the c-MET and / or EGFR-related disease comprises EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification, optionally, the c-MET and / or EGFR-related disease includes cancer.

114. The method of claim 113, wherein the cancer is selected from epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

115. The method of claim 113 or 114, wherein the method further comprises administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy is administered simultaneously, separately, or sequentially.

116. An antibody-drug conjugate of any one of claims 1-99, a composition of any one of claims 100-105, a pharmaceutical composition of claim 106 or 107, or a pharmaceutical combination of claim 108 for the prevention, treatment, and / or adjunctive treatment of a subject with c-MET and / or EGFR-related diseases. The c-MET and / or EGFR-related diseases comprise EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification. Optionally, the c-MET and / or EGFR-related diseases are cancer.

117. The antibody-drug conjugate of claim 116, wherein the cancer is selected from epithelial carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

118. The antibody-drug conjugate of claim 116 or 117, wherein the method further comprises administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; optionally, the second therapy may be administered simultaneously, alone, or sequentially.

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