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] This application is a divisional application of the Chinese Invention Patent Application No. 202411638831.1, filed on November 15, 2024, entitled “Antibody-drug conjugates targeting EGFR / c-MET and methods of making and using the same”. TECHNICAL FIELD

[0002] The present disclosure relates to targeted therapy, and more particularly, the present disclosure relates to antibody-drug conjugates targeting EGFR and c-MET and methods of making and using the same. BACKGROUND

[0003] EGFR (epidermal growth factor receptor, also known as EGFR, ErbB-1 or HER1) is one of the members of the epidermal growth factor receptor (HER) family. The family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3) and HER4 (erbB4). The HER family plays an important regulatory role in cell physiological processes. EGFR is a type I transmembrane glycoprotein composed of 1210 amino acids, with a molecular weight of about 170 kDa. Its main structure includes a ligand-binding extracellular domain, a hydrophobic transmembrane region and an intracellular region containing tyrosine kinase. EGFR is highly expressed in colorectal cancer, head and neck cancer, non-small cell lung cancer and other tumors, and has certain expression in normal epithelial tissues such as skin and lung. High expression of EGFR in tumor tissues leads to homodimerization or heterodimerization by binding to ligands such as EGF or TGFa, which activates tyrosine kinase in tumor cells, protein phosphorylation, mediates gene transcription and cell cycle. Activation of various cell signaling pathways plays an important role in promoting tumor cell survival, proliferation and migration, and tumor angiogenesis.

[0004] Tyrosine-protein kinase 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 of 1390 amino acids with a molecular weight of approximately 190 kDa, which is ultimately formed by two polypeptide chains, alpha chain (50 kDa) and beta chain (140 kDa), connected by disulfide bonds, through cleavage. The MET protein can be divided from the outside of the membrane to the inside of the cell into SEMA domain (Semaphorin domain, SEMA), PSI domain (Plexin-semaphorin-integrin domain, PSI), 4 immunoglobulin-like repeat domains (Immunoglobulin-plexins-transcription domain, IPT), a transmembrane domain (Transmembrane region), a juxtamembrane domain (Juxtamembrane region, JM), a tyrosine kinase domain (Tyrosine kinase domain, TK) and a carboxyl terminal region (Carboxyl terminal region, CT). The natural ligand of c-MET receptor is hepatocyte growth factor (HGF), which is an inactive protein that is converted to an active form by proteolytic cleavage. Abnormal regulation of c-MET has been reported to exist in a variety of cancers, including colorectal cancer, non-small cell lung cancer, gastric cancer and breast cancer. High activation of c-MET and its downstream signaling pathway has been shown to trigger hyperproliferation, tumor invasion, and angiogenesis, and is associated with low survival rate.

[0005] In tumors resistant to EGFR tyrosine kinase inhibitors (TKIs), nearly 60% increase c-MET expression, amplify c-MET or increase c-MET ligand HGF (Turke et al., Cancer Cell, 17:77-88, 2010), indicating that the tumor c-MET signaling pathway will compensate 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 is expected to simultaneously inhibit both the EGFR and c-MET signaling pathways, avoiding the development of tumors caused by the activation of the c-MET signaling pathway after EGFR inhibition, thereby improving overall efficacy and safety.

[0006] Anti-c-MET and anti-EGFR antibodies are known in the art, for example, Telisotuzumab vedotin (Teliso-v or ABBV399) disclosed in US Patent No. 10603389 is an antibody-drug conjugate of vc-MMAE (vedotin) conjugated to the anti-c-MET antibody ABT-700, MRG003 (ADC-3 or MYK-3) disclosed in WO2023040941 is an antibody-drug conjugate of vedotin conjugated to the anti-EGFR antibody BA03.

[0007] Several EGFR / c-Met bispecific antibodies have been disclosed in the art. The bispecific antibody BSAB01 disclosed in WO2010115551 comprises the EGFR binding VH / VL pair (Fab) of cetuximab and the c-Met binding VH / VL pair (Fab) of onartuzumab, which is currently in phase III trials. US Patent No. 9593164 discloses Amivantamab, trade name of Rybrevant®, a bispecific antibody. Amivantamab comprises an anti-EGFR Fab comprising the VH and VL of Zalutumumab (see US Patent No. 7247301 and US Patent No. 7595378) and an anti-cMET Fab comprising the VH and VL of antibody 069 disclosed in US Patent No. 9068011. Sellman et al. (J Biol Chem. 291(48):25106-25119 (2016)) discloses EGFR / c-Met bispecific antibodies conjugated to a cytotoxin, which discloses EGFR / c-Met bispecific antibodies conjugated to vedotin. WO2023083846 discloses AZD9592, wherein the EGFR / c-Met bispecific antibody is conjugated to a camptothecin derivative. There is a need in the art for antibody-drug conjugates (ADC) that bind to EGFR and c-MET and comprise a cytotoxic drug. SUMMARY

[0008] The present application relates to an antibody-drug conjugate for use in the prevention and / or treatment and / or as an adjunct medication for c-MET and / or EGFR related diseases. The ADC of the present application comprises a bispecific antibody conjugated to a cytotoxic drug, said antibody comprising an arm that specifically binds EGFR and an arm that specifically binds c-MET. In some particular embodiments, the bispecific antibody comprises a scFv that binds EGFR and a Fab that binds c-MET. In other embodiments, the bispecific antibody comprises a Fab that binds EGFR and a scFV that binds c-MET, or comprises a scFv that binds EGFR and a scFv that binds c-MET. In yet other embodiments, the anti-EGFR Fab and / or anti-c-MET Fab comprises a CrossMab format selected from the group consisting of CrossMab Fab , CrossMab VH-VL and CrossMab CH1-CL . The ADC and the drug thereof are useful for the prevention and / or treatment and / or as an adjunct medication for c-MET and / or EGFR related diseases, such as cancer.

[0009] Exemplary ADCs comprising the bispecific antibodies 07B, 10B, 38B, 41B, 49B, 55B or 56B conjugated to a camptothecin derivative disclosed herein show affinity to EGFR and c-MET and better affinity to EGFR and / or c-MET positive tumor cells, such as gastric cancer, non-small cell lung cancer (e.g. lung adenocarcinoma), epidermal squamous carcinoma cells, etc., and in vivo and in vitro killing activity.

[0010] The present application provides an antibody-drug conjugate comprising the structure shown below:

[0011] Ab-[M-L-E-D]x

[0012] wherein:

[0013] Ab is a bispecific antibody or an antigen binding fragment thereof comprising a first antigen binding domain that specifically binds c-MET and a second antigen binding domain that specifically binds 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 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;

[0014] M is a linker moiety attached to the bispecific antibody or antigen binding fragment thereof;

[0015] L is a structural fragment connecting the linker moiety M and E;

[0016] E is a structural fragment connecting L and D;

[0017] D is a cytotoxic drug or fragment thereof; and

[0018] x is selected from any integer from 1 to 10.

[0019] In another embodiment of the antibody-drug conjugate, the first and second antigen binding domains are each independently selected from the group consisting of scFv, Fab, and scFab.

[0020] In another embodiment of the antibody-drug conjugate, the bispecific antibody or bispecific antigen binding fragment thereof further comprises an 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.

[0021] In another embodiment of the antibody-drug conjugate, the Fc domain comprises a first Fc domain monomer comprising a modification forming a knob structure and a second Fc domain monomer comprising a modification forming a hole structure, wherein the hole structure can pair with the knob structure to form a heterodimerized Fc domain.

[0022] In another embodiment of the antibody-drug conjugate, the first Fc domain monomer comprises an amino acid sequence as set forth in SEQ ID NO: 49 or 51 and the second Fc domain monomer comprises an amino acid sequence as set forth in SEQ ID NO: 50 or 52.

[0023] In another embodiment of the antibody-drug conjugate, the first and second antigen binding domains are each connected to one of the first and second Fc domain monomers of the Fc domain.

[0024] In another embodiment of the antibody-drug conjugate, the first antigen binding domain is connected to the first Fc domain monomer and the second antigen binding domain is connected to the second Fc domain monomer; or the first antigen binding domain is connected to the second Fc domain monomer and the second antigen binding domain is connected to the first Fc domain monomer.

[0025] In another embodiment of the antibody-drug conjugate, the first VL comprises the amino acid sequences of the complementarity determining regions (CDRs) -L1, -L2, and -L3 of the VL amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 59; and / or the first VH comprises the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 60.

[0026] In another embodiment of the antibody-drug conjugate, the first VL comprises:

[0027] (i) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 34, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 36, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 38; or,

[0028] (ii) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 35, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 37, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 38;

[0029] and / or,

[0030] the first VH comprises:

[0031] (i) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 39, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 43, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 47;

[0032] (ii) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 40, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 44, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 47;

[0033] (iii) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 42, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 46, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 47; or,

[0034] (iv) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 41, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 45, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 48.

[0035] In another embodiment of the antibody-drug conjugate, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 17 or 59, and / or the first VH comprises the amino acid sequence set forth in SEQ ID NO: 18 or 60; or

[0036] the first VL comprises the amino acid sequence set forth in SEQ ID NO: 17, and the first VH comprises the amino acid sequence set forth in SEQ ID NO: 18; or the first VL comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first VH comprises the amino acid sequence set forth in SEQ ID NO: 60.

[0037] 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 set forth 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 set forth in SEQ ID NO: 16.

[0038] In another embodiment of the antibody-drug conjugate, the second VL comprises:

[0039] (i) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 19, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 21, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23; or,

[0040] (ii) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 20, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 22, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23; or,

[0041] and / or,

[0042] the second VH comprises:

[0043] (i) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 24, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 28, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 32.

[0044] (ii) a CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 25, a CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 29, and a CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 32;

[0045] (iii) a CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 27, a CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 31, and a CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 32; or,

[0046] (iv) a CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 26, a CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 30, and a CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 33.

[0047] In another embodiment of the antibody-drug conjugate, the second VL comprises an amino acid sequence as set forth in SEQ ID NO: 15, and / or the second VH comprises an amino acid sequence as set forth in SEQ ID NO: 16.

[0048] In another embodiment of the antibody-drug conjugate, the first antigen binding domain is a Fab and the second antigen binding domain is a scFv.

[0049] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises a polypeptide chain I-A, a polypeptide chain I-B, and a polypeptide chain I-C; wherein the polypeptide chain I-A comprises the first VL and a light chain constant region, the polypeptide chain I-B comprises the first VH, a heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and the polypeptide chain I-C comprises the second VL, the second VH, and the second Fc domain monomer (the first Fc domain monomer).

[0050] In another embodiment of the antibody-drug conjugate, the polypeptide chain I-A comprises from N-terminus to C-terminus the first VL and a light chain constant region, the polypeptide chain I-B comprises from N-terminus to C-terminus the first VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and / or the polypeptide chain I-C comprises from N-terminus to 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).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] In another embodiment of the antibody-drug conjugate, the polypeptide chain IA comprises an amino acid sequence as shown in SEQ ID NO:1, the polypeptide chain IB comprises an amino acid sequence as shown in SEQ ID NO:2 or 9, and / or the polypeptide chain IC comprises an amino acid sequence as shown in SEQ ID NO:3 or 10.

[0055] In another embodiment of the antibody-drug conjugate, the first antigen-binding domain is scFv and the second antigen-binding domain is Fab.

[0056] 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 a second Fc domain monomer), and polypeptide chain II-C comprises the first VL, the first VH, and a second Fc domain monomer (or a first Fc domain monomer).

[0057] In another embodiment of the antibody-drug conjugate, the polypeptide chain II-A comprises from N- to C-terminal the second VL and light chain constant region, the polypeptide chain II-B comprises from N- to C-terminal the second VHand heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer), and / or the polypeptide chain II-C comprises from N- to C-terminal (i) the first VL, the first VHand the second Fc domain monomer (or the first Fc domain monomer), or (ii) the first VHand the first VL and the second Fc domain monomer (or the first Fc domain monomer).

[0058] In another embodiment of the antibody-drug conjugate, the adjacent domains of the polypeptide chain II-A are optionally connected by or without a linker, the adjacent domains of the polypeptide chain II-B are optionally connected by or without a linker, and / or the adjacent domains of the polypeptide chain II-C are optionally connected by or without a linker.

[0059] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., a rigid peptide linker or a flexible peptide linker); or the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., having the structure as shown in (GGGGS)n n wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NOs: 55-58 or 65-70); or the peptide linkers are each independently comprises an amino acid sequence as set forth in SEQ ID NOs: 55-58, 61, or 65-70.

[0060] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54.

[0061] In another embodiment of the antibody-drug conjugate, the polypeptide chain II-A comprises an amino acid sequence as set forth in SEQ ID NO: 4, the polypeptide chain II-B comprises an amino acid sequence as set forth in SEQ ID NO: 5 or 7, and / or the polypeptide chain II-C comprises an amino acid sequence as set forth in SEQ ID NO: 6 or 8.

[0062] In another embodiment of the antibody-drug conjugate, the first antigen binding domain is a Fab and the second antigen binding domain is a scFab.

[0063] 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 the polypeptide chain III-A comprises a first VL and a light chain constant region, the polypeptide chain III-B comprises a first VH, a heavy chain CH1 region, and a first Fc domain monomer (or the second Fc domain monomer), and / or the polypeptide chain III-C comprises a second VL, a light chain constant region, a second VH, a heavy chain CH1 region, and the second Fc domain monomer (or the first Fc domain monomer).

[0064] In another embodiment of the antibody-drug conjugate, the polypeptide chain III-A comprises, from N- to C-terminus, a first VL and a light chain constant region, the polypeptide chain III-B comprises, from N- to C-terminus, a first VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and / or the polypeptide chain III-C comprises, from N- to C-terminus, (i) the second VL, the light chain constant region, the second VH, the heavy chain CH1 region, and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the second VH, the heavy chain CH1 region, the second VL, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer).

[0065] In another embodiment of the antibody-drug conjugate, adjacent domains of the polypeptide chain III-A are optionally connected by or without a linker, adjacent domains of the polypeptide chain III-B are optionally connected by or without a linker, and / or adjacent domains of the polypeptide chain III-C are optionally connected by or without a linker.

[0066] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., a rigid peptide linker or a flexible peptide linker); or the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., having the structure set forth in (GGGGS) n wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NOs: 55-58 or 65-70); or the peptide linkers are each independently comprises an amino acid sequence set forth in SEQ ID NOs: 55-58, 61, or 65-70.

[0067] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54.

[0068] In another embodiment of the antibody-drug conjugate, the first antigen binding domain is a scFab and the second antigen binding domain is a Fab.

[0069] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises a polypeptide chain IV-A, a polypeptide chain IV-B, and a polypeptide chain IV-C; wherein the polypeptide chain IV-A comprises the second VL and a light chain constant region, the polypeptide chain IV-B comprises the second VHand a heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and / or the polypeptide chain IV-C comprises the first VL, a light chain constant region, the first VHand a heavy chain CH1 region, and the second Fc domain monomer (or the first Fc domain monomer).

[0070] In another embodiment of the antibody-drug conjugate, the polypeptide chain IV-A comprises, from N-terminal to C-terminal, the second VL and a light chain constant region, the polypeptide chain IV-B comprises, from N-terminal to C-terminal, the second VHand a heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), and / or the polypeptide chain IV-C comprises, from N-terminal to C-terminal, (i) the first VL, the light chain constant region, the first VHand the heavy chain CH1 region, and the second Fc domain monomer (or the first Fc domain monomer), or (ii) the first VHand 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).

[0071] In another embodiment of the antibody-drug conjugate, adjacent domains of the polypeptide chain IV-A are optionally connected by or without a linker, adjacent domains of the polypeptide chain IV-B are optionally connected by or without a linker, and / or adjacent domains of the polypeptide chain IV-C are optionally connected by or without a linker.

[0072] In another embodiment of the antibody-drug conjugate, the linker is each independently the same peptide linker or different peptide linkers (e.g., a rigid peptide linker or a flexible peptide linker); or the peptide linker is each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., having the structure of (GGGGS )n n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NOs: 55-58 or 65-70); or the peptide linker comprises the amino acid sequence set forth in SEQ ID NOs: 55-58, 61, or 65-70.

[0073] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54.

[0074] In another embodiment of the antibody-drug conjugate, the polypeptide chain IV-A comprises an amino acid sequence as set forth in SEQ ID NO: 4, the polypeptide chain IV-B comprises an amino acid sequence as set forth in SEQ ID NO: 7, and / or the polypeptide chain IV-C comprises an amino acid sequence as set forth in SEQ ID NO: 11.

[0075] In another embodiment of the antibody-drug conjugate, the first and second antigen binding domains are Fabs, and the Fab of the second antigen binding domain comprises a domain swap in the format of a CrossMab.

[0076] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises a polypeptide chain V-A, a polypeptide chain V-B, a polypeptide chain V-C, and a polypeptide chain V-D; wherein the polypeptide chain V-A comprises the first VL and a light chain constant region, the polypeptide chain V-B comprises the first VH, a heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), the polypeptide chain V-C comprises the second VH, a light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer), and / or the polypeptide chain V-D comprises the second VL and a heavy chain CH1 region.

[0077] In another embodiment of the antibody-drug conjugate, the polypeptide chain V-A comprises the first VL and the light chain constant region from N-terminus to C-terminus, the polypeptide chain V-B comprises the first VH, the heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer) from N-terminus to C-terminus, the polypeptide chain V-C comprises the second VH, the light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer) from N-terminus to C-terminus, and / or the polypeptide chain V-D comprises the second VL and a heavy chain CH1 region from N-terminus to C-terminus.

[0078] In another embodiment of the antibody-drug conjugate, adjacent domains of the polypeptide chain V-A are optionally connected with or without a linker, adjacent domains of the polypeptide chain V-B are optionally connected with or without a linker, adjacent domains of the polypeptide chain V-C are optionally connected with or without a linker, and / or adjacent domains of the polypeptide chain V-D are optionally connected with or without a linker.

[0079] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., a rigid peptide linker or a flexible peptide linker); or the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., having the structure shown in SEQ ID NOs: 55-58 or 65-70, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or the peptide linkers each independently comprise the amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 65-70. n In another embodiment of the antibody-drug conjugate, the peptide linker is a flexible peptide linker, e.g., having the structure shown in SEQ ID NO: 55, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0080] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0081] In another embodiment of the antibody-drug conjugate, the polypeptide chain V-A comprises the amino acid sequence set forth in SEQ ID NO: 1, the polypeptide chain V-B comprises the amino acid sequence set forth in SEQ ID NO: 9, the polypeptide chain V-C comprises the amino acid sequence set forth in SEQ ID NO: 13, and / or the polypeptide chain V-D comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0082] In another embodiment of the antibody-drug conjugate, the first and second antigen binding domains are Fabs, and the Fab of the first antigen binding domain comprises a domain swap in the format of a CrossMab.

[0083] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises a polypeptide chain VI-A, a polypeptide chain VI-B, a polypeptide chain VI-C, and a polypeptide chain VI-D; wherein the polypeptide chain VI-A comprises the second VL and a light chain constant region, the polypeptide chain VI-B comprises the second VH, a heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), the polypeptide chain VI-C comprises the first VH, a light chain constant region, and the second Fc domain monomer (or the first Fc domain monomer); and the polypeptide chain VI-D comprises the first VL and a heavy chain CH1 region.

[0084] In another embodiment of the antibody-drug conjugate, the polypeptide chain VI-A comprises from N- to C-terminus the second VL and the light chain constant region, the polypeptide chain VI-B comprises from N- to C-terminus the second VHand the heavy chain CH1 region and the first Fc domain monomer (or the second Fc domain monomer), the polypeptide chain VI-C comprises from N- to C-terminus the first VHand the light chain constant region and the second Fc domain monomer (or the first Fc domain monomer), and / or the polypeptide chain VI-D comprises from N- to C-terminus the first VL and heavy chain CH1 region.

[0085] In another embodiment of the antibody-drug conjugate, adjacent domains of the polypeptide chain VI-A are optionally connected by or without a linker, adjacent domains of the polypeptide chain VI-B are optionally connected by or without a linker, adjacent domains of the polypeptide chain VI-C are optionally connected by or without a linker, and / or adjacent domains of the polypeptide chain VI-D are optionally connected by or without a linker.

[0086] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., a rigid peptide linker or a flexible peptide linker); or the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., having the structure shown in (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NOs: 55-58 or 65-70); or the peptide linkers are each independently comprises an amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 65-70. n In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence shown in SEQ ID NO: 54.

[0087] In another embodiment of the antibody-drug conjugate, the light chain constant region comprises an amino acid sequence shown in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence shown in SEQ ID NO: 54.

[0088] In another embodiment of the antibody-drug conjugate, the first and the second antigen binding domain are each a scFv.

[0089] In another embodiment of the antibody-drug conjugate, the bispecific antibody comprises a polypeptide chain VII-A and a polypeptide chain VII-B; wherein the polypeptide chain VII-A comprises the first VL, the first VH, and the first Fc domain monomer (or the second Fc domain monomer), the polypeptide chain VII-B comprises the second VL, the second VH, and the second Fc domain monomer (or the first Fc domain monomer).

[0090] In another embodiment of the antibody-drug conjugate, the polypeptide chain VII-A comprises, from N-terminal to C-terminal, (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 N-terminal to C-terminal, (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).

[0091] In another embodiment of the antibody-drug conjugate, the adjacent domains of the polypeptide chain VII-A are optionally connected by or without a linker, and / or the adjacent domains of the polypeptide chain VII-B are optionally connected by or without a linker.

[0092] In another embodiment of the antibody-drug conjugate, the linkers are each independently the same peptide linker or different peptide linkers (e.g., a rigid peptide linker or a flexible peptide linker); or the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., having the structure shown in (GGGGS) n wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NOs: 55-58 or 65-70); or the peptide linkers are each independently comprises an amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 65-70.

[0093] In another embodiment of the antibody-drug conjugate, the peptide linkers are each independently comprises an amino acid sequence shown in SEQ ID NOs: 55-58, 61, or 65-70.

[0094] 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.

[0095] In another embodiment of the antibody-drug conjugate, the bispecific antibody or antigen binding fragment thereof comprises:

[0096] (1) polypeptide chain I-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, polypeptide chain I-B comprising an amino acid sequence as set forth in SEQ ID NO: 2, and, polypeptide chain I-C comprising an amino acid sequence as set forth in SEQ ID NO: 3;

[0097] (2) polypeptide chain I-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, polypeptide chain I-B comprising an amino acid sequence as set forth in SEQ ID NO: 9, and, polypeptide chain I-C comprising an amino acid sequence as set forth in SEQ ID NO: 10;

[0098] (3) polypeptide chain II-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, polypeptide chain II-B comprising an amino acid sequence as set forth in SEQ ID NO: 5, and, polypeptide chain II-C comprising an amino acid sequence as set forth in SEQ ID NO: 6;

[0099] (4) polypeptide chain II-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, polypeptide chain II-B comprising an amino acid sequence as set forth in SEQ ID NO: 7, and, polypeptide chain II-C comprising an amino acid sequence as set forth in SEQ ID NO: 8;

[0100] (5) polypeptide chain IV-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, polypeptide chain IV-B comprising an amino acid sequence as set forth in SEQ ID NO: 7, and, polypeptide chain IV-C comprising an amino acid sequence as set forth in SEQ ID NO: 11;

[0101] (6) polypeptide chain V-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, polypeptide chain V-B comprising an amino acid sequence as set forth in SEQ ID NO: 9, polypeptide chain V-C comprising an amino acid sequence as set forth in SEQ ID NO: 13, and, polypeptide chain V-D comprising an amino acid sequence as set forth in SEQ ID NO: 12; or,

[0102] (7) polypeptide chain VII-A comprising an amino acid sequence as set forth in SEQ ID NO: 14, and, polypeptide chain VII-B comprising an amino acid sequence as set forth in SEQ ID NO: 8.

[0103] In another embodiment of the antibody-drug conjugate, the antibody-drug conjugate possesses enhanced tumor inhibition relative to a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody; wherein the CDR amino acid sequences of the monospecific anti-c-MET antibody are identical to the CDR amino acid sequences of the first antigen binding domain, and the CDR amino acid sequences of the monospecific anti-EGFR antibody are identical to the CDR amino acid sequences of the second antigen binding domain.

[0104] In another embodiment of the antibody-drug conjugate, the tumor inhibition comprises inhibition of EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0105] In a further embodiment of the antibody-drug conjugate, M comprises

[0106] wherein ring A is a 5-6 membered aliphatic heterocyclic ring, or a 5-20 membered aromatic ring system, said aliphatic heterocyclic ring and aromatic ring system being optionally substituted with one or more groups independently selected from oxo (=0), halogen, cyano, amino, carboxyl, thiol, and C 1-6 alkyl; M1is selected from a single bond, C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene, or amine.

[0107] In a further embodiment of the antibody-drug conjugate, M comprises wherein ring A is a 5 membered aliphatic heterocyclic ring, a 6 membered heteroaromatic ring, or a polycyclic ring formed by one or more 6 membered aromatic heterocyclic rings and a benzene ring connected by a single bond, or a polycyclic ring formed by multiple 6 membered heteroaromatic rings connected by a single bond, said aliphatic heterocyclic ring being optionally substituted with one or more groups selected from oxo (=0), halogen, and C 1-4 alkyl; M1is selected from a single bond, C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene, or amine.

[0108] In a further embodiment of the antibody-drug conjugate, M is wherein ring A is selected from M1is selected from a single bond, C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene, or amine.

[0109] In a further embodiment of the antibody-drug conjugate, M is selected from

[0110] In further embodiments of the antibody-drug conjugate, M is

[0111] In further embodiments of the antibody-drug conjugate, M is selected from

[0112] In further embodiments of the antibody-drug conjugate, M is selected from

[0113] In further embodiments of the antibody-drug conjugate, L is selected from the structure consisting of one or more of: 1-6 alkylene, -N(R’)-, carbonyl, -0-, a natural amino acid or a non-natural amino acid selected from Ala, Arg, Asn, Asp, Cit, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2) s OCH3) and analogs thereof, and a short peptide comprising 1, 2, 3 or 4 amino acids,

[0114] wherein R’ represents hydrogen, C 1-6 alkyl or a polyethylene glycol fragment comprising 1 to 10 EO units; s is selected from an integer between 1 and 20.

[0115] In further embodiments of the antibody-drug conjugate, the short peptide is selected from the group consisting of 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)).

[0116] In further embodiments of the antibody-drug conjugate, the L is selected from the group consisting of structures comprising one or more of: 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-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)),

[0117] wherein s is selected from an integer from 1 to 20.

[0118] In further embodiments of the antibody-drug conjugate, L is selected from a structure comprising one or more of:

[0119]

[0120]

[0121] In further embodiments of the antibody-drug conjugate, L is selected from a structure comprising one or more of:

[0122]

[0123]

[0124] In further embodiments of the antibody-drug conjugate, L is selected from a structure comprising one or more of:

[0125]

[0126] In further embodiments of the antibody-drug conjugate, L is selected from a structure comprising one or more of:

[0127] In further embodiments of the antibody-drug conjugate, L is selected from a structure comprising one or more of:

[0128] In further embodiments of the antibody-drug conjugate, E is a single bond or selected from a structure comprising one or more of: In further embodiments of the antibody-drug conjugate, E is a single bond, -NHCH2-,

[0129] In further embodiments of the antibody-drug conjugate, E is -NHCH2- or In further embodiments of the antibody-drug conjugate, E is -NHCH2- or a single bond.

[0130] In further embodiments of the antibody-drug conjugate, E is -NHCH2-.

[0131] In further embodiments of the antibody-drug conjugate, E is a single bond.

[0132] In further embodiments of the antibody-drug conjugate, E is In further embodiments of the antibody-drug conjugate, selected from a structure comprising one or more of:

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] In further embodiments of the antibody-drug conjugate, selected from the following structures:

[0142]

[0143]

[0144] In further embodiments of the antibody-drug conjugate, the cytotoxic drug is selected from the group consisting of a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor.

[0145] In further embodiments of the antibody-drug conjugate, the tubulin inhibitor is an auristatin or a maytansinoid.

[0146] In further embodiments of the antibody-drug conjugate, the DNA intercalator is a pyrrolobenzodiazepine (PBD).

[0147] In further embodiments of the antibody-drug conjugate, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0148] In further embodiments of the antibody-drug conjugate, the topoisomerase I inhibitor is selected from the group consisting of camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, and pharmaceutically acceptable salts, esters, or analogs thereof; and the topoisomerase II inhibitor is selected from the group consisting of doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, and pharmaceutically acceptable salts, esters, or analogs thereof.

[0149] In further embodiments of the antibody-drug conjugate, the RNA polymerase inhibitor is a-amanitin and pharmaceutically acceptable salts, esters, or analogs thereof.

[0150] In further embodiments of the antibody-drug conjugate, the cytotoxic drug is selected from the group consisting of compounds of Formula I and Formula II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of the compounds of Formula I and Formula II:

[0151]

[0152] wherein R1and R2are each independently selected from C 1-6 alkyl and halogen;

[0153] R3is selected from H and -CO-CH2OH;

[0154] R4and R5are each independently selected from H, halogen, and hydroxyl; or R4and R5are joined to form a 5-6 membered oxygen-containing heterocycle;

[0155] R6is selected from hydrogen or -C 1-4 alkylene-NR a R b ; and

[0156] R7is 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 ; wherein R a , R b and R c are each independently selected at each occurrence from H, C 1-6 alkyl, -SO2-C 1-6 alkyl and -CO-C 1-6 alkyl; wherein optionally R a and R b together with the attached atoms form a 5-6 membered nitrogen-containing heterocycle.

[0157] In further embodiments of the antibody-drug conjugate, the cytotoxic drug is selected from the group consisting of the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of the compounds:

[0158]

[0159]

[0160] wherein the fragment corresponding to the cytotoxic drug after the cytotoxic drug is linked to the linker is D in the above formula.

[0161] In further embodiments of the antibody-drug conjugate, the cytotoxic drug is selected from the following compounds or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or prodrug of the compound:

[0162]

[0163] wherein the fragment corresponding to the cytotoxic drug after the cytotoxic drug is linked to the linker is D in the above formula.

[0164] In further embodiments of the antibody-drug conjugate, D is a monovalent structure resulting from the loss of one H from -OH, -NH2, or a secondary amine group on the cytotoxic drug.

[0165] In further embodiments of the antibody-drug conjugate, the antibody-drug conjugate is selected from the group consisting of: ADC A-01

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181] wherein Ab is a bispecific antibody or an antigen binding fragment thereof as defined above;

[0182] represents the specific linking mode of the thiol group of the cysteine residue in the bispecific antibody or the antigen binding fragment thereof to M in the antibody-drug conjugate; and

[0183] x represents the number of drug loads.

[0184] In further embodiments of the antibody-drug conjugate, the thiol group in the bispecific antibody or the antigen binding fragment thereof and M in the antibody-drug conjugate form the antibody-drug conjugate by addition reaction or substitution reaction to form a thioether bond.

[0185] In further embodiments of the antibody-drug conjugate, Ab is a bispecific antibody selected from the group consisting of BsAb 07B, BsAb 10B, BsAb 38B, BsAb 41B, BsAb 49B, BsAb 55B, and BsAb 56B.

[0186] The present application also provides an antibody-drug conjugate selected from the group consisting of 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.

[0187] The present application also provides a composition comprising one or more antibody-drug conjugates described herein. In further embodiments of the composition, the DAR value (drug antibody coupling ratio) of the composition is 1-10. In further embodiments 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 further embodiments of the composition, the DAR value of the composition is 3-9.

[0188] In further embodiments of the composition, the DAR value of the composition is from 4 to 8. In further embodiments of the composition, the DAR value of the composition is from 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 6.5, 3.5 to 7.0, 3.5 to 7.5, 3.5 to 8.0, 4.0 to 4.5, 4.0 to 5.0, 4.0 to 5.5, 4.0 to 6.0, 4.0 to 6.5, 4.0 to 7.0, 4.0 to 7.5, 4.0 to 8.0, 4.5 to 5.0, 4.5 to 5.5, 4.5 to 6.0, 4.5 to 6.5, 4.5 to 7.0, 4.5 to 7.5, 4.5 to 8.0, 5.0 to 5.5, 5.0 to 6.0, 5.0 to 6.5, 5.0 to 7.0, 5.0 to 7.5, 5.0 to 8.0, 5.5 to 6.0, 5.5 to 6.5, 5.5 to 7.0, 5.5 to 7.5, 5.5 to 8.0, 6.0 to 6.5, 6.0 to 7.0, 6.0 to 7.5, 6.0 to 8.5, 6.5 to 7.0, 6.5 to 7.5, 6.5 to 8.5, 7.0 to 7.5, 7.0 to 9.0, or 7.5 to 9.0.

[0189] The present application further provides a pharmaceutical composition comprising one or more antibody-drug conjugates disclosed herein or a composition 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 an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-MET or VEGF inhibitor, a chemotherapeutic drug, and any combination thereof.

[0190] The present application further provides 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, the first therapeutic agent is selected from one or more antibody-drug conjugates or compositions of any of the preceding embodiments; and the second therapeutic agent is selected from an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-MET or VEGF inhibitor, a chemotherapeutic drug, or any combination thereof.

[0191] The present application further provides use of the antibody-drug conjugate, the composition, the pharmaceutical composition or the pharmaceutical combination as described above in the preparation of a medicament for preventing, treating and / or as an adjuvant therapy in a subject for a disease associated with c-MET and / or EGFR, and / or for inhibiting the activity of c-MET and / or EGFR in vitro or in a subject; wherein the disease associated with c-MET and / or EGFR is associated with EGFR activating mutation, EGFR gene amplification, elevated circulating HGF level, c-MET activating mutation and / or c-MET gene amplification, and optionally, the disease associated with c-MET and / or EGFR comprises cancer.

[0192] In further embodiments of use, the cancer is selected from the group consisting of an 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, pharynx cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular cancer, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.

[0193] In further embodiments of use, the antibody-drug conjugate, or the pharmaceutical composition is administered in combination, e.g. simultaneously, separately or sequentially, with another pharmaceutically active agent selected from the group consisting of an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-MET or VEGF inhibitor, a chemotherapeutic drug or any combination thereof.

[0194] The present application further provides a method of inhibiting the activity of c-MET and / or EGFR in a cell, comprising contacting the cell with the antibody-drug conjugate, the composition, the pharmaceutical composition or the pharmaceutical combination as described in any one of the preceding embodiments; wherein the cell is a cell expressing c-MET and / or EGFR.

[0195] The present application further provides a method for preventing, treating and / or as an adjuvant therapy in a subject for a disease associated with c-MET and / or EGFR, comprising administering to a subject in need thereof an effective amount of the antibody-drug conjugate, the composition, the pharmaceutical composition or the pharmaceutical combination as described in any one of the preceding embodiments; wherein the disease associated with c-MET and / or EGFR comprises EGFR activating mutation, EGFR gene amplification, elevated circulating HGF level, c-MET activating mutation and / or c-MET gene amplification, and optionally, the disease associated with c-MET and / or EGFR comprises cancer.

[0196] In further method embodiments, the cancer is selected from the group consisting of an 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, pharynx cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular cancer, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0197] In further method embodiments, the method further comprises administering to the subject a second therapy 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; optionally, the second therapy is administered simultaneously, separately, or sequentially.

[0198] The present application further provides the antibody-drug conjugate, composition, pharmaceutical composition, or drug combination of any one of the preceding, for use in preventing, treating, and / or as an adjuvant therapy for a disease associated with c-MET and / or EGFR in a subject, wherein the disease associated with c-MET and / or EGFR comprises an EGFR activating mutation, an EGFR gene amplification, an elevated circulating HGF level, a disease with c-MET activating mutation, and / or a c-MET gene amplification, optionally, the disease associated with c-MET and / or EGFR is a cancer. In further embodiments, the cancer is selected from the group consisting of an 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, pharynx cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular cancer, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer. In further embodiments, the method further comprises administering to the subject a second therapy 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; optionally, the second therapy is administered simultaneously, separately, or sequentially. BRIEF DESCRIPTION OF DRAWINGS

[0199] Figure 1 A: Fab-scFv-Fc (KIH) bispecific antibody structure schematic.

[0200] Figure 1 B: Fab-scFab-Fc (KIH) bispecific antibody structure schematic.

[0201] Figure 1 C: Crossmab VH-VL Bispecific antibody structure schematic.

[0202] Figure 1 D: Schematic diagram of scFv-Fc(KIH) bispecific antibody structure.

[0203] Figure 2 : Pharmacodynamic detection of different antibody conjugated drugs on MKN45 model.

[0204] Figure 3 : Pharmacodynamic detection of different antibody conjugated drugs on MKN45-EGFR model.

[0205] Figure 4 : Pharmacodynamic detection of different antibody conjugated drugs on MKN45 model.

[0206] Figure 5 : Pharmacodynamic detection of different antibody conjugated drugs on HCC827 model.

[0207] Figure 6 : Pharmacodynamic detection of different antibody conjugated drugs on EBC-1-EGFR model.

[0208] Figure 7 : Pharmacodynamic detection of different antibody conjugated drugs on HCC827 model.

[0209] Figure 8 : Pharmacodynamic detection of different antibody conjugated drugs on PC-9 model.

[0210] Figure 9 : Bystander effect detection of different antibody conjugated drugs. DETAILED DESCRIPTION

[0211] Terminology Definition

[0212] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. And the cell culture, biochemical, nucleic acid chemistry, immunology laboratory and other operation steps used in the present disclosure are all conventional steps widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0213] As used herein, the term "antibody" is used in the broadest sense, and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can be comprised of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be assigned to a kind of kappa (kappa) and lambda (lambda) light chain. The heavy chains can be assigned to mu, delta, gamma, alpha, or epsilon, and define a class of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains are not involved directly in binding of an antibody to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antigen binding site. Assignment of amino acids to each region or domain can follow the definition of 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.

[0214] Herein, unless the context clearly indicates otherwise, the term "antibody" includes not only intact antibodies, but also antigen binding fragments of antibodies.

[0215] The term "antibody" also includes embodiments in which the heavy chain constant region comprises 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 variable region of the antibody has cyclized to a pyroglutamate. Thus, in a composition comprising antibodies disclosed herein, the various antibodies therein can independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid or an N-terminal amino acid cyclized to a pyroglutamate.

[0216] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, e.g., according to the definitions in the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), the MacCallum numbering system (MacCallum et al., (1996) J Mol Biol 262:732-745, see also Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Duibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)), the AHo numbering system (Honegger and Pluckthun, A., J. Mol. Biol. 309:657-670 (2001)), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill in the art will readily be able to identify the CDRs defined by each numbering system. And, the correspondence between different numbering systems is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0217] 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.

[0218] The following general rules (published at www.bioinf.org.uk: Professor Andrew C. 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.

[0219]

[0220]

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] As used herein, the term “bispecific antibody” or “BsAb” refers to an antibody having binding specificity for two different antigens (or epitopes), which comprises antigen binding domains having binding specificity for different antigens (or epitopes), e.g., two antigen binding domains having binding specificity for different antigens (or epitopes), enabling binding to two different binding sites and / or target molecules. The individual antigen binding domains of a bispecific antibody can each independently be selected from a full-length antibody (e.g., an IgG antibody) or an antigen binding fragment thereof (e.g., Fv, Fab, scFab, or scFv). In some cases, the individual antigen binding domains are connected by a peptide linker.

[0226] As used herein, the term “Fv fragment” means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. An Fv fragment is generally considered to be the smallest antibody fragment that is capable of forming a complete antigen binding site. It is believed that the six CDRs confer the antigen binding specificity of an antibody. However, even a single variable domain (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although it may

[0227] As used herein, the term “Fc fragment” means an antibody fragment formed by disulfide bond linkage of the second, third constant regions of the first heavy chain with the second, third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.

[0228] As used herein, the term “scFv” refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art peptide linkers consist of repeating GGGGS amino acid sequences (SEQ ID NO: 57) or variants thereof, e.g., the amino acid sequence (GGGGS)4(SEQ ID NO: 58), although variants thereof can also be used. In some cases, a disulfide bond can also be present between the VH and VL of a scFv.

[0229] As used herein, the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains, which typically consists of one peptide chain comprising VL and CL and another peptide chain comprising VH and CH1, however, the skilled person understands that the Fab domain can be arranged according to the above-mentioned natural orientation, but can also comprise domain substitutions or swaps that promote correct VH and VL pairing (e.g. in the form of Crossmab). The term "scFab" refers to a single polypeptide chain comprising VL, VH, CL and CH1 domains, wherein each adjacent domain is optionally connected by a linker, in a typical structure, the scFab contains a single polypeptide chain comprising from N- to C-terminus: (1) VL, CL, VH and CH1, wherein CL and VH are typically connected by a peptide linker (e.g. a flexible peptide linker), or (1) VH, CH1, VL and CL, wherein CH1 and VL are typically connected by a peptide linker (e.g. a flexible peptide linker).

[0230] As used herein, the terms "monoclonal antibody", "monoclonal", "mAb" have the same meaning and are used interchangeably to refer to an antibody or fragment thereof from a population of highly homogenous antibody molecules, i.e. a population of antibody molecules that are identical except for naturally occurring mutations that can arise during production. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are in contrast to monoclonal antibodies and typically refer to antibodies or fragments thereof from a population of antibodies comprising at least 2 or more different antibodies, which typically recognize different epitopes on the antigen. Furthermore, the modifier "monoclonal" is intended to refer to the character of the antibody as being derived from a population of identical antibodies, not to the method by which it is made.

[0231] As used herein, the term "CrossMab" refers to a method of constructing bispecific antibodies by swapping the heavy and light chain domains within the antigen-binding fragments (Fabs) of one half of the bispecific antibody, so that the light chains and their cognate heavy chains are correctly paired. This "swap" preserves the affinity of antigen binding but makes the two arms so different that light chain mispairing no longer occurs. The three possible "CrossMab" formats are: CrossMab Fab , which refers to a cross or swap of all VH-CH1 and VL-CL domain positions of one half of the bispecific antibody; CrossMab VH-VL , which refers to a cross or swap of only VH and VL domain positions of one half of the bispecific antibody; and CrossMab CH1-CLCrossMab, CrossMAb, CrossMab®: refers to the crossover or exchange of CH1 and CL domain positions within the Fab region of a half of a bispecific antibody. CrossMab antibodies have been described or claimed in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010136172, WO2010145792, and WO2013026831. The term "CrossMab" antibody is art-recognized; see, e.g., 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); Klein et al., MAbs 8(6):1010-1020 (2016); and Klein et al., MAbs 4(6):653-663 (2012).

[0232] As used herein, the term "specifically binds" refers to a nonrandom binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) or half maximal effective concentration (EC50) of the interaction. 50

[0233] The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of formation and dissociation of antigen binding site / antigen complex. Both the "association rate constant" (k a or k on ) and the "dissociation rate constant" (k dis or k off ) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k dis / k on equals the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). KD, k on and k dis values can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using bioluminescence interferometry (e.g., ForteBio Octet method). In addition to this, the dissociation constant can be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0234] ​As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is said to be an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, and the resulting integrants have the genetic material of the vector reproduced along with the genetic material of the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector can contain a variety of control elements to control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can contain a replication origin.

[0235] Expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is the origin of replication, or autonomously replicating sequence, which permits the vector to replicate independent of the host chromosomal DNA. The term "expression vector" as used herein refers to a vector that comprises a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors comprise sufficient cis-acting elements for expression; additional elements can be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art such as cosmids, plasmids (e.g., naked or contained within liposomes), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0236] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, NS0 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.

[0237] As used herein, the term "identity" is used in the context of a match between sequences of two polypeptides or two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is the number of matching positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Generally, the comparison is made over the full length of the sequences being compared. Such a comparison can be conveniently performed by means of the algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443-453, implemented as the Align program (DNAstar, Inc.), using, for example. The percentage of 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)) as integrated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined using the Needleman algorithm as implemented in the GAP program, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0238] The nomenclature used to designate the twenty conventional amino acids in accordance with the present disclosure follows conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present disclosure, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present disclosure, amino acids are generally represented by their one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0239] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, in pharmacology and / or physiology, with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Stabilizers have the meaning commonly understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium 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), and the like.

[0240] As used herein, the term "DAR" or "drug-to-antibody ratio" or "drug-to-antibody coupling ratio" (as used interchangeably herein) refers to the average number of linker / payload moieties attached to each antibody present in a composition. For compositions comprising an ADC of the disclosure, the DAR of the composition is the average of all DARs (linker-payload moieties present in all ADC molecules in the composition), which can be expressed as a decimal or an integer. As such, in some embodiments of compositions comprising an ADC of the disclosure, the DAR of the composition is an integer or a decimal of 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 comprising an ADC of the disclosure, the DAR of the composition is an integer or a decimal of 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. As used above, the term "composition" is understood to include a pharmaceutical composition. Average DAR can be determined by a variety of conventional methods, such as UV spectroscopy, mass spectrometry, ELISA, radioassay, hydrophobic interaction chromatography (HIC), electrophoresis, and HPLC.

[0241] As used herein, the term "prevent" refers to an approach taken to stop or delay the occurrence of a disease or disorder or a symptom (e.g., a tumor) in a subject. As used herein, the term "treat" refers to an approach taken to achieve a beneficial or desired clinical result. For the purposes of the present disclosure, a beneficial or desired clinical result includes, but is not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0242] As used herein, the term "subject" refers to a mammal, such as a primate, e.g., a human. In certain embodiments, the subject (e.g., human) has a tumor, or is at risk of having a disease described herein.

[0243] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount refers to an amount that is sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); a therapeutically effective amount refers to an amount that is sufficient to cure, or at least partially arrest, a disease and its complications. Determining such an effective amount is well within the capability of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., the age, weight, and sex of the patient, the mode of administration of the drug, and other therapies that the patient may be undergoing, etc.

[0244] As used herein, the term "effector function" refers to those biological activities attributable to an antibody Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with the antibody 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 cellular phagocytosis (ADCP), binding of cell surface receptors (e.g., B cell receptor), B cell activation, cytokine secretion, half-life / clearance of antibodies and antigen-antibody complexes, etc. Methods of altering the effector function of an antibody are known in the art, e.g., by introducing mutations into the Fc region.

[0245] 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 certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, or macrophages) to enable these cytotoxic effector cells to bind to target cells that display antigen and subsequently kill the target cells through secretion of cytotoxins.

[0246] In the present context, combination therapy includes the use of the bispecific antibody or pharmaceutical composition of the present disclosure in combination with one or more additional active therapeutic agents (e.g., chemotherapeutic agents) or other prophylactic or therapeutic modalities (e.g., radiotherapy) of a second therapy.

[0247] In such combination therapies, the various active agents often have different mechanisms of complementary action. The combination therapy can result in a synergistic effect. Combination therapies include agents that affect the immune response (e.g., enhance or activate the response) and 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 can allow for a reduction in the dose of one or more of the agents to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies can have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder, or condition.

[0248] As used herein, "combination" includes therapies that can be administered separately, e.g., separately formulated (e.g., can be provided in a kit), and therapies that can be administered together as a single formulation (i.e., "co-formulation"). In certain embodiments, the bispecific antibody of the present disclosure can be administered sequentially. In other embodiments, the bispecific antibody can be administered simultaneously. The bispecific antibody of the present disclosure can be used in combination with at least one other (active) agent in any manner.

[0249] 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. Unregulated cell division can lead to the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.

[0250] The term "alkyl" denotes a straight or branched hydrocarbon group derived from a straight or branched alkane (e.g., "C 1-20 The term "alkyl" denotes a straight or branched hydrocarbon group derived from a straight or branched alkane (e.g., "C 1-10 The term "alkyl" denotes a straight or branched hydrocarbon group derived from a straight or branched alkane (e.g., "C 1-6 The term "alkyl" denotes a straight or branched hydrocarbon group derived from a straight or branched alkane (e.g., "C 1-4 The term "alkyl" denotes a straight or branched hydrocarbon group derived from a straight or branched alkane (e.g., "C 1-3Examples, without limitation, include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, i-hexyl, 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, and the like.

[0251] The term "alkylene" denotes a straight or branched chain hydrocarbon group of loss of two hydrogen atoms from an alkyl group, for example, "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", and the like. Examples, without limitation, include methylene, ethylene, 1,3- propylene, 1,4-butylene, 1,5-pentylene, or 1,6-hexylene, and the like.

[0252] The term "alkenylene" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond, which is a divalent group of loss of two hydrogen atoms from an alkenyl group, including, for example, "C 2-20 alkenylene", "C 3-10 alkenylene", "C 5-8 alkenylene", and the like. Examples, without limitation, include ethenylene, 1- propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butenylene, 1-pentenylene, 2- pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2- hexenylene, 3-hexenylene, 1,4-hexadienylene, and the like.

[0253] The term "alkynylene" refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond, which is a divalent group of loss of two hydrogen atoms from an alkynyl group. Examples, without limitation, include "C 2-20 alkynylene", "C 3-10 alkynylene", "C 5-8 alkynylene", and the like. Examples, without limitation, include ethynylene, 1- propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2- pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2- hexynylene, 3-hexynylene, 1,4-hexadiynylene, and the like.

[0254] The term "aliphatic heterocycle" refers to a saturated or partially saturated 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 aliphatic heterocycles, 5-6 membered nitrogen-containing aliphatic heterocycles, 5-6 membered oxygen-containing aliphatic heterocycles, and the like, such as tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, and the like.

[0255] The term "aromatic heterocycle" 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, 5-6 membered oxygen-containing aromatic heterocycles, and the like, 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-tetrazine, and the like.

[0256] The term "aromatic ring system" refers to a monocyclic or polycyclic system comprising at least one aromatic ring (e.g., benzene ring, etc.) or heteroaromatic ring (e.g., pyrimidine ring, etc.), two or more aromatic and / or heteroaromatic rings can form a fused ring or be connected by a single bond (e.g., bispyrimidylphenyl, etc.), the aromatic ring system can be divalent or higher valence (e.g., trivalent or tetravalent), such as a 5-20 membered aromatic ring system.

[0257] abbreviation

[0258] The abbreviations used herein have the following meanings:

[0259]

[0260]

[0261]

[0262]

[0263] Antibody-drug conjugate

[0264] In one aspect, the present application provides an antibody-drug conjugate having the structure of Formula Ab-[M-L-E-D] x wherein:

[0265] Ab is a bispecific antibody or antigen-binding fragment thereof comprising a first antigen binding domain that specifically binds c-MET and a second antigen binding domain that specifically binds EGFR;

[0266] M is a linker moiety attached to the bispecific antibody or antigen-binding fragment thereof;

[0267] L is a structural fragment connecting the linker moiety M and E;

[0268] E is a structural fragment connecting L and D;

[0269] D is a cytotoxic drug or a fragment thereof; and

[0270] x is selected from any integer from 1 to 10.

[0271] Antibody of the ADC

[0272] In particular embodiments, the bispecific antibody of the ADC is a bispecific antibody with high affinity to both EGFR and c-MET, which is capable of specifically recognizing / binding to EGFR and c-MET, can block the binding of EGF to EGFR and / or the binding of HGF to c-MET, and thus can simultaneously block the downstream signaling pathways of EGFR and / or c-MET, leading to cell proliferation inhibition.

[0273] In some embodiments, the first antigen binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), which together form a domain capable of specifically binding to c-MET; and the second antigen binding domain comprises a second VL and a second VH, which together form a domain capable of specifically binding to EGFR.

[0274] In certain embodiments, the first and second antigen binding domains are each independently selected from scFv, Fab and scFab.

[0275] Unless explicitly stated otherwise, a Fab domain can be arranged according to its native orientation or can comprise a domain substitution or swap (e.g. in the form of a CrossMab) to promote correct VH and VL pairing.

[0276] CrossMab is a method to prevent light chain mispairing with heavy chains, see US patent US9266967B2, which is incorporated herein by reference in its entirety. The method is mainly based on the principle that the same part of an antibody repels each other, i.e. VH repels VH, CH1 repels CH1, CL repels CL, VL repels VL, by regionally swapping the heavy and light chains of one of the Fabs in the bispecific antibody (e.g. swapping light chain CL-VL with heavy chain CH1-VH, swapping VL and VH or swapping CH and CL) to achieve the purpose of preventing light chain mispairing.

[0277] In certain embodiments, the domain swap in the form of CrossMab is selected from:

[0278] CrossMab Fab : in the Fab, the antibody light chain CL-VL is exchanged with the antibody heavy chain CH1-VH;

[0279] CrossMab VH-VL : in the Fab, the VL and VH of the antibody are exchanged; and

[0280] CrossMab CH1-CL : in the Fab, the CH and CL of the antibody are exchanged.

[0281] In certain embodiments, the bispecific antibody further comprises an Fc domain. In certain embodiments, the Fc domain is an Fc domain dimer comprising a first Fc domain monomer and a second Fc domain monomer. In certain embodiments, the first and second Fc domain monomers each independently comprise one or more amino acid modifications that are capable of promoting heterodimerization of the first and second Fc domain monomers relative to homodimerization.

[0282] In certain embodiments, the Fc domain comprises a first Fc domain monomer comprising an amino acid modification capable of forming a knob structure and a second Fc domain monomer comprising an amino acid modification capable of forming a hole structure, wherein the hole structure is capable of pairing with the knob structure to form a heterodimerized Fc domain (Fc heterodimer).

[0283] As will be readily understood by one skilled in the art, the hole structure is capable of pairing with the knob structure to form a "knob in hole structure (KIH)." A "knob in hole structure" is a method for reducing heavy chain mispairing in a bispecific antibody by mutating amino acid modifications capable of forming a "knob" structure 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 entireties of which are hereby incorporated by reference), which is capable of effectively reducing heavy chain mispairing in a bispecific antibody due to the mutual attraction between the "knob" structure and the "hole" structure and the mutual repulsion between the "knob" structure and the "knob" structure.

[0284] WO9850431 discloses amino acid modifications that promote heterodimer formation, which discloses a KIH method for producing monomeric heterodimers of Fc domains. In the KIH method described, one Fc domain monomer of a heterodimer pair comprises an amino acid substitution that creates a protuberance (knob) extending outward from the surface of the Fc domain monomer that is adapted to a recess (hole) created by an appropriate amino acid substitution in the other Fc domain monomer of the heterodimer pair, which promotes formation of the heterodimer over homodimer formation. Examples of amino acid substitutions include S354C:T366W amino acid substitutions in a first Fc domain monomer to form a knob, including Y349C:T366S:L368A:Y407V amino acid substitutions in a second Fc domain monomer to form a hole (amino acids numbered according to the EU index), 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 comprising a K409W amino acid substitution to form a knob and a second Fc domain monomer comprising D399V and F405T amino acid substitutions to form a hole (amino acid numbering 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 numbering according to the EU index), wherein the first and second Fc domain monomers form a heterodimer pair.

[0285] In certain embodiments, the Fc domain monomers are derived from, and modified based on, an Fc domain of a human immunoglobulin to obtain Fc domain monomers capable of forming a knob structure or a hole structure. For example, the amino acid sequence of a first Fc domain monomer is modified in a particular region thereof to provide a knob 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 the knob structure on the first Fc domain monomer, thereby allowing the first and second Fc domain monomers to form a heterodimer pair while preventing the first and second Fc domain monomers from forming a homodimer pair. In certain embodiments, the human immunoglobulin is an IgGl, IgG2, IgG3, or IgG4. In certain embodiments, the Fc domain monomers comprise one or more modifications to form a knob structure, wherein the one or more modifications comprise S354C and / or T366W amino acid substitutions, wherein numbering is according to the Eu numbering scheme. In certain embodiments, the Fc domain monomers comprise S354 and T366W amino acid substitutions, wherein numbering is according to the Eu numbering scheme. In some embodiments, the Fc domain monomers comprise one or more modifications to form a hole structure, wherein the one or more modifications comprise Y349C, T366S, L368A, and / or Y407V amino acid substitutions, wherein numbering is according to the Eu numbering scheme. In certain embodiments, the Fc domain monomers comprise Y349C, T366S, L368A, and Y407V amino acid substitutions, wherein numbering is according to the Eu numbering scheme.

[0286] In certain embodiments, the Fc domain further has altered effector function (e.g., enhanced ADCC activity) compared to a wild-type Fc region. The altered effector function can be introduced by, e.g., mutation or chemical modification of the Fc domain monomers in the Fc heterodimer.

[0287] In further embodiments, the first and second Fc domain monomers further comprise one or more amino acid substitutions that reduce or eliminate Fc region effector functions. In certain particular embodiments, the first and second Fc domain monomers further comprise: (i) E233A and L235A (EALA) amino acid substitutions, wherein numbering is according to the Eu numbering scheme; (ii) L234A and L235A (LALA) amino acid substitutions, wherein numbering is according to the Eu numbering scheme; (iii) L234A L235A D265S (LALADS) amino acid substitutions, wherein numbering is according to the Eu numbering scheme; (iv) L234A L235A P329G (LALAPG) amino acid substitutions, wherein numbering is according to the Eu numbering scheme; (v) L235E (LE) amino acid substitution, wherein numbering is according to the Eu numbering scheme; (vi) D265A (DS) amino acid substitution, wherein numbering is according to the Eu numbering scheme; (vii) D265A N297G (DANG) amino acid substitution, wherein numbering is according to the Eu numbering scheme; (viii) N297X amino acid substitution, wherein X is any amino acid other than N, wherein numbering is according to the Eu numbering scheme; or (ix) N297A / D356E / L358M (NADELM) amino acid substitutions, wherein numbering is according to the Eu numbering scheme; or (x) D356E L358M (DELM) amino acid substitutions, wherein numbering is according to the Eu numbering scheme.

[0288] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth as SEQ ID NO: 49 or 51, and the second Fc domain monomer comprises the amino acid sequence set forth as SEQ ID NO: 50 or 52.

[0289] In certain embodiments, the first and second antigen binding domains are each linked to one of the first and second Fc domain monomers of the Fc heterodimer.

[0290] In certain 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.

[0291] In certain 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.

[0292] The individual CDRs of the antigen binding domain of the bispecific antibody of the ADC provided herein can be determined according to any CDR numbering scheme known in the art.

[0293] In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a light chain CDR 1 (CDR-L1), CDR-L2, and / or CDR-L3 of the VL amino acid sequence of SEQ ID NO: 15, 17, and 59 as determined by the Kabat numbering scheme. In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a heavy chain CDR 1 (CDR-H1), CDR-H2, and / or CDR-H3 of the VH amino acid sequence of SEQ ID NO: 16, 18, and 60 as determined by the Kabat numbering scheme.

[0294] In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-L1, CDR-L2, and CDR-L3 of the VL amino acid sequence set forth in any one of SEQ ID NO: 15, 17, and 59 as determined by the Chothia numbering scheme. In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-H1, CDR-H2, and / or CDR-H3 of the VH amino acid sequence set forth in any one of SEQ ID NO: 16, 18, and 60 as determined by the Chothia numbering scheme.

[0295] In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-L1, CDR-L2, and / or CDR-L3 of the VL amino acid sequence set forth in any one of SEQ ID NO: 15, 17, and 59 as determined by the MacCallum numbering scheme. In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-H1, CDR-H2, and / or CDR-H3 of the VH amino acid sequence set forth in any one of SEQ ID NO: 16, 18, and 60 as determined by the MacCallum numbering scheme.

[0296] In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-L1, CDR-L2, and / or CDR-L3 of the VL amino acid sequence set forth in any one of SEQ ID NO: 15, 17, and 59 as determined by the IMGT numbering scheme. In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-H1, CDR-H2, and / or CDR-H3 of the VH amino acid sequence set forth in any one of SEQ ID NO: 16, 18, and 60 as determined by the IMGT numbering scheme.

[0297] In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-L1, CDR-L2, and / or CDR-L3 of a VL amino acid sequence as set forth in any of SEQ ID NOs: 15, 17, and 59, as determined by the AbM numbering scheme. In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-H1, CDR-H2, and / or CDR-H3 of a VH amino acid sequence as set forth in any of SEQ ID NOs: 16, 18, and 60, as determined by the AbM numbering scheme.

[0298] In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-L1, CDR-L2, and / or CDR-L3 of a VL amino acid sequence as set forth in any of SEQ ID NOs: 15, 17, and 59, as determined by the AHo numbering scheme. In certain embodiments, the antigen binding domain of the bispecific antibody of the ADC provided herein comprises a CDR-H1, CDR-H2, and / or CDR-H3 of a VH amino acid sequence as set forth in any of SEQ ID NOs: 16, 18, and 60, as determined by the AHo numbering scheme.

[0299] In certain embodiments, each CDR of the antigen binding domain of the bispecific antibody of the ADC provided herein is each independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering scheme, or by structural analysis of the antigen binding domain, wherein the structural analysis identifies residues in the variable region expected to contact the epitope region of c-MET or EGFR.

[0300] In certain embodiments, the present disclosure provides an ADC comprising a bispecific antibody comprising a first antigen binding domain that specifically binds c-MET and a second antigen binding domain that specifically binds EGFR, wherein the first antigen binding domain comprises a VL comprising CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 17 or 59, and a VH comprising CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 18 or 60, and / or wherein the second antigen binding domain comprises a VL comprising CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 16. Wherein each CDR is each independently identified according to one of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering scheme, respectively, or by structural analysis of the antigen binding domain, wherein structural analysis identifies residues in the variable region expected to contact the epitope region of c-MET or EGFR.

[0301] In certain embodiments, the present disclosure provides an ADC comprising a bispecific antibody comprising a first antigen binding domain that specifically binds c-MET and a second antigen binding domain that specifically binds EGFR, wherein the first antigen binding domain comprises a VL comprising CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 17 or 59, and a VH comprising CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 18 or 60, and / or wherein the second antigen binding domain comprises a VL comprising CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 15, and a VH comprising CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 16. Wherein each CDR is each independently identified according to one of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering scheme, respectively, or by structural analysis of the antigen binding domain, wherein structural analysis identifies residues in the variable region expected to contact the epitope region of c-MET or EGFR.

[0302] In certain embodiments, the present disclosure provides an ADC comprising a bispecific antibody comprising a first antigen binding domain that specifically binds c-MET and a second antigen binding domain that specifically binds EGFR, wherein the first antigen binding domain comprises a VL of the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 59, and a VH of the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 60, and / or the second antigen binding domain comprises a VL of the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 15, and a VH of the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 16. Wherein each CDR is each independently identified according to one of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering scheme, respectively, or by structural analysis of the antigen binding domain, wherein structural analysis identifies residues in the variable region expected to contact the epitope region of c-MET or EGFR.

[0303] In certain embodiments, the present disclosure provides an ADC comprising a bispecific antibody comprising a first antigen binding domain that specifically binds c-MET and a second antigen binding domain that specifically binds EGFR, wherein the first antigen binding domain comprises a VL of the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 17, and a VH of the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 60, and / or the second antigen binding domain comprises a VL of the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 15, and a VH of the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 16. Wherein each CDR is each independently identified according to one of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering scheme, respectively, or by structural analysis of the antigen binding domain, wherein structural analysis identifies residues in the variable region expected to contact the epitope region of c-MET or EGFR.

[0304] In certain embodiments, the present disclosure provides an ADC comprising a bispecific antibody comprising a first antigen binding domain that specifically binds c-MET and a second antigen binding domain that specifically binds EGFR, wherein the first antigen binding domain comprises a VL of the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 59, and a VH of the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 18, and / or the second antigen binding domain comprises a VL of the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 15, and a VH of the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 16. Wherein each CDR is each independently identified according to one of Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering scheme, respectively, or by structural analysis of the antigen binding domain, wherein structural analysis identifies residues in the variable region that are expected to contact the epitope region of c-MET or EGFR.

[0305] In certain embodiments of the ADC provided herein, the first VL of the bispecific antibody of the ADC comprises:

[0306] a CDR-L1 comprising an amino acid sequence set forth in SEQ ID NO: 34, a CDR-L2 comprising an amino acid sequence set forth in SEQ ID NO: 36, and a CDR-L3 comprising an amino acid sequence set forth in SEQ ID NO: 38; wherein the CDRs are defined by the Chothia numbering system;

[0307] a CDR-L1 comprising an amino acid sequence set forth in SEQ ID NO: 34, a CDR-L2 comprising an amino acid sequence set forth in SEQ ID NO: 36, and a CDR-L3 comprising an amino acid sequence set forth in SEQ ID NO: 38; wherein the CDRs are defined by the Chothia numbering system;

[0308] a CDR-L1 comprising an amino acid sequence set forth in SEQ ID NO: 34, a CDR-L2 comprising an amino acid sequence set forth in SEQ ID NO: 36, and a CDR-L3 comprising an amino acid sequence set forth in SEQ ID NO: 38; wherein the CDRs are defined by the Abm numbering system; or

[0309] CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 35, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 37, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 38; wherein the CDRs are defined by the IMGT numbering system;

[0310] and / or

[0311] the first VH comprises:

[0312] CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 39, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 43, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 47; wherein the CDRs are defined by the Kabat numbering system;

[0313] CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 40, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 44, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 47; wherein the CDRs are defined by the Chothia numbering system;

[0314] CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 42, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 46, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 47; wherein the CDRs are defined by the Abm numbering system; or,

[0315] CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 41, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 45, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 48; wherein the CDRs are defined by the IMGT numbering system.

[0316] In certain 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 set forth in SEQ ID NO: 17 or 59. In certain embodiments, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 17 or 59. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 or 59.

[0317] In certain 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 set forth in SEQ ID NO: 17 or 59. In certain embodiments, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 17 or 59. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 or 59.

[0318] In certain 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 set forth in SEQ ID NO: 17 or 59, and 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 set forth in SEQ ID NO: 18 or 60. In certain embodiments, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 17 or 59, and the first VH comprises the amino acid sequence set forth in SEQ ID NO: 18 or 60. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17 or 59, and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18 or 60.

[0319] In certain 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 set forth in SEQ ID NO: 17, and 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 set forth in SEQ ID NO: 18. In certain embodiments, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 17, and the first VH comprises the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17, and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18.

[0320] In certain 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 set forth in SEQ ID NO: 59, and 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 set forth in SEQ ID NO: 60. In certain embodiments, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first VH comprises the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 59, and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 60.

[0321] In certain 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 set forth in SEQ ID NO: 17, and 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 set forth in SEQ ID NO: 60. In certain embodiments, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 17, and the first VH comprises the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 17, and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 60.

[0322] In certain 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 set forth in SEQ ID NO: 59, and 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 set forth in SEQ ID NO: 18. In certain embodiments, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first VH comprises the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the first VL consists of the amino acid sequence set forth in SEQ ID NO: 59, and the first VH consists of the amino acid sequence set forth in SEQ ID NO: 18.

[0323] In certain embodiments of the ADC provided herein, the second VL comprises:

[0324] (i) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 19, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 21, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23; wherein the CDRs are defined by the Kabat numbering system;

[0325] (ii) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 19, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 21, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23; wherein the CDRs are defined by the Chothia numbering system;

[0326] (iii) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 19, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 21, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23; wherein the CDRs are defined by the Abm numbering system; or

[0327] (iv) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 20, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 22, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23; wherein the CDRs are defined by the IMGT numbering system;

[0328] and / or

[0329] the second VH of the bispecific antibody of the ADC comprises:

[0330] (i) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 24, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 28, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 32; wherein the CDRs are defined by the Kabat numbering system;

[0331] (ii) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 25, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 29, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 32; wherein the CDRs are defined by the Chothia numbering system;

[0332] (iii) a CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 27, a CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 31, and a CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 32; wherein the CDRs are defined by the Abm numbering system; or

[0333] (iv) a CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 26, a CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 30, and a CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 33; wherein the CDRs are defined by the IMGT numbering system.

[0334] In certain embodiments, the second 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 set forth in SEQ ID NO: 15. In certain embodiments, the second VL comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the second VL consists of the amino acid sequence set forth in SEQ ID NO: 15.

[0335] In certain embodiments, the second 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 set forth in SEQ ID NO: 16. In certain embodiments, the second VH comprises the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the second VH consists of the amino acid sequence set forth in SEQ ID NO: 16.

[0336] In certain embodiments, the second 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 set forth in SEQ ID NO: 15, and the second 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 set forth in SEQ ID NO: 16. In certain embodiments, the second VL comprises the amino acid sequence set forth in SEQ ID NO: 15, and the second VH comprises the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the second VL consists of the amino acid sequence set forth in SEQ ID NO: 15, and the second VH consists of the amino acid sequence set forth in SEQ ID NO: 16.

[0337] In certain embodiments, the present disclosure provides an ADC comprising a bispecific antibody that cross-competes with any of the antibodies described herein for binding to c-MET and / or EGFR. In certain embodiments, the present disclosure provides an ADC comprising a bispecific antibody that binds to the same or overlapping epitope of c-MET and / or EGFR as an antibody described herein.

[0338] In certain embodiments, the epitope of an antibody can be determined by, for example, NMR spectroscopy, surface plasmon resonance X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping) to determine. For X-ray crystallography, crystallization can be accomplished 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 entireties). 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, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, Eds. Wyckoff HW, et al.; U.S. Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr. D. Biol. Crystallogr. 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 entireties). Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, e.g., Champe M, et al. (1995) and Cunningham BC and Wells JA (1989). In a particular embodiment, the epitope of an antibody is determined using alanine scanning mutagenesis studies. Additionally, bispecific antibodies that recognize and bind the same or overlapping epitopes of c-MET and / or EGFR can be identified using routine techniques such as immunoassays, e.g., by showing 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 for an epitope. Competitive binding can be determined in an assay in which the test immunoglobulin inhibits the specific binding of a reference antibody to a common antigen (e.g., c-MET or EGFR).A variety of competitive binding assay formats are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid phase direct biotin-streptavidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid phase direct label, solid phase direct label sandwich (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid phase direct biotin-streptavidin enzyme immunoassay (see Chang RC et al., (1990) Virology 176:546-52); and direct label RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein in their entirety by reference. Typically, such assays involve the use of purified antigen (e.g., c-MET or EGFR) bound to a solid surface, or cells containing any of the above, unlabeled test immunoglobulin, and labeled reference immunoglobulin. Competitive inhibition is determined by measuring the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, the competing antibody 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 when present in excess. Competition binding assays can be configured in a large number of different formats using either labeled antigen or labeled antibody. In a common version of such assays, antigen is immobilized on a 96 well plate. Radioactive or enzymatic labels are then used to measure the ability of unlabeled antibody to block the binding of labeled antibody to the antigen. See, e.g., Wagener C et al., (1983) J Immunol 130:2308-2315; Weinberger et al., (1984) Immunol Methods 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, Cold Spring Harbor Press, pp. 386-389, all of which are incorporated herein in their entirety by reference.

[0339] In certain embodiments of the ADCs 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 IgGl)) and / or the CH3 domain (residues 341-447 of human IgGl, numbered according to the EU numbering system) and / or the hinge region (residues 216-230, numbered according to the EU numbering system) of the bispecific antibody of the ADC to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0340] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region to, e.g., reduce or eliminate effector function of the Fc region. In certain embodiments, the antibody comprises an Fc region comprising: (i) E233A and L235A (EALA) amino acid substitutions, wherein numbered according to the Eu numbering scheme; (ii) L234A and L235A (LALA) amino acid substitutions, wherein numbered according to the Eu numbering scheme; (iii) L234A, L235A, D265S (LALADS) amino acid substitutions, wherein numbered according to the Eu numbering scheme; (iv) L234A L235A P329G (LALAPG) amino acid substitutions, wherein numbered according to the Eu numbering scheme; (v) L234A L235A P329A (LALAPA) amino acid substitutions, wherein numbered according to the Eu numbering scheme; (vi) L235E (LE) amino acid substitution, wherein numbered according to the Eu numbering scheme; (vii) D265A (DS) amino acid substitution, wherein numbered according to the Eu numbering scheme; (viii) D265A N297G (DANG) amino acid substitutions, wherein numbered according to the Eu numbering scheme; (ix) N297X amino acid substitution, wherein X is any amino acid other than N, wherein numbered according to the Eu numbering scheme; (x) N297A / D356E / L358M (NADELM) amino acid substitutions, wherein numbered according to the Eu numbering scheme; or (xi) D356E L358M (DELM) amino acid substitutions, wherein numbered according to the Eu numbering scheme.

[0341] In certain embodiments, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of the antibodies described herein having two heavy chain constant regions.

[0342] Fab-scFv structure

[0343] In certain embodiments, the first antigen binding domain is a Fab and the second antigen binding domain is a scFv.

[0344] In certain embodiments, the bispecific antibody comprises polypeptide chain I-A, polypeptide chain I-B, and polypeptide chain I-C; wherein (a) the elements comprised in polypeptide chain I-A comprise a first VL and a first CL; the elements comprised in polypeptide chain I-B comprise a first VH, a first heavy chain CH1 region, and a first Fc domain monomer; and the elements comprised in polypeptide chain I-C comprise a second VL, a second VH, a second heavy chain CH1 hinge region, and a second Fc domain monomer; or (b) the elements comprised in polypeptide chain I-A comprise a first VL and a first CL; the elements comprised in polypeptide chain I-B comprise a first VH, a first heavy chain CH1, and a second Fc domain monomer; and the elements comprised in polypeptide chain I-C comprise a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc domain monomer.

[0345] In certain embodiments, (a) the elements comprised in polypeptide chain I-A comprise, from N-terminal to C-terminal, a first VL and a first CL; the elements comprised in polypeptide chain I-B comprise, from N-terminal to C-terminal, a first VH, a first heavy chain CH1 region, and a first Fc domain monomer; and / or the elements comprised in polypeptide chain I-C comprise, from N-terminal to C-terminal: (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 elements comprised in polypeptide chain I-A comprise, from N-terminal to C-terminal, a first VL and a first CL; the elements comprised in polypeptide chain I-B comprise, from N-terminal to C-terminal, a first VH, a first heavy chain CH1, and a second Fc domain monomer; and / or the elements comprised in polypeptide chain I-C comprise, from N-terminal to C-terminal: (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.

[0346] In certain embodiments, adjacent elements comprised in polypeptide chain I-A are connected to each other with or without a peptide linker; adjacent elements comprised in polypeptide chain I-B are connected to each other with or without a peptide linker; and / or adjacent elements comprised in polypeptide chain I-C are connected to each other with or without a peptide linker.

[0347] In certain embodiments, the peptide linker is each independently the same peptide linker or a different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, each peptide linker independently comprises 3 to 55 (e.g., 3-10, 10-25, 10-20, 10-17, 10-15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linker is each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S) residues. In certain embodiments, each peptide linker can independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues. In certain embodiments, each peptide linker can independently comprise the peptide GGGG (SEQ ID NO: 61). In certain embodiments, the peptide linker is each 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 can be represented by (GGGGS)n, where 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 certain embodiments, the peptide linker is each independently comprises the amino acid sequence set forth in any one of SEQ ID NOs: 55-58 or 65-70. n In certain embodiments, the first CL comprises the amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 63. In certain embodiments, the first heavy chain CH1 region comprises the amino acid sequence set forth in SEQ ID NO: 54. In certain embodiments, the second heavy chain CH1 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 62.

[0348] In certain embodiments, the first CL comprises the amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 63. In certain embodiments, the first heavy chain CH1 region comprises the amino acid sequence set forth in SEQ ID NO: 54. In certain embodiments, the second heavy chain CH1 hinge region comprises the amino acid sequence set forth in SEQ ID NO: 62.

[0349] In certain embodiments, the first VH comprises the amino acid sequence set forth in SEQ ID NO: 18 and the first VL comprises the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the first VH comprises the amino acid sequence set forth in SEQ ID NO: 60 and the first VL comprises the amino acid sequence set forth in SEQ ID NO: 59. In certain embodiments, the second VH comprises the amino acid sequence set forth in SEQ ID NO: 16 and the second VL comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence set forth in SEQ ID NO: 18, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 17, the second VH comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second VL comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first VH comprises the amino acid sequence set forth in SEQ ID NO: 60, the first VL comprises the amino acid sequence set forth in SEQ ID NO: 59, the second VH comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second VL comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0350] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49 and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51 and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52.

[0351] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50 and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52 and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0352] In certain embodiments, the polypeptide chain I-A comprises the amino acid sequence set forth in SEQ ID NO: 1, the polypeptide chain I-B comprises the amino acid sequence set forth in SEQ ID NO: 2 or 9, and / or the polypeptide chain I-C comprises the amino acid sequence set forth in SEQ ID NO: 3 or 10.

[0353] scFv-Fab structure

[0354] In certain embodiments, the first antigen binding domain is an scFv and the second antigen binding domain is a Fab.

[0355] In certain embodiments, the bispecific antibody comprises a peptide chain II-A, a peptide chain II-B, and a peptide chain II-C; wherein (a) the elements included in the polypeptide chain II-A comprise a second VL and a second CL; the elements included in the polypeptide chain II-B comprise a second VH, a second heavy chain CH1 region, and a first Fc domain monomer; and the elements included in the polypeptide chain II-C comprise a first VL, a first VH, a first heavy chain CH1 hinge region, and a second Fc domain monomer, or (b) the elements included in the polypeptide chain II-A comprise a second VL and a second CL; the elements included in the polypeptide chain II-B comprise a second VH, a second heavy chain CH1 region, and a second Fc domain monomer; and the elements included in the polypeptide chain II-C comprise a first VL, a first VH, a first heavy chain CH1 hinge region, and a first Fc domain monomer.

[0356] In certain embodiments, (a) the elements included in the polypeptide chain II-A comprise, from N-terminal to C-terminal, a second VL and a second CL; the elements included in the polypeptide chain II-B comprise, from N-terminal to C-terminal, a second VH, a second heavy chain CH1 region, and a first Fc domain monomer; and / or, the elements included in the polypeptide chain II-C comprise, from N-terminal to C-terminal: (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 elements included in the polypeptide chain II-A comprise, from N-terminal to C-terminal, a second VL and a second CL; the elements included in the polypeptide chain II-B comprise, from N-terminal to C-terminal, a second VH, a second heavy chain CH1 region, and a second Fc domain monomer; and / or, the elements included in the polypeptide chain II-C comprise, from N-terminal to C-terminal: (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.

[0357] In certain embodiments, each adjacent elements included in the polypeptide chain II-A is optionally connected by or without a linker, each adjacent elements included in the polypeptide chain II-B is optionally connected by or without a linker, and / or, each adjacent elements included in the polypeptide chain II-C is optionally connected by or without a linker.

[0358] In certain embodiments, the linkers are each independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, the peptide linkers are each independently composed of 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, the peptide linkers are each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which can be represented by (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as set forth in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, 70, respectively). In certain embodiments, the peptide linkers are each independently comprise an amino acid sequence as set forth in one of SEQ ID NOs: 55-58 or 65-70. n In certain embodiments, the second CL comprises an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54, and / or the heavy chain CH1 hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 62.

[0359] In certain embodiments, the second CL comprises an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54, and / or the heavy chain CH1 hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 62.

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

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

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

[0363] In certain embodiments, the polypeptide chain II-A comprises an amino acid sequence as set forth in SEQ ID NO: 4, the polypeptide chain II-B comprises an amino acid sequence as set forth in SEQ ID NO: 5 or 7, and / or, the polypeptide chain II-C comprises an amino acid sequence as set forth in SEQ ID NO: 6 or 8.

[0364] Fab-scFab structure

[0365] In certain embodiments, the first antigen binding domain is a Fab and the second antigen binding domain is a scFab (single chain Fab).

[0366] In certain embodiments, the bispecific antibody comprises a polypeptide chain III-A, a polypeptide chain III-B, and a polypeptide chain III-C; wherein (a) the elements comprised in the polypeptide chain III-A comprise a first VL and a first CL; the elements comprised in the polypeptide chain III-B comprise a first VH, a first heavy chain CH1 region, and a first Fc domain monomer; and the elements comprised in the polypeptide chain III-C comprise a second VL, a second CL, a second VH, a second heavy chain CH1 region, and a second Fc domain monomer; or, (b) the elements comprised in the polypeptide chain III-A comprise a first VL and a first CL; the elements comprised in the polypeptide chain III-B comprise a first VH, a first heavy chain CH1 region, and a second Fc domain monomer; and the elements comprised in the polypeptide chain III-C comprise a second VL, a second CL, a second VH, a second heavy chain CH1 region, and a first Fc domain monomer.

[0367] In certain embodiments, (a) the elements comprised in the polypeptide chain III-A contain a first VL and a first CL from N-terminus to C-terminus; the elements comprised in the polypeptide chain III-B contain a first VH, a first heavy chain CH1 region, and a first Fc domain monomer from N-terminus to C-terminus; and / or, the elements comprised in the polypeptide chain III-C contain (i) a second VL, a second CL, a second VH, a second heavy chain CH1 region, and a second Fc domain monomer or (ii) a second VH, a second heavy chain CH1 region, a second VL, a CL, and a second Fc domain monomer from N-terminus to C-terminus; or, (b) the elements comprised in the polypeptide chain III-A contain a first VL and a first CL from N-terminus to C-terminus; the elements comprised in the polypeptide chain III-B contain a first VH, a first heavy chain CH1 region, and a second Fc domain monomer from N-terminus to C-terminus; and / or, the elements comprised in the polypeptide chain III-C contain (i) a second VL, a second CL, a second VH, a second heavy chain CH1 region, and a first Fc domain monomer or (ii) a second VH, a second heavy chain CH1 region, a second VL, a second CL, and a first Fc domain monomer from N-terminus to C-terminus.

[0368] In certain embodiments, each adjacent element comprised in the polypeptide chain III-A is optionally connected by or without a linker, each adjacent element comprised in the polypeptide chain III-B is optionally connected by or without a linker, and / or, each adjacent element comprised in the polypeptide chain III-C is optionally connected by or without a linker.

[0369] In certain embodiments, the linkers are each independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, the peptide linker is each independently composed of 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linker is each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, the peptide linker is each 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 can be represented by (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as set forth in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, 70, respectively). In certain embodiments, the peptide linker is each independently comprises an amino acid sequence as set forth in one of SEQ ID NOs: 55-58 or 65-70. n In certain embodiments, the peptide linker is each independently comprises an amino acid sequence as set forth in one of SEQ ID NOs: 55-58 or 65-70.

[0370] In certain embodiments, the first CL and the second CL comprises an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, the second heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 62.

[0371] In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0372] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52.

[0373] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0374] scFab-Fab structure

[0375] In certain embodiments, the first antigen binding domain is a scFab and the second antigen binding domain is a Fab.

[0376] In certain embodiments, the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C, wherein (a) the elements comprised in the polypeptide chain IV-A comprise a second VL and a second CL; the elements comprised in the polypeptide chain IV-B comprise a second VH, a second heavy chain CH1 region, and a first Fc domain monomer; and the elements comprised in the polypeptide chain IV-C comprise 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 elements comprised in the polypeptide chain IV-A comprise a second VL and a second CL; the elements comprised in the polypeptide chain IV-B comprise a second VH, a second heavy chain CH1 region, and a second Fc domain monomer; and the elements comprised in the polypeptide chain IV-C comprise a first VL, a first CL, a first VH, a first heavy chain CH1 hinge region, and a first Fc domain monomer.

[0377] In certain embodiments, (a) the elements comprised in the polypeptide chain IV-A comprise from N-terminal to C-terminal a second VL and a second CL; the elements comprised in the polypeptide chain IV-B comprise from N-terminal to C-terminal a second VH, a second heavy chain CH1 region, and a first Fc domain monomer; and / or, the elements comprised in the polypeptide chain IV-C comprise from N-terminal to C-terminal 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 elements comprised in the polypeptide chain IV-A comprise from N-terminal to C-terminal a second VL and a second CL; the elements comprised in the polypeptide chain IV-B comprise from N-terminal to C-terminal a second VL and a second LC, a second VH, a second heavy chain CH1 region, and a second Fc domain monomer; and / or, the elements comprised in the polypeptide chain IV-C comprise from N-terminal to C-terminal (i) a first VL, a first CL, a first VH, a first heavy chain CH1 hinge region, and a first Fc domain monomer.

[0378] In certain embodiments, each adjacent elements comprised in the polypeptide chain IV-A is optionally connected by or without a linker, each adjacent elements comprised in the polypeptide chain IV-B is optionally connected by or without a linker, and / or, each adjacent elements comprised in the polypeptide chain IV-C is optionally connected by or without a linker.

[0379] In certain embodiments, the linkers are each independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, the peptide linkers are each independently composed of 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, the peptide linkers are each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which can be represented by (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as set forth in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, 70, respectively). In certain embodiments, the peptide linkers are each independently comprise an amino acid sequence as set forth in one of SEQ ID NOs: 55-58 or 65-70. n In certain embodiments, the first and second CL comprise an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54, and / or the first heavy chain CH1 hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 62.

[0380] In certain embodiments, the first and second CL comprise an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54, and / or the first heavy chain CH1 hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 62.

[0381] In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0382] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52.

[0383] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0384] In certain embodiments, the polypeptide chain IV-A comprises the amino acid sequence set forth in SEQ ID NO: 4, the polypeptide chain IV-B comprises the amino acid sequence set forth in SEQ ID NO: 7, and / or, the polypeptide chain IV-C comprises the amino acid sequence set forth in SEQ ID NO: 11.

[0385] Fab-Fab (including crossMab-style domain exchange) structure

[0386] In certain embodiments, the first and the second antigen binding domain are Fabs, and the Fab of the second antigen binding domain comprises a domain swap in the format of a CrossMab.

[0387] In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the VH and VL in the format of a CrossMab VH-VL In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the CH1 and CL in the format of a CrossMab

[0388] In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the CH1 and CL in the format of a CrossMab CH1-CL In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the CH1 and CL in the format of a CrossMab

[0389] In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the CH1 and CL in the format of a CrossMab Fab In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the CH1 and CL in the format of a CrossMab

[0390] In certain embodiments, the CrossMab CH1-CL In certain embodiments, the CrossMab (a) the elements comprised in the polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in the polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a first Fc domain monomer; the elements comprised in the polypeptide chain V-C comprise a second VH, a second CL, a second heavy chain CH1 hinge region, and a second Fc domain monomer; and the elements comprised in the polypeptide chain V-D comprise a second VL and a second heavy chain CH1 region; or (b) the elements comprised in the polypeptide chain V-A comprise a first VL and a first CL; the elements comprised in the polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a second Fc domain monomer; the elements comprised in the polypeptide chain V-C comprise a second VH, a second CL, a second heavy chain CH1 hinge region, and a first Fc domain monomer; and the elements comprised in the polypeptide chain V-D comprise a second VL and a second heavy chain CH1 region.

[0391] In certain embodiments, (a) the elements included in the polypeptide chain V-A comprise, from N- to C-terminal, a first VL and a first CL; the elements included in the polypeptide chain V-B comprise, from N- to C-terminal, a first VH, a first heavy chain CH1 region, and a first Fc domain monomer; the elements included in the polypeptide chain V-C comprise, from N- to C-terminal, a second VH, a second CL, a second heavy chain CH1 hinge region, and a second Fc domain monomer, and / or, the elements included in the polypeptide chain V-D comprise, from N- to C-terminal, a second VL and a second heavy chain CH1 region; or, (b) the elements included in the polypeptide chain V-A comprise, from N- to C-terminal, a first VL and a first CL; the elements included in the polypeptide chain V-B comprise, from N- to C-terminal, a first VH, a first heavy chain CH1 region, and a second Fc domain monomer; the elements included in the polypeptide chain V-C comprise, from N- to C-terminal, a second VH, a second CL, a second heavy chain CH1 hinge region, and a first Fc domain monomer, and / or, the elements included in the polypeptide chain V-D comprise, from N- to C-terminal, a second VL and a second heavy chain CH1 region.

[0392] In certain embodiments, each adjacent pair of elements included in the polypeptide chain V-A is optionally connected by or without a linker, each adjacent pair of elements included in the polypeptide chain V-B is optionally connected by or without a linker, each adjacent pair of elements included in the polypeptide chain V-C is optionally connected by or without a linker, and / or, each adjacent pair of elements included in the polypeptide chain V-D is optionally connected by or without a linker.

[0393] In certain embodiments, each of the linkers is independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, each of the peptide linkers independently consists of 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, each of the peptide linkers is independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, each of the peptide linkers 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 can be represented by (GGGGS) n wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as set forth in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, 70, respectively). In certain embodiments, each of the peptide linkers independently comprises an amino acid sequence as set forth in one of SEQ ID NOs: 55-58 or 65-70.

[0394] In certain embodiments, the first and second CL

[0395] comprises an amino acid sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 63, the heavy chain CH1 region comprises an amino acid sequence set forth in SEQ ID NO: 54, and / or the heavy chain CH1 hinge region comprises an amino acid sequence set forth in SEQ ID NO: 62.

[0396] In certain embodiments, the first heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 18, and the first light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the first light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the second heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 18, and the first light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 17, the second heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 60, the second heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 15.

[0397] In certain embodiments, the first Fc domain monomer comprises an amino acid sequence set forth in SEQ ID NO: 49, and the second Fc domain monomer comprises an amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the first Fc domain monomer comprises an amino acid sequence set forth in SEQ ID NO: 51, and the second Fc domain monomer comprises an amino acid sequence set forth in SEQ ID NO: 52.

[0398] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0399] In certain embodiments, the polypeptide chain V-A comprises the amino acid sequence set forth in SEQ ID NO: 1, the polypeptide chain V-B comprises the amino acid sequence set forth in SEQ ID NO: 9, the polypeptide chain V-C comprises the amino acid sequence set forth in SEQ ID NO: 13, and / or, the polypeptide chain V-D comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0400] Fab (including crossMab-style domain exchanges) - Fab structure

[0401] In certain embodiments, the first and second antigen binding domains are Fabs, and the Fab of the first antigen binding domain comprises a domain crossover in the format of a CrossMab.

[0402] In certain embodiments, the Fab of the first antigen binding domain comprises a CH1 and CL domain crossover in the format of a CrossMab CH1-CL

[0403] In certain embodiments, the Fab of the first antigen binding domain comprises a VH and VL domain crossover in the format of a CrossMab VH-VL

[0404] In certain embodiments, the Fab of the first antigen binding domain comprises a domain crossover in the format of a CrossMab Fab

[0405] ​​​In certain embodiments, the bispecific antibody comprises a polypeptide chain VI-A, a polypeptide chain VI-B, a polypeptide chain VI-C, and a polypeptide chain VI-D; wherein (a) the elements included in the polypeptide chain VI-A comprise the second light chain variable region and second CL, the elements included in the polypeptide chain VI-B comprise: the second heavy chain variable region, second heavy chain CH1 region, and the first Fc domain monomer, the elements included in the polypeptide chain VI-C comprise: the first heavy chain variable region, first CL, and the second Fc domain monomer, and the elements included in the polypeptide chain VI-D comprise: the first light chain variable region and first heavy chain CH1 region; or (b) the elements included in the polypeptide chain VI-A comprise the second light chain variable region and second CL, the elements included in the polypeptide chain VI-B comprise: the second heavy chain variable region, second heavy chain CH1 region, and the second Fc domain monomer, the elements included in the polypeptide chain VI-C comprise: the first heavy chain variable region, first CL, and the first Fc domain monomer, and the elements included in the polypeptide chain VI-D comprise: the first light chain variable region and first heavy chain CH1 region.

[0406] In certain embodiments, the polypeptide chain VI-A comprises from N-terminal to C-terminal: the second light chain variable region and second CL, the polypeptide chain VI-B comprises from N-terminal to C-terminal: the second heavy chain variable region, second heavy chain CH1 region, and the first Fc domain monomer (or the second Fc domain monomer), the polypeptide chain VI-C comprises from N-terminal to C-terminal: the first heavy chain variable region, first CL, and the second Fc domain monomer (or the first Fc domain monomer), and / or, the polypeptide chain VI-D comprises from N-terminal to C-terminal: the first light chain variable region and first heavy chain CH1 region.

[0407] In certain embodiments, (a) the elements included in the polypeptide chain VI-A comprise, from N-terminal to C-terminal, a second VL and a second CL; the elements included in the polypeptide chain VI-B comprise, from N-terminal to C-terminal, a second VL and a second CL, a second VH, a heavy chain CH1 region, and a first Fc domain monomer; the elements included in the polypeptide chain VI-C comprise, from N-terminal to C-terminal, a first VH, a first CL, a first heavy chain CH1 hinge region, and a second Fc domain monomer; and / or, the elements included in the polypeptide chain VI-D comprise, from N-terminal to C-terminal, a first VL and a heavy chain CH1 region; or, (b) the elements included in the polypeptide chain VI-A comprise, from N-terminal to C-terminal, a second VL and a second CL; the elements included in the polypeptide chain VI-B comprise, from N-terminal to C-terminal, a second VH, a heavy chain CH1 region, and a second Fc domain monomer; the elements included in the polypeptide chain VI-C comprise, from N-terminal to C-terminal, a first VH, a first CL, a first heavy chain CH1 hinge region, and a first Fc domain monomer; and / or, the elements included in the polypeptide chain VI-D comprise, from N-terminal to C-terminal, a first VL and a heavy chain CH1 region.

[0408] In certain embodiments, each adjacent pair of elements included in the polypeptide chain VI-A is optionally connected by or without a linker, each adjacent pair of elements included in the polypeptide chain VI-B is optionally connected by or without a linker, each adjacent pair of elements included in the polypeptide chain VI-C is optionally connected by or without a linker, and / or, each adjacent pair of elements included in the polypeptide chain VI-D is optionally connected by or without a linker.

[0409] In certain embodiments, each of the linkers is independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, each of the peptide linkers independently consists of 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, each of the peptide linkers is independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, each of the peptide linkers 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 can be represented by (GGGGS) n wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as set forth in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, 70, respectively). In certain embodiments, each of the peptide linkers independently comprises an amino acid sequence as set forth in one of SEQ ID NOs: 55-58 or 65-70.

[0410] In certain embodiments, the first CL and second CL comprise the amino acid sequence set forth as SEQ ID NO: 53 or SEQ ID NO: 63, the heavy chain CHI region comprises the amino acid sequence set forth as SEQ ID NO: 54, and / or the first heavy chain CHI hinge region comprises the amino acid sequence set forth as SEQ ID NO: 62.

[0411] In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 17. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 60. In certain embodiments, the second heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15. In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth as SEQ ID NO: 15.

[0412] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth as SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence set forth as SEQ ID NO: 50. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth as SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence set forth as SEQ ID NO: 52.

[0413] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0414] In certain embodiments, the CrossMab VH-VL In certain embodiments, the CrossMab

[0415] In certain embodiments, (a) the elements comprised in the polypeptide chain V-A comprise from N-terminal to C-terminal a first VL and a first CL; the elements comprised in the polypeptide chain V-B comprise from N-terminal to C-terminal a first VH, a first heavy chain CH1 region, and a first Fc domain monomer; the elements comprised in the polypeptide chain V-C comprise from N-terminal to C-terminal a second VL, a second heavy chain CH1 hinge region, and a second Fc domain monomer, and / or, the elements comprised in the polypeptide chain V-D comprise from N-terminal to C-terminal a second VH and a second CL; or, (b) the elements comprised in the polypeptide chain V-A comprise from N-terminal to C-terminal a first VL and a first CL; the elements comprised in the polypeptide chain V-B comprise from N-terminal to C-terminal a first VH, a first heavy chain CH1 region, and a second Fc domain monomer; the elements comprised in the polypeptide chain V-C comprise from N-terminal to C-terminal a second VL, a second heavy chain CH1 hinge region, and a first Fc domain monomer, and / or, the elements comprised in the polypeptide chain V-D comprise from N-terminal to C-terminal a second VH and a second CL.

[0416] In certain embodiments, each adjacent element comprised in said polypeptide chain V-A is optionally connected by or without a linker, each adjacent element comprised in said polypeptide chain V-B is optionally connected by or without a linker, each adjacent element comprised in said polypeptide chain V-C is optionally connected by or without a linker, and / or, each adjacent element comprised in said polypeptide chain V-D is optionally connected by or without a linker.

[0417] In certain embodiments, said linkers are each independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, said peptide linker is each independently composed of 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, said peptide linker is each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, said peptide linker is each independently comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which can be represented by (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as set forth in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, 70, respectively). In certain embodiments, said peptide linker is each independently comprising an amino acid sequence as set forth in one of SEQ ID NOs: 55-58 or 65-70. n In certain embodiments, said peptide linker is each independently comprising an amino acid sequence as set forth in one of SEQ ID NOs: 55-58 or 65-70.

[0418] In certain embodiments, said first and second CL comprise an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, said first and second heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54, and / or said second heavy chain CH1 hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 62.

[0419] In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0420] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52.

[0421] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0422] In certain embodiments, the CrossMab FabThe format of the bispecific antibody comprises a polypeptide chain V-A, a polypeptide chain V-B, a polypeptide chain V-C, and a polypeptide chain V-D; wherein (a) the elements comprised in the polypeptide chain V-A comprise a first VL and a first CL, the elements comprised in the polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a first Fc domain monomer, the elements comprised in the polypeptide chain V-C comprise a second VL, a second CL region, a second heavy chain CH1 hinge region, and a second Fc domain monomer, and the elements comprised in the polypeptide chain V-D comprise a second VH and a second heavy chain CH1 region; or (b) the elements comprised in the polypeptide chain V-A comprise a first VL and a second CL, the elements comprised in the polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a second Fc domain monomer, the elements comprised in the polypeptide chain V-C comprise a second VL, a second heavy chain CH1 hinge region, and a first Fc domain monomer, and the elements comprised in the polypeptide chain V-D comprise a second VH and a second heavy chain CH1 region.

[0423] In certain embodiments, (a) the elements comprised in the polypeptide chain V-A comprise a first VL and a first CL from N- to C-terminus; the elements comprised in the polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a first Fc domain monomer from N- to C-terminus; the elements comprised in the polypeptide chain V-C comprise a second VL, a second heavy chain CH1 hinge region, and a second Fc domain monomer from N- to C-terminus; and / or, the elements comprised in the polypeptide chain V-D comprise a second VH and a second heavy chain CH1 region from N- to C-terminus; or (b) the elements comprised in the polypeptide chain V-A comprise a first VL and a second CL from N- to C-terminus; the elements comprised in the polypeptide chain V-B comprise a first VH, a first heavy chain CH1 region, and a second Fc domain monomer from N- to C-terminus; the elements comprised in the polypeptide chain V-C comprise a second VL, a second heavy chain CH1 hinge region, and a first Fc domain monomer from N- to C-terminus; and / or, the elements comprised in the polypeptide chain V-D comprise a second VH and a second heavy chain CH1 region from N- to C-terminus.

[0424] In certain embodiments, each adjacent elements comprised in the polypeptide chain V-A is optionally connected by or without a linker, each adjacent elements comprised in the polypeptide chain V-B is optionally connected by or without a linker, each adjacent elements comprised in the polypeptide chain V-C is optionally connected by or without a linker, and / or, each adjacent elements comprised in the polypeptide chain V-D is optionally connected by or without a linker.

[0425] In certain embodiments, the linkers are each independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, the peptide linkers are each independently composed of 3 to 55 (e.g., 3-10, 10-25, 10-20, 10 to 17, 10 to 15, 10-25, 10-35, 10-45, 10-55) amino acid residues. In certain embodiments, the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, the peptide linkers are each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of the peptide linker subunit GGGGS (SEQ ID NO: 57), which can be represented by (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (as set forth in SEQ ID NOs: 57, 65, 55, 58, 66-68, 56, 69, 70, respectively). In certain embodiments, the peptide linkers are each independently comprise an amino acid sequence as set forth in one of SEQ ID NOs: 55-58 or 65-70. n In certain embodiments, the first and second CL comprise an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, the first and second heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 62.

[0426] In certain embodiments, the first and second CL comprise an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, the first and second heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54, and / or the second heavy chain CH1 hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 62.

[0427] In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18, and the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the first light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 59, and the first heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60, the second heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the second light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0428] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52.

[0429] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0430] scFv-scFv structure

[0431] In certain embodiments, the first and second antigen binding domains are scFvs.

[0432] In certain embodiments, the bispecific antibody comprises a polypeptide chain VII-A and a polypeptide chain VII-B; wherein (a) the elements included in the polypeptide chain VII-A comprise the first VL, the first VH, a first heavy chain CH1 hinge region, and the first Fc domain monomer; the elements included in the polypeptide chain VII-B comprise: the second VL, the second VH, a second heavy chain CH1 hinge region, and the second Fc domain monomer; or (b) the elements included in the polypeptide chain VII-A comprise a first VL, a first VH, a first heavy chain CH1 hinge region, and a second Fc domain monomer; and the elements included in the polypeptide chain VII-B comprise a second VL, a second VH, a second heavy chain CH1 hinge region, and a first Fc domain monomer

[0433] In certain embodiments, (a) the elements included in the polypeptide chain VII-A comprise, from N-terminal to C-terminal, (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 elements included in the polypeptide chain VII-B comprise, from N-terminal to C-terminal, (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 elements included in the polypeptide chain VII-A comprise, from N-terminal to C-terminal, (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 elements included in the polypeptide chain VII-B comprise, from N-terminal to C-terminal, (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.

[0434] In certain embodiments, each adjacent elements included in the polypeptide chain VII-A are connected by or without a linker, and / or, each adjacent elements included in the polypeptide chain VII-B are connected by or without a linker.

[0435] 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 a 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.

[0436] In some embodiments, the first and second heavy chain CH1 hinge regions contain the amino acid sequence shown in SEQ ID NO:62.

[0437] 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.

[0438] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52.

[0439] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 50, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the first Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 52, and the second Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0440] In certain embodiments, the polypeptide chain VII-A comprises the amino acid sequence set forth in SEQ ID NO: 14, and / or, the polypeptide chain VII-B comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0441] In certain example embodiments, the bispecific antibody in the ADC provided herein comprises:

[0442] a polypeptide chain I-A comprising the amino acid sequence set forth in SEQ ID NO: 1, a polypeptide chain I-B comprising the amino acid sequence set forth in SEQ ID NO: 2, and, a polypeptide chain I-C comprising the amino acid sequence set forth in SEQ ID NO: 3;

[0443] a polypeptide chain I-A comprising the amino acid sequence set forth in SEQ ID NO: 1, a polypeptide chain I-B comprising the amino acid sequence set forth in SEQ ID NO: 9, and, a polypeptide chain I-C comprising the amino acid sequence set forth in SEQ ID NO: 10;

[0444] a polypeptide chain II-A comprising the amino acid sequence set forth in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence set forth in SEQ ID NO: 5, and, a polypeptide chain II-C comprising the amino acid sequence set forth in SEQ ID NO: 6;

[0445] a polypeptide chain II-A comprising the amino acid sequence set forth in SEQ ID NO: 4, a polypeptide chain II-B comprising the amino acid sequence set forth in SEQ ID NO: 7, and, a polypeptide chain II-C comprising the amino acid sequence set forth in SEQ ID NO: 8;

[0446] a polypeptide chain IV-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, a polypeptide chain IV-B comprising an amino acid sequence as set forth in SEQ ID NO: 7, and, a polypeptide chain IV-C comprising an amino acid sequence as set forth in SEQ ID NO: 11;

[0447] a polypeptide chain V-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, a polypeptide chain V-B comprising an amino acid sequence as set forth in SEQ ID NO: 9, a polypeptide chain V-C comprising an amino acid sequence as set forth in SEQ ID NO: 13, and, a polypeptide chain V-D comprising an amino acid sequence as set forth in SEQ ID NO: 12; or,

[0448] a polypeptide chain VII-A comprising an amino acid sequence as set forth in SEQ ID NO: 14, and, a polypeptide chain VII-B comprising an amino acid sequence as set forth in SEQ ID NO: 8.

[0449] In certain embodiments, the bispecific antibody has enhanced tumor inhibition relative to a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody; wherein the CDR amino acid sequences of the monospecific anti-c-MET antibody are identical to the CDR amino acid sequences of the first antigen binding domain, and the CDR amino acid sequences of the monospecific anti-EGFR antibody are identical to the CDR amino acid sequences of the second antigen binding domain.

[0450] In certain embodiments, the tumor inhibition comprises inhibition of EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0451] Drug-linker of ADC

[0452] The ADC provided herein comprises a bispecific antibody as described herein, further comprising a cytotoxic drug moiety D linked to the antibody through a linker -M-L-E-.

[0453] In some embodiments, linkers described in European Patent Publication EP 4349372 are incorporated herein by reference. In some embodiments, linkers (M-L-E) and individual components M, L, and E as described in European Patent Publication EP 4349372 are incorporated herein by reference. In some embodiments, cytotoxic drugs described in European Patent Publication EP 4349372 are incorporated herein by reference. In some embodiments, linkers / cytotoxic drugs described in European Patent Publication EP 4349372 are incorporated herein by reference.

[0454] In some embodiments, M in the ADC comprises wherein ring A is a 5-6 membered aliphatic heterocycle, or a 5-20 membered aromatic ring system, said aliphatic heterocycle and aromatic ring system being optionally substituted with one or more groups independently selected from oxo (=0), halogen, cyano, amino, carboxyl, thiol, and C 1-6 alkyl; M1is selected from a single bond, C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene, or amine.

[0455] In some embodiments, M comprises wherein ring A is a 5 membered aliphatic heterocycle, a 6 membered heteroaromatic ring, or a polycycle formed by one or more 6 membered aromatic heterocycles connected to a benzene ring by a single bond, or a polycycle formed by multiple 6 membered heteroaromatic rings connected by a single bond, said aliphatic heterocycle being optionally substituted with one or more groups selected from oxo (=0), halogen, and C 1-4 alkyl; M1is selected from a single bond, C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene, or amine.

[0456] In some embodiments, M is wherein ring A is selected from M1is selected from a single bond and C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene, and amine.

[0457] In some embodiments, M is selected from

[0458] In some embodiments, M is

[0459] In some embodiments, M is selected from

[0460] In further embodiments of the antibody-drug conjugate, M is selected from

[0461] In some embodiments, L in the antibody-drug conjugate is selected from one or more of the following structures: 1-6alkylene, -N(R')-, carbonyl, -0-, a natural amino acid or a non-natural amino acid and analogs thereof (such as Ala, Arg, Asn, Asp, Cit, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2) s OCH3)), 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)),

[0462] wherein R' represents hydrogen, C 1-6 alkyl or a polyethylene glycol fragment containing 1-10 EO units; s is selected from an integer from 1-20.

[0463] In some embodiments, L in the ADC is selected from the structure consisting of one or more of the following: 1-6alkylene, 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-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)), Gly-Gly-Gly-Gly (GGGG (SEQ ID NO: 76)), Gly-Gly-Gly (GGG (SEQ ID NO: 77)), Gly-Gly (GG (SEQ ID NO: 78)), Gly (SEQ ID NO: 79), Ala (SEQ ID NO: 80), Phe (SEQ ID NO: 81), Leu (SEQ ID NO: 82), Val (SEQ ID NO: 83), Lys (SEQ ID NO: 84), Cit (SEQ ID NO: 85), Ser (SEQ ID NO: 86), Pro (SEQ ID NO: 87), Asn (SEQ ID NO: 88), Glu (SEQ ID NO: 89), and Arg (SEQ ID NO: 90).

[0464] wherein s is selected from an integer from 1-20.

[0465] In some embodiments, L is selected from the structure consisting of one or more of the following:

[0466]

[0467]

[0468] In some embodiments, L is selected from the following structure:

[0469]

[0470]

[0471] In some embodiments, L is selected from the following structure:

[0472]

[0473] In some embodiments, L is selected from the following structure:

[0474] In some embodiments, L is selected from the following structure:

[0475]

[0476] In some embodiments, E in the antibody-drug conjugate is a single bond or is selected from the group consisting of:

[0477] -NHCH2-,

[0478] In some embodiments, E is a single bond, -NHCH2-,

[0479] In some embodiments, E is -NHCH2- or

[0480] In some embodiments, E is -NHCH2-.

[0481] In some embodiments, E is a single bond.

[0482] In some embodiments, E is

[0483] In some embodiments, M is selected from

[0484] L is selected from

[0485] E is selected from -NHCH2- and

[0486] In some embodiments, the antibody-drug conjugate is selected from the group consisting of:

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494] In some embodiments, selected from the group consisting of:

[0495]

[0496]

[0497] In some embodiments, the cytotoxic drug is selected from microtubule inhibitors, DNA intercalating agents, DNA topoisomerase inhibitors, and RNA polymerase inhibitors. In some embodiments, the microtubule 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.

[0498] In some embodiments, the cytotoxic agent 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.

[0499]

[0500] Among them, R1 and R2 are each independently selected from C. 1-6 Alkyl and halogen;

[0501] R3 is selected from H and -CO-CH2OH;

[0502] 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.

[0503] 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

[0504] R7 is selected from C 1-6 Alkyl, -C 1-4 Alkylene-NR a R b -C 1-4 Alkylene-SiR a Rb R c , -SiR a R b R c , -C 1-4 alkylene = N-OR a ; wherein R a , R b and R c are each independently at each occurrence selected from H, C 1-6 alkyl, -SO2-C 1-6 alkyl and -CO-C 1-6 alkyl; wherein optionally R a , R b and the atom to which they are attached collectively form a 5-6 membered nitrogen containing heterocyclic ring.

[0505] In further embodiments of the antibody-drug conjugate, the cytotoxic drug is selected from the following compounds or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of the compound:

[0506]

[0507]

[0508] The cytotoxic drugs disclosed herein typically contain a variety of functional groups such as hydroxyl (-OH), carboxyl (-COOH), thiol (-SH), primary amine (-NH2), secondary amine (-NR A H) or tertiary amine groups (-NR B R C ), while R A , R B and R C herein represent only non-hydrogen substituents on N and the cytotoxic drug can be linked to the linker in the conjugate through these functional groups.

[0509] In some embodiments, the cytotoxic drug is selected from the following compounds or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of the compound:

[0510]

[0511]

[0512] Linkage between linker and drug (payload)

[0513] In some embodiments, the cytotoxic drug is linked to E in the antibody-drug conjugate through -OH, -SH, -NH2, secondary amine groups or tertiary amine groups.

[0514] In some embodiments, D is -OH, -NH2, or a monovalent structure resulting from the loss of one H from a secondary amine group on the cytotoxic drug.

[0515] Examples of ADCs

[0516] 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 ADCs are as follows:

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534] wherein Ab is a bispecific antibody or antigen binding fragment thereof as defined above;

[0535] represents the specific linkage mode of the thiol group in the bispecific antibody or antigen binding fragment thereof to the other moiety in the antibody-drug conjugate; and x represents the number of drug loads.

[0536] In some embodiments, the thiol group in the bispecific antibody or antigen binding fragment thereof forms a thioether linkage with the precursor of the other moiety of the antibody-drug conjugate via an addition reaction or a substitution reaction to obtain the antibody-drug conjugate.

[0537] In some embodiments, the Ab comprises:

[0538] (1) polypeptide chain I-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, polypeptide chain I-B comprising an amino acid sequence as set forth in SEQ ID NO: 2 or 9, and / or, polypeptide chain I-C comprising an amino acid sequence as set forth in SEQ ID NO: 3 or 10;

[0539] (2) polypeptide chain II-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, polypeptide chain II-B comprising an amino acid sequence as set forth in SEQ ID NO: 5 or 7, and / or, polypeptide chain II-C comprising an amino acid sequence as set forth in SEQ ID NO: 6 or 8;

[0540] (3) polypeptide chain IV-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, polypeptide chain IV-B comprising an amino acid sequence as set forth in SEQ ID NO: 7, and / or, polypeptide chain IV-C comprising an amino acid sequence as set forth in SEQ ID NO: 11;

[0541] (4) polypeptide chain V-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, polypeptide chain V-B comprising an amino acid sequence as set forth in SEQ ID NO: 9, polypeptide chain V-C comprising an amino acid sequence as set forth in SEQ ID NO: 13, and / or, polypeptide chain V-D comprising an amino acid sequence as set forth in SEQ ID NO: 12; or

[0542] (5) polypeptide chain VII-A comprising an amino acid sequence as set forth in SEQ ID NO: 14, and / or, polypeptide chain VII-B comprising an amino acid sequence as set forth in SEQ ID NO: 8.

[0543] In some embodiments, the Ab comprises:

[0544] (1) polypeptide chain I-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, polypeptide chain I-B comprising an amino acid sequence as set forth in SEQ ID NO: 2, and, polypeptide chain I-C comprising an amino acid sequence as set forth in SEQ ID NO: 3;

[0545] (2) a polypeptide chain I-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, a polypeptide chain I-B comprising an amino acid sequence as set forth in SEQ ID NO: 9, and, a polypeptide chain I-C comprising an amino acid sequence as set forth in SEQ ID NO: 10;

[0546] (3) a polypeptide chain II-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, a polypeptide chain II-B comprising an amino acid sequence as set forth in SEQ ID NO: 5, and, a polypeptide chain II-C comprising an amino acid sequence as set forth in SEQ ID NO: 6;

[0547] (4) a polypeptide chain II-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, a polypeptide chain II-B comprising an amino acid sequence as set forth in SEQ ID NO: 7, and, a polypeptide chain II-C comprising an amino acid sequence as set forth in SEQ ID NO: 8;

[0548] (5) a polypeptide chain IV-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, a polypeptide chain IV-B comprising an amino acid sequence as set forth in SEQ ID NO: 7, and, a polypeptide chain IV-C comprising an amino acid sequence as set forth in SEQ ID NO: 11;

[0549] (6) a polypeptide chain V-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, a polypeptide chain V-B comprising an amino acid sequence as set forth in SEQ ID NO: 9, a polypeptide chain V-C comprising an amino acid sequence as set forth in SEQ ID NO: 13, and, a polypeptide chain V-D comprising an amino acid sequence as set forth in SEQ ID NO: 12; or,

[0550] (7) a polypeptide chain VII-A comprising an amino acid sequence as set forth in SEQ ID NO: 14, and, a polypeptide chain VII-B comprising an amino acid sequence as set forth in SEQ ID NO: 8.

[0551] In particular embodiments, Ab is selected from the group consisting of bispecific antibodies 07B, 10B, 38B, 41B, 49B, 55B, and 56B.

[0552] In particular embodiments of the antibody-drug conjugates disclosed herein, the heavy chain constant region of the bispecific antibody of the antibody-drug conjugate can comprise a C-terminal lysine or lack a C-terminal lysine 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 can comprise a glutamic acid or glutamine residue or cyclized to pyroglutamic acid.

[0553] In some embodiments, the N-terminal amino acid of the variable region of the bispecific antibody or antigen-binding fragment thereof of the antibody-drug conjugate can be cyclized to pyroglutamic acid. Accordingly, the present disclosure provides compositions of antibody-drug conjugates, wherein each antibody-drug conjugate independently can comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid residue or an N-terminal glutamine or glutamic acid cyclized to pyroglutamate.

[0554] Accordingly, in some embodiments, the present application further provides compositions comprising an antibody-drug conjugate described herein, wherein the predominant antibody-drug conjugate in the composition comprises (i) an antibody wherein the heavy chain C-terminus lacks a lysine residue; (ii) an antibody wherein the heavy chain N-terminus comprises a glutamine, glutamic acid or pyroglutamic acid residue; (iii) an antibody wherein the heavy chain C-terminus lacks a lysine residue and the heavy chain N-terminus is a glutamine, glutamic acid or pyroglutamic acid residue; (iv) an antibody wherein the heavy chain C-terminus lacks a lysine residue and the heavy chain N-terminus is a pyroglutamic acid residue; or, (v) an antibody wherein the heavy chain C-terminus lacks a lysine residue and the heavy chain N-terminus is a glutamine or glutamic acid residue.

[0555] As known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution. Accordingly, the present application also provides a composition comprising one or more antibody-drug conjugates of any of the preceding. For example, the present application provides an ADC embodiment comprising the amino acid sequence set forth in SEQ ID NO: 2, 9, 18 or 60, wherein the N-terminal glutamic acid has been converted to pyroglutamate, and / or an ADC embodiment comprising the amino acid sequence set forth in SEQ ID NO: 5, 7, 13 or 16, wherein the N-terminal glutamine has been converted to pyroglutamate.

[0556] In some embodiments, the DAR (drug to antibody ratio) of the composition is 1-10, e.g., 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.

[0557] In some embodiments, the DAR value of the composition is 3-9, or 4-8, e.g., 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.

[0558] coupling

[0559] In another aspect, the application provides a method of conjugating a linker-payload described herein to an antibody described herein to make an antibody-drug conjugate (ADC) described herein.

[0560] In certain embodiments, an antibody described herein is conjugated to a linker-payload described herein by binding to a lysine in the antibody.

[0561] In certain embodiments, an antibody described herein is conjugated to a linker-payload described herein by binding to a cysteine residue in the antibody. In certain embodiments, the cysteine residue is from a reduced intra-chain disulfide bond in the antibody. In certain embodiments, the cysteine residue is from a reduced inter-chain disulfide bond in the antibody.

[0562] In some embodiments, the antibody is conjugated to the linker-payload by conjugation to reduced interchain disulfide bonds in the antibody. For example, an IgGl antibody is composed of four polypeptide chains, two heavy chains comprising VH, CH1, and Fc (e.g., hinge, CH2, and CH3) domains, and two light chains comprising VL and CL domains, linked by interchain cysteine disulfide bonds (-S-S-) (e.g., two heavy chain-light chain interchain disulfide bonds and two hinge heavy chain-heavy chain interchain disulfide bonds). In certain embodiments, when these disulfide bonds are broken under reducing conditions, eight (8) reactive cysteine thiol moieties are produced. In certain embodiments, each of the eight reactive cysteine thiol moieties is a point of attachment for a linker-drug, such that up to eight (x = 8) linker-payloads can be attached to the reduced antibody. In certain embodiments, any one of the four disulfide bonds is broken under reducing conditions, producing two (2) reactive cysteine thiol moieties. In further embodiments, each of the two reactive cysteine thiol moieties is a point of attachment for a linker-drug, such that two (x = 2) linker-drugs can be attached to the reduced antibody. In certain embodiments, any two of the four disulfide bonds are broken under reducing conditions, producing four (4) reactive cysteine thiol moieties. In further embodiments, each of the four reactive cysteine thiol moieties is a point of attachment for a linker-drug, such that four (x = 4) linker-drugs can be attached to the reduced antibody. In certain embodiments, any three of the four disulfide bonds are broken under reducing conditions, producing six (6) reactive cysteine thiol moieties. In further embodiments, each of the six reactive cysteine thiol moieties is a point of attachment for a linker-drug, such that six (x = 6) linker-drugs can be attached to the reduced antibody.

[0563] In some embodiments, the interchain disulfide bond is between two cysteine residues that are cleaved under reducing conditions to produce two reactive cysteine thiol moieties. In further embodiments, the interchain disulfide bond in the antibody is between a heavy chain and a light chain, e.g., 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 between two heavy chains, e.g., between C226 and / or C229 of a first heavy chain and C226 and / or C229 of a second heavy chain according to EU numbering. In some embodiments, the cysteine residues are in the hinge region of the antibody. In some embodiments, the cysteine residues are in the heavy chain at any one or more of positions 220, 226, or 229 according to EU numbering (also referred to herein as C220, C226, or C229, respectively). In some embodiments, the cysteine residue is in the light chain at position 214 according to EU and / or Kabat numbering (also referred to herein as C214, e.g., position 214 in a kappa light chain according to EU and Kabat numbering or position 214 in a lambda light chain according to EU and Kabat numbering). In one embodiment, the cysteine residues are at each of positions 220, 226, and 229 in the heavy chain according to EU numbering and at position 214 in the light chain according to EU or Kabat numbering. In one embodiment, the cysteine residues are at each of positions 220, 226, and 229 in the heavy chain according to EU numbering and at position 214 in a kappa light chain according to EU and Kabat numbering. In one embodiment, the cysteine residues are at each of positions 220, 226, and 229 in the heavy chain according to EU numbering and at position 214 in a lambda light chain according to EU and Kabat numbering. In one embodiment, the cysteine residues are at any one or more of:

[0564] (i) any one, or any two or three of positions 220, 226, and 229 in a first heavy chain according to EU numbering;

[0565] (ii) any one, or any two or three of positions 220, 226, and 229 in a second heavy chain according to EU numbering;

[0566] (iii) position 214 in a first light chain according to Kabat numbering and / or EU numbering; and / or

[0567] (iv) position 214 in a second light chain according to Kabat numbering and / or EU numbering.

[0568] As used herein, C220, C226, and C229 refer to the amino acid residue (cysteine, Cys, C) of an immunoglobulin identified according to EU numbering. Those skilled in the art will appreciate that such numbering correspondingly represents alignment of the amino acid residues of a polypeptide with the amino acid residues identified in an immunoglobulin, e.g., the amino acid residues shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0569] As used herein, the cysteine residue at position 214 of a kappa light chain refers to the amino acid residue (cysteine, Cys, C) of an immunoglobulin identified according to Eu or Kabat numbering. Those skilled in the art will appreciate that such numbering correspondingly represents alignment of the amino acid residues of a polypeptide with the amino acid residues identified in an immunoglobulin, e.g., the amino acid residues shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html.

[0570] As used herein, the cysteine residue at position 214 of a lambda light chain refers to the amino acid residue (cysteine, Cys, C) of an immunoglobulin identified according to Eu or Kabat numbering. Those skilled in the art will appreciate that such numbering correspondingly represents alignment of the amino acid residues of a polypeptide with the amino acid residues identified in an immunoglobulin, e.g., the amino acid residues shown in www.imgt.org / IMGTScientificChart / Numbering / Hu_IGLCnber.html.

[0571] In certain embodiments, the antibodies described herein comprise four interchain disulfide bonds in the hinge region, which disulfide bonds can be reduced, thereby breaking the bonds and exposing reactive thiol moieties that can bind to maleimide moieties on a linker-payload, e.g., maleimide moieties on linker-payloads described herein.

[0572] In certain embodiments, the antibodies described herein comprise lysine residues, wherein the reactive amine side chains of the lysine residues can bind to a linker-payload, e.g., maleimide moieties on linker-payloads described herein.

[0573] In one embodiment, the present disclosure provides a method of making an ADC described herein, comprising the steps of: a) providing a solution comprising an antibody; b) contacting the solution of a) with a reducing agent; c) contacting the solution of b) with a solution comprising a linker-payload or a salt thereof, as described herein, to make the ADC.

[0574] In one embodiment, the reducing agent is tris(2 carboxyethyl)phosphine (TCEP).

[0575] Pharmaceutical compositions or combinations

[0576] In another aspect, the present application provides a pharmaceutical composition comprising one or more antibody-drug conjugates or compositions of any of the preceding, and a pharmaceutically acceptable carrier and / or excipient.

[0577] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In some embodiments, the additional pharmaceutically active agent is a drug having anti-tumor activity. In some embodiments, the additional pharmaceutically active agent is selected from the group consisting of: an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-MET or VEGF inhibitor, a chemotherapeutic drug, or any combination thereof. In certain embodiments, the ADC or composition as described herein and the additional pharmaceutically active agent are provided as separate components or as a mixed component.

[0578] In certain embodiments, the ADC or composition in the pharmaceutical composition of the present application is sufficient to exert a tumor inhibitory effect (e.g., superior to that of a mono-specific anti-c-MET antibody and / or a mono-specific anti-EGFR antibody, wherein the CDRs of the mono-specific anti-c-MET antibody have the same amino acid sequences as the CDRs of the first antigen binding domain, and the CDRs of the mono-specific anti-EGFR antibody have the same amino acid sequences as the CDRs of the second antigen binding domain) (e.g., in a subject).

[0579] In certain embodiments, the tumor inhibitory effect comprises: inhibition of EGFR and c-MET signaling, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0580] In some embodiments, the antibody-drug conjugates described herein are typically formulated in dosage unit form for parenteral administration together with a pharmaceutically acceptable parentally acceptable vehicle, carrier, diluent or excipient. Optionally, the antibody-drug conjugates having the desired degree of purity are mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer to produce the dosage forms of this application, which are in unit- or multi-dose sealed containers, for example, ampoules, vials, pre-filled syringes, or test tubes (Remington's Pharmaceutical Sciences (1980) 16th Ed., Osol, A. Ed.). The antibody-drug conjugates described herein or pharmaceutical compositions comprising the antibody-drug conjugates can be administered to a subject by any appropriate route.

[0581] Therapeutic uses of ADCs

[0582] In another aspect, the disclosure provides use of the antibody-drug conjugate, composition, pharmaceutical composition or combination of the disclosure in the manufacture of a medicament for preventing and / or treating and / or as an adjunct medicament for treating a disease associated with c-MET and / or EGFR in a subject, and / or for inhibiting the activity of c-MET and / or EGFR in vitro or in a subject; wherein the disease associated with c-MET and / or EGFR includes, but is not limited to, a cancer associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations and / or c-MET gene amplification.

[0583] In some embodiments, the cancer is selected from the group consisting of an 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, pharynx cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular cancer, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer or head and neck cancer.

[0584] In some embodiments, the antibody-drug conjugate, or the pharmaceutical composition is administered in combination, e.g., simultaneously, separately or sequentially, with another pharmaceutically active agent. In some embodiments, the other pharmaceutically active agent is an agent having anti-tumor activity. In some embodiments, the other pharmaceutically active agent is selected from the group consisting of an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-MET or VEGF inhibitor, a chemotherapeutic drug or any combination thereof.

[0585] In another aspect, the present disclosure provides a method of inhibiting the activity of c-MET and / or EGFR in a cell, comprising contacting the cell with the antibody-drug conjugate or the pharmaceutical composition described herein. The cell is a cell expressing c-MET and / or EGFR, e.g., a tumor cell.

[0586] In another aspect, the present disclosure provides a method for preventing, treating and / or as an adjunct medicament for treating a disease associated with c-MET and / or EGFR in a subject, comprising administering to a subject in need thereof an effective amount of the antibody-drug conjugate, composition and / or pharmaceutical composition of any of the preceding.

[0587] In some embodiments, the disease associated with c-MET and / or EGFR is a cancer. In certain 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.

[0588] In some embodiments, the cancer is an 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, pharynx cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer. In certain embodiments, the method further comprises administering to the subject a second therapy 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.

[0589] In some embodiments, the second therapy can be administered simultaneously, separately, or sequentially with the above-described methods.

[0590] In particular embodiments, the present application provides an ADC, composition, or pharmaceutical composition as described herein for use in a method of preventing, treating, and / or as an adjunct medication for treating a disease associated with c-MET and / or EGFR in a subject. In some embodiments, the disease associated with c-MET and / or EGFR is a 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.

[0591] In some embodiments, the cancer is selected from the group consisting of an 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, pharynx cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, and head and neck cancer.

[0592] In some embodiments, the method further comprises administering to the subject a second therapy 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. In some embodiments, the second therapy can be administered simultaneously, separately, or sequentially.

[0593] The ADCs of the present application and the pharmaceutical compositions of the present application can be formulated into any dosage form known in the medical arts, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including solutions for injection, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present application should be sterile and stable under the conditions of manufacture and storage. The preferred dosage form is an injection. Such injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by adding the desired dosage of the ADCs or the pharmaceutical compositions of the present application to a suitable solvent, and optionally adding other desired ingredients (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) at the same time, followed by sterilization filtration. In addition, to facilitate storage and use, the sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying). Such sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0594] In addition, the ADCs of the present application can be present in unit dosage form in the pharmaceutical compositions to facilitate administration.

[0595] The ADCs and the pharmaceutical compositions of the present application 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 web, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will appreciate that the route and / or means of administration will vary depending on the intended purpose. In some preferred embodiments, the ADCs and the pharmaceutical compositions of the present application are administered by intravenous injection or injection.

[0596] The pharmaceutical compositions of the present application can comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the bispecific antibodies of the present application. A "prophylactically effective amount" refers to an amount that is sufficient to prevent, preclude, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount that is sufficient to cure, or at least partially arrest, the disease and its complications in an individual already suffering from the disease. The therapeutically effective amount of the bispecific antibodies of the present application can vary depending on the severity of the disease to be treated, the general state of the patient's immune system, the general condition of the patient, such as age, body weight, and sex, the mode of administration of the drug, and other therapeutic agents concurrently administered, and the like.

[0597] In the present application, the dosing regimen can be adjusted to achieve the optimal desired response (e.g., therapeutic or prophylactic response). For example, a single dose can be administered, multiple doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0598] In the present application, the subject can be a mammal, e.g., a human.

[0599] Kit

[0600] Also provided are kits comprising one or more ADCs described herein or a pharmaceutical composition thereof. In particular embodiments, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of a pharmaceutical composition described herein, e.g., one or more ADCs provided herein. In certain embodiments, the kits comprise a pharmaceutical composition described herein and any prophylactic or therapeutic agents, e.g., described herein. In certain embodiments, the kits can include a T cell mitogen, e.g., phytohemagglutinin (PHA) and / or phorbol myristate (PMA), or a TCR complex stimulating antibody, e.g., an anti-CD3 antibody and an anti-CD28 antibody. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of manufacture, use or sale of the item.

[0601] Also provided are kits useful in the methods described herein. In certain embodiments, the kits comprise an ADC described herein, preferably a purified ADC, disposed in one or more containers. In particular embodiments, the kits described herein comprise a substantially isolated c-MET and / or EGFR antigen as a control. In another particular embodiment, the kits described herein further comprise a control antibody that does not react with the c-MET or EGFR antigen. In another particular embodiment, the kits described herein comprise one or more elements for detecting binding of the antibody to the c-MET and / or EGFR antigen (e.g., the antibody can be conjugated to a detectable substrate (e.g., a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound), or a second antibody that recognizes the first antibody can be conjugated to a detectable substrate). In particular embodiments, the kits provided herein can include a recombinantly produced or chemically synthesized c-MET and / or EGFR antigen. The antigen provided in the kits can also be attached to a solid support. In a more particular embodiment, the detection means of the above-described kits comprises a solid phase support to which c-MET and / or EGFR antigen is attached. Such kits can also include unattached reporter-labeled anti-human or anti-mouse / rat antibodies. In this embodiment, binding of the bispecific antibody to the antigen can be detected by binding of the reporter-labeled antibodies.

[0602] Embodiments of the application include:

[0603] Embodiment 1. An antibody-drug conjugate comprising a structure as shown below:

[0604] Ab-[M-L-E-D] x

[0605] wherein:

[0606] Ab

[0607] is a bispecific antibody or antigen-binding fragment thereof comprising a first antigen binding domain that specifically binds c-MET and a second antigen binding domain that specifically binds EGFR; M is a linker moiety attached to the bispecific antibody or antigen-binding fragment thereof; L is a structural moiety linking between the linker moiety M and E; E is a structural moiety linking L and D; D is a cytotoxic drug or fragment thereof; and x is an integer selected from 1 to 10.

[0608] Embodiment 2. The antibody-drug conjugate of embodiment 1, wherein the first antigen binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), which together form a domain capable of specifically binding c-MET; and the second antigen binding domain comprises a second VL and a second VH, which together form a domain capable of specifically binding EGFR.

[0609] wherein the first VL comprises:

[0610] (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 36, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38, wherein the CDRs are defined by the Kabat numbering system;

[0611] (ii) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 36, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38, wherein the CDRs are defined by the Chothia numbering system;

[0612] (iii) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 36, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38, wherein the CDRs are defined by the Abm numbering system; or,

[0613] (iv) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 35, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 37, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 38, wherein the CDRs are defined by the IMGT numbering system;

[0614] and / or the first heavy chain (HC) variable region (VH) comprises:

[0615] (i) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 39, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 43, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 47, wherein the CDRs are defined by the Kabat numbering system;

[0616] (ii) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 40, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 44, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 47, wherein the CDRs are defined by the Chothia numbering system;

[0617] (iii) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 42, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 46, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 47, wherein the CDRs are defined by the Abm numbering system; or,

[0618] (iv) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 41, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 45, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 48, wherein the CDRs are defined by the IMGT numbering system.

[0619] Embodiment 3. The antibody-drug conjugate of embodiment 2, wherein the second VL comprises:

[0620] (i) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 19, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 21, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23, wherein the CDRs are defined by the Kabat numbering system;

[0621] (ii) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 19, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 21, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23, wherein the CDRs are defined by the Chothia numbering system;

[0622] (iii) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 19, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 21, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23, wherein the CDRs are defined by the Abm numbering system; or,

[0623] (iv) CDR-L1 comprising an amino acid sequence as set forth in SEQ ID NO: 20, CDR-L2 comprising an amino acid sequence as set forth in SEQ ID NO: 22, and CDR-L3 comprising an amino acid sequence as set forth in SEQ ID NO: 23, wherein the CDRs are defined by the IMGT numbering system;

[0624] and / or the second VH comprises:

[0625] (i) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 24, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 28, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 32

[0626] , wherein the CDRs are defined by the Kabat numbering system;

[0627] (ii) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 25, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 29, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 32, wherein the CDRs are defined by the Chothia numbering system;

[0628] (iii) CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 27, CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 31, and CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 32, wherein the CDRs are defined by the Abm numbering system; or,

[0629] (iv) a CDR-H1 comprising an amino acid sequence as set forth in SEQ ID NO: 26, a CDR-H2 comprising an amino acid sequence as set forth in SEQ ID NO: 30, and a CDR-H3 comprising an amino acid sequence as set forth in SEQ ID NO: 33, wherein the CDRs are defined by the IMGT numbering system.

[0630] Embodiment 4. The antibody-drug conjugate of any one of embodiments 2-3, wherein (i) the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 17 and the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 18; or (ii) the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 59 and the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 60.

[0631] Embodiment 5. The antibody-drug conjugate of any one of embodiments 2-4, wherein the second VL comprises an amino acid sequence as set forth in SEQ ID NO: 15 and / or the second VH comprises an amino acid sequence as set forth in SEQ ID NO: 16.

[0632] Embodiment 6. The antibody-drug conjugate of any one of embodiments 1-5, wherein the bispecific antibody or antigen binding fragment thereof further comprises an Fc domain comprising first and second Fc domain monomers, and each of the first and second Fc domain monomers independently comprises one or more modifications of amino acids that promote heterodimerization of the first and second Fc domain monomers.

[0633] Embodiment 7. The antibody-drug conjugate of embodiment 6, wherein the Fc domain comprises a first Fc domain monomer comprising amino acid modifications that form a knob structure and a second Fc domain monomer comprising amino acid modifications that form a hole structure, wherein the hole structure pairs with the knob structure to form a heterodimerized Fc domain dimer.

[0634] Embodiment 8. The antibody-drug conjugate of embodiment 6 or 7, wherein (a) the first Fc domain monomer comprises an amino acid sequence as set forth in SEQ ID NO: 49 and the second Fc domain monomer comprises an amino acid sequence as set forth in SEQ ID NO: 50; or (b) the first Fc domain monomer comprises an amino acid sequence as set forth in SEQ ID NO: 51 and the second Fc domain monomer comprises an amino acid sequence as set forth in SEQ ID NO: 52.

[0635] Embodiment 9. The antibody-drug conjugate of any one of embodiments 6-8, wherein the first antigen binding domain is linked to the first Fc domain monomer, 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, the second antigen binding domain is linked to the first Fc domain monomer.

[0636] Embodiment 10. The antibody-drug conjugate of any one of embodiments 1-9, wherein the first antigen binding domain is a Fab, and the second antigen binding domain is a scFv.

[0637] Embodiment 11. The antibody-drug conjugate of embodiment 10, wherein the bispecific antibody comprises a polypeptide chain I-A, a polypeptide chain I-B, and a polypeptide chain I-C; wherein,

[0638] (a) the elements comprised in the polypeptide chain I-A comprise from N- to C- terminus the first VL and light chain constant region, the elements comprised in the polypeptide chain I-B comprise from N- to C-terminus the first VHand heavy chain CH1 region and the first Fc domain monomer, and / or, the elements comprised in the polypeptide chain I-C comprise from N- to C-terminus (i) the second VL, the second VHand the second Fc domain monomer, or, (ii) the second VHand the second VL and the second Fc domain monomer; or,

[0639] (b) the elements comprised in the polypeptide chain I-A comprise from N- to C- terminus the first VL and light chain constant region, the elements comprised in the polypeptide chain I-B comprise from N- to C-terminus the first VHand heavy chain CH1 region and the second Fc domain monomer, and / or, the elements comprised in the polypeptide chain I-C comprise from N- to C-terminus (i) the second VL, the second VHand the first Fc domain monomer, or, (ii) the second VHand the second VL and the first Fc domain monomer.

[0640] Embodiment 12. The antibody-drug conjugate of embodiment 10 or 11, wherein (a) the polypeptide chain I-A comprises an amino acid sequence as set forth in SEQ ID NO: 1, the polypeptide chain I-B comprises an amino acid sequence as set forth in SEQ ID NO: 2, and / or, the polypeptide chain I-C comprises an amino acid sequence as set forth in SEQ ID NO: 3; or, (b) the polypeptide chain I-A comprises an amino acid sequence as set forth in SEQ ID NO: 1, the polypeptide chain I-B comprises an amino acid sequence as set forth in SEQ ID NO: 9, and / or the polypeptide chain I-C comprises an amino acid sequence as set forth in SEQ ID NO: 10.

[0641] Embodiment 13. The antibody-drug conjugate of any one of embodiments 1-9, wherein the first antigen binding domain is a scFv and the second antigen binding domain is a Fab.

[0642] Embodiment 14. The antibody-drug conjugate of embodiment 13, wherein (a) the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein the elements comprised in the polypeptide chain II-A comprise from N- to C-terminus the second VL and light chain constant region, the elements comprised in the polypeptide chain II-B comprise from N- to C-terminus the second VHand heavy chain CH1 region and the first Fc domain monomer, and / or the elements comprised in the polypeptide chain II-C comprise from N- to C-terminus (i) the first VL, the first VHand the second Fc domain monomer, or (ii) the first VHand the first VL and the second Fc domain monomer; or

[0643] (b) the bispecific antibody comprises polypeptide chain II-A, polypeptide chain II-B, and polypeptide chain II-C; wherein the elements comprised in the polypeptide chain II-A comprise from N- to C-terminus the second VL and light chain constant region, the elements comprised in the polypeptide chain II-B comprise from N- to C-terminus the second VHand heavy chain CH1 region and the second Fc domain monomer, and / or the elements comprised in the polypeptide chain II-C comprise from N- to C-terminus (i) the first VL, the first VHand the first Fc domain monomer, or, (ii) the first VHand the first VL and the first Fc domain monomer.

[0644] Embodiment 15. The antibody-drug conjugate of embodiment 13 or 14, wherein (a) the polypeptide chain II-A comprises the amino acid sequence of SEQ ID NO: 4, the polypeptide chain II-B comprises the amino acid sequence of SEQ ID NO: 5, and / or the polypeptide chain II-C comprises the amino acid sequence of SEQ ID NO: 6; or (b) the polypeptide chain II-A comprises the amino acid sequence of SEQ ID NO: 4, the polypeptide chain II-B comprises the amino acid sequence of SEQ ID NO: 7, and / or the polypeptide chain II-C comprises the amino acid sequence of SEQ ID NO: 8.

[0645] Embodiment 16. The antibody-drug conjugate of any one of embodiments 1-9, the first antigen binding domain is a scFab and the second antigen binding domain is a Fab.

[0646] Embodiment 17. The antibody-drug conjugate of embodiment 16, wherein (a) the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C; wherein the elements comprised in the polypeptide chain IV-A comprise from N- to C-terminus the second VL and light chain constant region, the elements comprised in the polypeptide chain IV-B comprise from N- to C-terminus the second VHand heavy chain CH1 region and the first Fc domain monomer, and / or, the elements comprised in the polypeptide chain IV-C comprise from N- to C-terminus (i) the first VL, light chain constant region, the first VHand heavy chain CH1 region and the second Fc domain monomer, or, (ii) the first VHand heavy chain CH1 region, the first VL, light chain constant region and the second Fc domain monomer; or,

[0647] (b) the bispecific antibody comprises polypeptide chain IV-A, polypeptide chain IV-B, and polypeptide chain IV-C; wherein the elements comprised in the polypeptide chain IV-A comprise from N- to C-terminus the second VL and light chain constant region, the elements comprised in the polypeptide chain IV-B comprise from N- to C-terminus the second VHand heavy chain CH1 region and the second Fc domain monomer, and / or, the elements comprised in the polypeptide chain IV-C comprise from N- to C-terminus (i) the first VL, light chain constant region, the first VHand heavy chain CH1 region and the first Fc domain monomer, or, (ii) the first VHand heavy chain CH1 region, the first VL, light chain constant region and the first Fc domain monomer.

[0648] Embodiment 18. The antibody-drug conjugate of embodiment 16 or 17, wherein the polypeptide chain IV-A comprises the amino acid sequence of SEQ ID NO: 4, the polypeptide chain IV-B comprises the amino acid sequence of SEQ ID NO: 7, and / or, the polypeptide chain IV-C comprises the amino acid sequence of SEQ ID NO: 11.

[0649] Embodiment 19. The antibody-drug conjugate of any one of embodiments 1-9, wherein the first and the second antigen binding domain are Fabs, and the Fab of the second antigen binding domain comprises a domain swap in the format of a CrossMab.

[0650] Embodiment 20. The antibody-drug conjugate of embodiment 19, wherein (a) the bispecific antibody comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D; wherein the elements comprised in the polypeptide chain V-A comprise from N- to C-terminus the first VL and a light chain constant region, the elements comprised in the polypeptide chain V-B comprise from N- to C-terminus the first VHand a heavy chain CH1 region and the first Fc domain monomer, the elements comprised in the polypeptide chain V-C comprise from N- to C-terminus the second VHand a light chain constant region and the second Fc domain monomer, and / or the elements comprised in the polypeptide chain V-D comprise from N- to C-terminus the second VL and a heavy chain CH1 region; or

[0651] (b) the bispecific antibody comprises polypeptide chain V-A, polypeptide chain V-B, polypeptide chain V-C, and polypeptide chain V-D; wherein the elements comprised in the polypeptide chain V-A comprise from N- to C-terminus the first VL and a light chain constant region, the elements comprised in the polypeptide chain V-B comprise from N- to C-terminus the first VHand a heavy chain CH1 region and the second Fc domain monomer, the elements comprised in the polypeptide chain V-C comprise from N- to C-terminus the second VHand a light chain constant region and the first Fc domain monomer, and / or the elements comprised in the polypeptide chain V-D comprise from N- to C-terminus the second VL and a heavy chain CH1 region.

[0652] Embodiment 21. The antibody-drug conjugate of embodiment 19 or 20, wherein the polypeptide chain V-A comprises the amino acid sequence of SEQ ID NO: 1, the polypeptide chain V-B comprises the amino acid sequence of SEQ ID NO: 9, the polypeptide chain V-C comprises the amino acid sequence of SEQ ID NO: 13, and / or the polypeptide chain V-D comprises the amino acid sequence of SEQ ID NO: 12.

[0653] Embodiment 22. The antibody-drug conjugate of any one of embodiments 1-9, wherein the first and the second antigen binding domain are scFvs.

[0654] Embodiment 23. The antibody-drug conjugate of embodiment 22, wherein (a) the bispecific antibody comprises polypeptide chain VII-A and polypeptide chain VII-B; wherein the elements comprised in the polypeptide chain VII-A comprise from N- to C-terminus:

[0655] (i) the first VL, the first VHand the first Fc domain monomer, or

[0656] (ii) the first VH, the first VL, and the second Fc domain monomer; and / or,

[0657] The elements included in the polypeptide chain VII-B comprise, from N- to C- terminus:

[0658] (x) the second VL, the second VH, and the first Fc domain monomer, or,

[0659] (y) the second VH, the second VL, and the first Fc domain monomer.

[0660] (b) the bispecific antibody comprises a polypeptide chain VII-A and a polypeptide chain VII-B; wherein the elements included in the polypeptide chain VII-A comprise, from N- to C-terminus:

[0661] (i) the first VL, the first VH, and the second Fc domain monomer, or,

[0662] (ii) the first VH, the first VL, and the second Fc domain monomer; and / or, the elements included in the polypeptide chain VII-B comprise, from N- to C-terminus:

[0663] (x) the second VL, the second VH, and the first Fc domain monomer, or,

[0664] (y) the second VH, the second VL, and the first Fc domain monomer.

[0665] Embodiment 24. The antibody-drug conjugate of embodiment 22 or 23, wherein the polypeptide chain VII-A comprises an amino acid sequence as set forth in SEQ ID NO: 14, and / or the polypeptide chain VII-B comprises an amino acid sequence as set forth in SEQ ID NO: 8.

[0666] Embodiment 25. The antibody-drug conjugate of embodiment 11, 14, 17, 20, or 23, wherein each adjacent elements included in each of the polypeptide chains are optionally connected by or without a linker.

[0667] Embodiment 26. The antibody-drug conjugate of embodiment 25, wherein the linkers are each independently the same or different peptide linker, wherein each of the peptide linkers is selected from a rigid peptide linker or a flexible peptide linker.

[0668] Embodiment 27. The antibody-drug conjugate of embodiment 26, wherein the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S).

[0669] Embodiment 28. The antibody-drug conjugate of embodiment 27, wherein the peptide linkers each independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 tandem copies of a peptide linker subunit comprising the amino acid sequence GGGGS (SEQ ID NO: 57).

[0670] Embodiment 29. The antibody-drug conjugate of embodiment 28, wherein the peptide linkers each independently comprise an amino acid sequence as set forth in SEQ ID NO: 55 or 56.

[0671] Embodiment 30. The antibody-drug conjugate of embodiment 11, 14, 17, 20, or 23, wherein the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 53 or SEQ ID NO: 63, and / or the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 54.

[0672] Embodiment 31. The antibody-drug conjugate of any one of embodiments 1-30, wherein the bispecific antibody or antigen-binding fragment thereof comprises:

[0673] (a) a polypeptide chain I-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, a polypeptide chain I-B comprising an amino acid sequence as set forth in SEQ ID NO: 2 or 9, and / or a polypeptide chain I-C comprising an amino acid sequence as set forth in SEQ ID NO: 3 or 10;

[0674] (b) a polypeptide chain II-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, a polypeptide chain II-B comprising an amino acid sequence as set forth in SEQ ID NO: 5 or 7, and / or a polypeptide chain II-C comprising an amino acid sequence as set forth in SEQ ID NO: 6 or 8;

[0675] (c) a polypeptide chain IV-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, a polypeptide chain IV-B comprising an amino acid sequence as set forth in SEQ ID NO: 7, and / or a polypeptide chain IV-C comprising an amino acid sequence as set forth in SEQ ID NO: 11;

[0676] (d) a polypeptide chain V-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, a polypeptide chain V-B comprising an amino acid sequence as set forth in SEQ ID NO: 9, a polypeptide chain V-C comprising an amino acid sequence as set forth in SEQ ID NO: 13, and / or a polypeptide chain V-D comprising an amino acid sequence as set forth in SEQ ID NO: 12; or

[0677] (e) a polypeptide chain VII-A comprising an amino acid sequence as set forth in SEQ ID NO: 14, and / or, a polypeptide chain VII-B comprising an amino acid sequence as set forth in SEQ ID NO: 8.

[0678] Embodiment 32. The antibody-drug conjugate of embodiment 28, wherein the bispecific antibody or antigen binding fragment thereof comprises:

[0679] (a) a polypeptide chain I-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, a polypeptide chain I-B comprising an amino acid sequence as set forth in SEQ ID NO: 2, and, a polypeptide chain I-C comprising an amino acid sequence as set forth in SEQ ID NO: 3;

[0680] (b) a polypeptide chain I-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, a polypeptide chain I-B comprising an amino acid sequence as set forth in SEQ ID NO: 9, and, a polypeptide chain I-C comprising an amino acid sequence as set forth in SEQ ID NO: 10;

[0681] (c) a polypeptide chain II-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, a polypeptide chain II-B comprising an amino acid sequence as set forth in SEQ ID NO: 5, and, a polypeptide chain II-C comprising an amino acid sequence as set forth in SEQ ID NO: 6;

[0682] (d) a polypeptide chain II-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, a polypeptide chain II-B comprising an amino acid sequence as set forth in SEQ ID NO: 7, and, a polypeptide chain II-C comprising an amino acid sequence as set forth in SEQ ID NO: 8;

[0683] (e) a polypeptide chain IV-A comprising an amino acid sequence as set forth in SEQ ID NO: 4, a polypeptide chain IV-B comprising an amino acid sequence as set forth in SEQ ID NO: 7, and, a polypeptide chain IV-C comprising an amino acid sequence as set forth in SEQ ID NO: 11;

[0684] (f) a polypeptide chain V-A comprising an amino acid sequence as set forth in SEQ ID NO: 1, a polypeptide chain V-B comprising an amino acid sequence as set forth in SEQ ID NO: 9, a polypeptide chain V-C comprising an amino acid sequence as set forth in SEQ ID NO: 13, and, a polypeptide chain V-D comprising an amino acid sequence as set forth in SEQ ID NO: 12; or,

[0685] (g) a polypeptide chain VII-A comprising an amino acid sequence as set forth in SEQ ID NO: 14, and, a polypeptide chain VII-B comprising an amino acid sequence as set forth in SEQ ID NO: 8.

[0686] Embodiment 33. The antibody-drug conjugate of any one of embodiments 1-30, wherein the bispecific antibody or antigen-binding fragment thereof has enhanced tumor inhibition relative to a monospecific anti-c-MET antibody and / or a monospecific anti-EGFR antibody; wherein,

[0687] (a) the CDR amino acid sequences of the monospecific anti-c-MET antibody are identical to the CDR amino acid sequences of the first antigen binding domain, and the CDR amino acid sequences of the monospecific anti-EGFR antibody are identical to the CDR amino acid sequences of the second antigen binding domain; or

[0688] (b) the CDR amino acid sequences of the monospecific anti-c-MET antibody are identical to the CDR amino acid sequences of the second antigen binding domain, and the CDR amino acid sequences of the monospecific anti-EGFR antibody are identical to the CDR amino acid sequences of the first antigen binding domain.

[0689] Embodiment 34. The antibody-drug conjugate of embodiment 33, wherein the tumor inhibition comprises inhibition of the EGFR and c-MET signaling pathways, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and / or complement-dependent cytotoxicity (CDC) activity.

[0690] Embodiment 35. The antibody-drug conjugate of any one of embodiments 1-34, M comprises

[0691] wherein ring A is a 5-6 membered aliphatic heterocyclic ring, or a 5-20 membered aromatic ring system, said aliphatic heterocyclic ring and aromatic ring system being optionally substituted with one or more groups independently selected from oxo (=0), halogen, cyano, amino, carboxyl, thiol, and C 1-6 alkyl. 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene, or amine.

[0692] Embodiment 36. The antibody-drug conjugate of any one of embodiments 1-34, M comprises wherein ring A is a 5 membered aliphatic heterocyclic ring, a 6 membered heteroaromatic ring, or a polycyclic ring formed by one or more 6 membered aromatic heterocyclic rings and a phenyl ring connected by a single bond, or a polycyclic ring formed by multiple 6 membered heteroaromatic rings connected by a single bond, said aliphatic heterocyclic ring being optionally substituted with one or more groups selected from oxo (=0), halogen, and C 1-4 alkyl. 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene, or amine.

[0693] Embodiment 37. The antibody-drug conjugate of any one of embodiments 1-34, wherein M is wherein ring A is selected from

[0694] M1is selected from a single bond and C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene or amine group.

[0695] Embodiment 38. The antibody-drug conjugate of any one of embodiments 1-34, wherein M is selected from

[0696] Embodiment 39. The antibody-drug conjugate of any one of embodiments 1-34, wherein M is

[0697] Embodiment 40. The antibody-drug conjugate of any one of embodiments 1-34, wherein M is selected from

[0698] Embodiment 41. The antibody-drug conjugate of any one of embodiments 1-34, wherein M is selected from

[0699] Embodiment 42. The antibody-drug conjugate of any one of embodiments 1-41, L is selected from one or more of the structures consisting of C 1-6 alkylene, -N(R’)-, carbonyl, -O-, a natural amino acid or non-natural amino acid selected from Ala, Arg, Asn, Asp, Cit, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH2CH2(OCH2CH2) s OCH3) and analogs thereof, and short peptides comprising 1, 2, 3 or 4 amino acids,

[0700]

[0701] wherein R’ represents hydrogen, C 1-6 alkyl or a polyethylene glycol fragment comprising 1-10 EO units; s is selected from an integer between 1 and 20.

[0702] Embodiment 43. The antibody-drug conjugate of embodiment 42, wherein the short peptide is selected from the group consisting of 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)).

[0703] Embodiment 44. The antibody-drug conjugate of any one of embodiments 1-41, wherein the L is selected from the group consisting of structures comprising one or more of: 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-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)),

[0704] wherein s is selected from an integer from 1 to 20.

[0705] Embodiment 45. The antibody-drug conjugate of any one of embodiments 1-41, wherein the L is selected from the structure consisting of one or more of the following:

[0706]

[0707]

[0708] Embodiment 46. The antibody-drug conjugate of any one of embodiments 1-41, wherein the L is selected from the following structure:

[0709]

[0710]

[0711] Embodiment 47. The antibody-drug conjugate of any one of embodiments 1-41, wherein the L is selected from the following structure:

[0712]

[0713] Embodiment 48. The antibody-drug conjugate of any one of embodiments 1-41, wherein the L is selected from the following structure:

[0714]

[0715] Embodiment 49. The antibody-drug conjugate of any one of embodiments 1-41, wherein the L is selected from the following structure:

[0716]

[0717] Embodiment 50. The antibody-drug conjugate of any one of embodiments 1-49, wherein E is a single bond or is selected from the following structure:

[0718] -NHCH2-,

[0719] Embodiment 51. The antibody-drug conjugate of any one of embodiments 1-49, wherein E is a single bond, -NHCH2-,

[0720] Embodiment 52. The antibody-drug conjugate of any one of embodiments 1-49, wherein E is -NHCH2- or

[0721] Embodiment 53. The antibody-drug conjugate of any one of embodiments 1-49, wherein E is -NHCH2-; preferably, E is a single bond.

[0722] Embodiment 54. The antibody-drug conjugate of any one of embodiments 1-49, wherein E is

[0723] Embodiment 55. The antibody-drug conjugate of any one of embodiments 1-55, selected from the following structures:

[0724]

[0725]

[0726]

[0727]

[0728]

[0729]

[0730]

[0731] Embodiment 56. The antibody-drug conjugate of any one of embodiments 1-54, wherein, selected from the following structures:

[0732]

[0733]

[0734] Embodiment 57. The antibody-drug conjugate of any one of embodiments 1-56, wherein the cytotoxic drug is selected from the group consisting of a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor.

[0735] Embodiment 58. The antibody-drug conjugate of embodiment 57, wherein the tubulin inhibitor is an auristatin or a maytansinoid.

[0736] Embodiment 59. The antibody-drug conjugate of embodiment 57, wherein the DNA intercalator is a pyrrolobenzodiazepine (PBD).

[0737] Embodiment 60. The antibody-drug conjugate of embodiment 57, wherein the DNA topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0738] Embodiment 61. The antibody-drug conjugate of embodiment 60, wherein the topoisomerase I inhibitor is selected from the group consisting of camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, and pharmaceutically acceptable salts, esters, or analogs thereof; and the topoisomerase II inhibitor is selected from the group consisting of doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, and pharmaceutically acceptable salts, esters, or analogs thereof.

[0739] Embodiment 62. The antibody-drug conjugate of embodiment 57, wherein the RNA polymerase inhibitor is a-amanitin or a pharmaceutically acceptable salt, ester, or analog thereof.

[0740] Embodiment 63. The antibody-drug conjugate of any one of embodiments 1-56, wherein the cytotoxic drug is selected from the group consisting of a compound of Formula I, Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or prodrug of a compound of Formula I, Formula II:

[0741]

[0742] wherein R1, R2are each independently selected from the group consisting of C 1-6 alkyl and halogen;

[0743] R3is selected from the group consisting of H and -CO-CH2OH;

[0744] R4and R5are each independently selected from the group consisting of H, halogen, and hydroxyl; or R4and R5are joined with the carbon atom to which they are attached to form a 5-6 membered oxygen-containing heterocycle;

[0745] R6is selected from the group consisting of hydrogen or -C 1-4 alkylene-NR a R b ;

[0746] R7is selected from the group consisting of 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 ;

[0747] wherein R a , R beach occurrence is independently selected from H, C 1-6 alkyl, -SO2-C 1-6 alkyl, and -CO-C 1-6 alkyl; wherein optionally Raand Rb, together with the atoms to which they are attached, form a 5-6 membered nitrogen-containing heterocyclic ring.

[0748] Embodiment 64. The antibody-drug conjugate of any one of embodiments 1-56, wherein the cytotoxic drug is selected from the following compounds or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or prodrug of the compound:

[0749]

[0750]

[0751] wherein the corresponding residue of the cytotoxic drug after the cytotoxic drug is linked to the linker is D in the general formula of embodiment 1.

[0752] Embodiment 65. The antibody-drug conjugate of any one of embodiments 1-56, wherein the cytotoxic drug is selected from the following compounds or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, or prodrug of the compound:

[0753]

[0754] wherein the corresponding fragment of the cytotoxic drug after the cytotoxic drug is linked to the linker is D in the general formula of embodiment 1.

[0755] Embodiment 66. The antibody-drug conjugate of embodiment 64 or 65, wherein D is a monovalent structure resulting from the loss of one H from -OH, -NH2, or a secondary amine group on the cytotoxic drug.

[0756] Embodiment 67. The antibody-drug conjugate of any one of embodiments 1-66, selected from:

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774] wherein Ab is a bispecific antibody or antigen-binding fragment thereof as defined in any one of embodiments 1-34;

[0775] represents the specific linkage mode of the thiol group of the cysteine residue in the bispecific antibody or antigen-binding fragment thereof to the other moiety in the antibody-drug conjugate; and

[0776] x represents the number of drug loads.

[0777] Embodiment 68. The antibody-drug conjugate of embodiment 67, wherein the thiol group in the bispecific antibody or antigen-binding fragment thereof and M in the antibody-drug conjugate form the antibody-drug conjugate through an addition reaction or a substitution reaction to form a thioether bond.

[0778] Embodiment 69. The antibody-drug conjugate of embodiments 67-68, wherein the Ab is a bispecific antibody selected from the group consisting of BsAb 07B, BsAb 10B, BsAb 38B, BsAb 41B, BsAb 49B, BsAb 55B, and BsAb 56B.

[0779] Embodiment 70. An antibody drug conjugate selected from the group consisting of 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, 5ADC 5B-A-14, and ADC 56B-A-14.

[0780] Embodiment 71. A composition comprising one or more antibody-drug conjugates according to any one of embodiments 1-70.

[0781] Embodiment 72. The composition of embodiment 71, wherein the DAR value (drug to antibody coupling ratio) of the composition is 1-10.

[0782] Embodiment 73. The composition of embodiment 71, wherein the DAR value (drug to antibody coupling 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.

[0783] Embodiment 74. The composition of embodiment 71, wherein the DAR value (drug to antibody coupling ratio) of the composition is 3-9.

[0784] Embodiment 75. The composition of embodiment 71, wherein the DAR value (drug to antibody coupling ratio) of the composition is 4-8.

[0785] Embodiment 76. The composition of embodiment 71, wherein the DAR value (drug antibody coupling 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.

[0786] Embodiment 77. A pharmaceutical composition comprising one or more antibody-drug conjugates of any one of embodiments 1-70 or the composition of embodiment 71, and a pharmaceutically acceptable carrier and / or excipient.

[0787] Embodiment 78. The pharmaceutical composition of embodiment 77, wherein the pharmaceutical composition further comprises one or more additional pharmaceutically active agents selected from the group consisting of an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-MET or VEGF inhibitor, a chemotherapeutic drug, or any combination thereof.

[0788] Embodiment 79. A pharmaceutical combination comprising a first therapeutic agent and a second therapeutic agent, wherein the first and second therapeutic agents are administered simultaneously or sequentially, the first therapeutic agent is selected from one or more antibody-drug conjugates of any one of embodiments 1-70 or the composition of any one of embodiments 71-76; and the second therapeutic agent is selected from an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-MET or VEGF inhibitor, a chemotherapeutic drug, or any combination thereof.

[0789] Embodiment 80. Use of the antibody-drug conjugate of any one of embodiments 1-70, the composition of any one of embodiments 71-76, the pharmaceutical composition of embodiment 77 or 78, or the combination of embodiment 80, in the manufacture of a medicament for preventing and / or treating and / or adjuvant treatment of a disease associated with c-MET and / or EGFR in a subject, and / or, for inhibiting the activity of c-MET and / or EGFR in a cell in vitro or in a subject; wherein the disease associated with c-MET and / or EGFR is a disease associated with EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations and / or c-MET gene amplification.

[0790] Embodiment 81. The use of embodiment 81, wherein the cancer is selected from the group consisting of an 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, pharynx cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular cancer, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0791] Embodiment 82. The use of embodiment 80 or 81, wherein the antibody-drug conjugate or the pharmaceutical composition is administered simultaneously, separately or sequentially in combination with another pharmaceutical agent; wherein the another pharmaceutical agent is selected from the group consisting of: an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-MET or VEGF inhibitor, a chemotherapeutic drug, or any combination thereof.

[0792] Embodiment 83. A method of inhibiting the activity of c-MET and / or EGFR in a cell, comprising contacting the cell with the antibody-drug conjugate of any one of embodiments 1-70, the composition of any one of embodiments 71-76, the pharmaceutical composition of embodiment 77 or 78, or the combination of embodiment 79; wherein the cell is a cell expressing c-MET and / or EGFR.

[0793] Embodiment 84. A method for preventing and / or treating and / or adjuvant therapy of a disease associated with c-MET and / or EGFR in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody-drug conjugate of any one of embodiments 1-70, the composition of any one of embodiments 71-76, the pharmaceutical composition of embodiment 77 or 78, or the pharmaceutical combination of embodiment 79; wherein the disease associated with c-MET and / or EGFR 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.

[0794] Embodiment 85. The method of embodiment 84, wherein the cancer is selected from the group consisting of an 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, pharynx cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular cancer, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0795] Embodiment 86. The method of embodiment 84 or 85, wherein the method further comprises administering to the subject a second therapy 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; optionally, the second therapy can be administered simultaneously, separately, or sequentially.

[0796] Embodiment 87. The antibody-drug conjugate of any one of embodiments 1-70, the composition of any one of embodiments 71-76, the pharmaceutical composition of embodiment 77 or 78, or the pharmaceutical combination of embodiment 79, for use in preventing and / or treating and / or adjuvant therapy of a disease associated with c-MET and / or EGFR in a subject, wherein the disease associated with c-MET and / or EGFR is a cancer comprising EGFR activating mutations, EGFR gene amplification, elevated circulating HGF levels, c-MET activating mutations, and / or c-MET gene amplification.

[0797] Embodiment 88. The antibody-drug conjugate of embodiment 87, wherein the cancer is selected from the group consisting of an epithelial cell carcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, oral cancer, colorectal cancer, anal cancer, and prostate cancer, bladder cancer, pharynx cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, thyroid cancer, hepatocellular cancer, sporadic or hereditary papillary renal cell carcinoma, colon cancer, liver cancer, kidney cancer, or head and neck cancer.

[0798] Embodiment 89. The antibody-drug conjugate of embodiment 87 or 88, wherein the prevention and / or treatment and / or adjunct therapy further comprises administering to the subject a second therapy selected from the group consisting of: surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjunct therapy, and any combination thereof; the second therapy can be applied simultaneously, separately, or sequentially as needed.

[0799] Embodiment 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; conjugated to one or more drug-linker 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.

[0800] Embodiment 91. 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; conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 molecules of a drug-linker, 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. Embodiment 92. A method for producing a bispecific antibody (BsAb) conjugate, comprising: providing a BsAb selected from the group consisting of: BsAb 07B, BsAb 10B, BsAb 38B, BsAb 41B, BsAb 49B, BsAb 55B, BsAb 56B, 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 to produce a BsAb conjugate; isolating the BsAb conjugate from the reaction mixture to produce the BsAb conjugate.

[0801] Embodiment 93. A method for producing a bispecific antibody (BsAb) conjugate having a DAR of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, comprising: providing a BsAb 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 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 an amount of the BsAb with an amount of the linker-payload to provide a reaction mixture capable of producing a BsAb conjugate having a DAR of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; incubating the reaction mixture under conditions suitable for producing a BsAb conjugate having a DAR of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; isolating the BsAb conjugate from the reaction mixture to produce a BsAb conjugate having a DAR of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0802] Sequence information

[0803] The description of the sequences to which this application relates is provided in the table below.

[0804]

[0805]

[0806]

[0807]

[0808]

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818] Example

[0819] 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.

[0820] The structures of the compounds described in the following examples were determined by NMR ( 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0821] Nuclear magnetic resonance (NMR) 1 The H NMR measurements were performed using a Bruker 400MHz NMR spectrometer; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0822] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0823] 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.

[0824] Mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0825] Example 1: Preparation of the compound

[0826] 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)

[0827]

[0828] Compound IM-1 (0.40 g, 640.59 µmol, its synthesis refer to patent application CN 111936169A), exatecan mesylate (0.37 g, 704.65 µmol) were dissolved in DMF (8 mL), HATU (0.32 g, 832.77 µmol) and DIPEA (0.25 g, 1.92 mmol) were added, and the reaction was carried out 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 a crude product, which was purified by preparative high performance liquid chromatography (conditions as follows) to obtain compound M-01 273 mg.

[0829] Column: Waters XBridge Prep C18 OBD 45mm×450mm×8.0µm

[0830] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

[0831] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0.00 30 70 70 5.00 30 70 70 60.00 70 30 70

[0832] The structural characterization data of M-01 are as follows:

[0833] ESI-MS (m / z): 1034.4 [M+H] + .

[0834] 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]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic amide (A-05)

[0835]

[0836] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (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 into DMF (19 mL), the system was warmed to 35 °C for 16 hours, then (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (1-4, 1.00 g, 1.77 mmol) was added, the system was cooled to 5-15 °C with ice water, DMTMM (0.98 g, 3.53 mmol) was added, then DIPEA (1.14 g, 8.84 mmol) was added dropwise, and the reaction was carried out 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), stirred for 10 minutes, and the DCM phase was separated, washed with brine (100 ml), and concentrated to obtain a crude product, which was purified by preparative high performance liquid chromatography and freeze-dried to obtain 0.98 g of compound A-05.

[0837] The separation and purification method of A-05 is as follows:

[0838] Chromatographic column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0839] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0840] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0.00 30 70 24 2.00 30 70 24 18.00 80 20 24

[0841] The structure characterization data of A-05 are as follows:

[0842] MS m / z (ESI): 1107.3 [M+H]+

[0843] 1H 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).

[0844] 1.3N-((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]pyrano[3',4':6,7]indolizine[1,2-b]quinoline- 1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-07) Preparation

[0845]

[0846] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5- yl)hex-5-ynoate (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 into DMF (1 mL), after the reaction system was warmed to 35 °C for 16 hours, (1S,9S)-1-amino-5-fluoro-9-ethyl-9-hydroxy-4-chloro-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[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,) were added, and the reaction system was reacted at 25 °C for 16 hours. The reaction solution was directly purified by preparative high performance liquid chromatography and freeze-dried to obtain 26.4 mg of compound A-07.

[0847] The separation and purification method of A-07 is as follows:

[0848] Column: SunFire Prep C18 OBD 19mmx150mmx5.0μm

[0849] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0850] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0 30 70 28 2 30 70 28 18 90 10 28

[0851] The structure characterization data of A-07 are as follows:

[0852] ESI-MS (m / z): 1111.3 [M+H] + .

[0853] 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]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1- yl)amino)-7,10-dimethyl-1,6,9,12-tetraoxo-3-oxa-5,8,11-triazatetradecyl)-6-(2-(methylsulfonyl) pyrimidin-5-yl)hex-5-ynoic amide (A-14)

[0854]

[0855] Step one:

[0856] Compound IM-4 (657 mg, 1.22 mmol) and compound 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 stirred at room temperature for 1 hour. After the completion of the reaction, the reaction solution was directly purified by preparative high-performance liquid chromatography and freeze-dried to obtain 700 mg of compound IM-5.

[0857] The preparative high-performance liquid chromatography purification method is as follows:

[0858] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0859] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0860] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0.00 30 70 28 2.00 30 70 28 18.00 90 10 28

[0861] Step two:

[0862] 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, and the reaction was allowed to proceed at room temperature for 1 hour. After the completion of the reaction, the reaction solution was directly purified by preparative high-performance liquid chromatography and freeze-dried to obtain 307 mg of compound IM-6.

[0863] The preparative high-performance liquid chromatography purification method is as follows:

[0864] Column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0865] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0866] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0.00 20 80 24 2.00 20 80 24 18.00 80 20 24

[0867] Step three:

[0868] 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 solution was stirred at room temperature for 16 hours. The reaction solution was directly purified by preparative high-performance liquid chromatography and freeze-dried to obtain 50.56 mg of compound A-14.

[0869] The structural characterization data are as follows:

[0870] MS m / z (ESI): 1002.4 [M+H]+

[0871] The preparation of high performance liquid chromatography purification method is as follows:

[0872] Chromatographic column: Waters SunFire Prep C18 OBD (5 μm*19 mm*150 mm)

[0873] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0874] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0.00 30 70 28 2.00 30 70 28 18.00 90 10 28

[0875] 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 = 7.2 Hz, 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).

[0876] 1.5 Synthesis of 4-((S)-2-(4-aminobutyl)-3-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanoylamino)benzyl ((S)-4-ethyl-11-(2-(N-(isopropyl)methylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) carbonate (B-01)

[0877]

[0878] Step one:

[0879] Compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis refer to patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL:0.5 mL) at room temperature, cuprous bromide (72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynoic amide (95.10 mg, 0.302 mmol) were added, the reaction was stirred for 1 h, then filtered, the filtrate was purified by preparative high performance liquid chromatography (conditions as follows), to obtain 30.00 mg of compound B-01-2.

[0880] Column: SunFire Prep C18 OBD 19mmx150mmx5.0um

[0881] Mobile phase A: acetonitrile; mobile phase B: water

[0882]

[0883]

[0884] Step two:

[0885] Compound B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL), trifluoroacetic acid (0.2 mL) was added, and the reaction was stirred at room temperature for 30 min. The reaction solution was concentrated under reduced pressure, and then purified by preparative high performance liquid chromatography (conditions as follows), to obtain 20.00 mg of trifluoroacetate salt of compound B-01.

[0886] Column: SunFire Prep C18 OBD 19mmx150mmx5.0um

[0887] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

[0888] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0.00 15 85 28 8.00 15 85 28 50.00 60 40 28

[0889] Structural characterization data as follows: ESI-MS (m / z): 1631.7 [M+H] + , 816.0 [M / 2+H] + .

[0890] 1.6 Preparation of 4-((S)-2-(4-aminobutyl)-3-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoylamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanoylamino)benzyl ((1S,9R)-9-ethyl-5-fluoro-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl) carbonate (B-02)

[0891]

[0892] Step one:

[0893] The methanesulfonate salt of 1-1 (30.00 mg, 56.44 μmol) was dissolved in N,N-dimethylformamide (1 mL) at 25 °C, and 1H-benzotriazol-1-yl oxytripyrrolidinophosphonium 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) were added successively, and the reaction was maintained at 25 °C for 1 h. The reaction was monitored by liquid chromatography-mass spectrometry. After the reaction was completed, water was added to the reaction solution, and ethyl acetate was used for extraction. 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.

[0894] Step two:

[0895] B-02-1 (20 mg, 27.33 μmol) was dissolved in dichloromethane (2 mL) at 0 °C, 4-dimethylaminopyridine (26.71 mg, 218.61 μmol) and a solution of triphosgene (8.11 mg, 27.33 μmol) in dichloromethane (0.5 mL) were added successively, and the reaction was kept at 0 °C for 0.5 h; after the residual triphosgene was replaced by nitrogen, a solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatetracosanamido)-N-(4- (hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (43.46 mg, 40.99 μmol) in dichloromethane (1 mL) was added dropwise, and the reaction was kept at 0 °C for 0.5 h; the reaction was monitored by LC-MS, and after completion of the reaction, the reaction solution was concentrated. The crude product was purified by thin layer chromatography (dichloromethane:methanol = 15:1) to obtain 30.00 mg of compound B-02-2.

[0896] Step three:

[0897] B-02-2 (250.00 mg, 137.51 μmol) was dissolved in a mixture of DMSO (2 mL) and water (0.4 mL) at 25 °C, 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynoic amide (62.98 mg, 206.26 μmol) and cuprous bromide (39.45 mg, 275.01 μmol) were added, and the reaction was kept at 25 °C for 1 h; the reaction was monitored by LC-MS; after completion of the reaction, the reaction solution was purified by preparative high performance liquid chromatography (conditions as follows), and the preparation was lyophilized to obtain 150.00 mg of compound B-02-3.

[0898] Column: SunFire Prep C18 OBD 19mm x 150mm x 5.0μm

[0899] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

[0900] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0 15 85 28 18 90 10 28

[0901] Step four:

[0902] B-02-3 (150 mg, 49.45 μmol) was dissolved in tetrahydrofuran (1 mL) at 25 °C, and a solution of tetrabutylammonium fluoride (1 M in tetrahydrofuran) / glacial acetic acid mixture (v / v = 13 / 1) (50 uL) was added dropwise, and the reaction was kept at 25 °C for 0.5 h; the reaction was monitored by LC-MS; after completion of the reaction, the reaction solution was purified by preparative high performance liquid chromatography (conditions as follows), and the preparation was lyophilized to obtain 50.00 mg of compound B-02-4.

[0903] Column: SunFire Prep C18 OBD 19mmx150mmx5.0um

[0904] Mobile Phase A: Acetonitrile; Mobile Phase B: Water (0.05% Formic Acid)

[0905] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0 15 85 28 20 90 10 28

[0906] Step five:

[0907] B-02-4 (50 mg, 26.52 μmol) was dissolved in dichloromethane (1 mL) at 25 °C, trifluoroacetic acid (60.49 mg, 530.49 μmol) was added, and the reaction was kept at 25 °C for 0.5 h; the reaction was monitored by LC-MS; after the reaction was completed, the reaction solution was concentrated, and the crude product was purified by preparative high performance liquid chromatography (conditions as follows), and the preparation was lyophilized to obtain 23.69 mg of compound B-02.

[0908] Column: SunFire Prep C18 OBD 19mmx150mmx5.0um

[0909] Mobile Phase A: Acetonitrile; Mobile Phase B: Water (0.05% Formic Acid)

[0910] Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [mL / min] 0 15 85 28 18 90 10 28

[0911] The structure characterization data of B-02 are as follows:

[0912] ESI-MS (m / z): 1613.6 [M+H] + .

[0913] 1.7 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]pyrano[3',4':6,7]indolizine[1,2-b]quinoline- 1-yl)aminol)-7,10,13-trimethyl-1,6,9,12,15-pentaoxo-3,17,20,23-tetraoxa-5,8,11,14- tetraazapentacosyl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzamide (C-07)

[0914]

[0915] Step one:

[0916] Compound C-07-2 (510...

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 containing 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), wherein the first VL and the first VH together form a domain capable of specifically binding c-MET; 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; or, the first VL comprises the amino acid sequence shown in SEQ ID NO: 59, and the first VH comprises the amino acid sequence shown in SEQ ID NO:

60. The second antigen-binding domain comprises a second VL and a second VH, which together form a domain capable of specifically binding to EGFR; the second VL comprises the amino acid sequence shown in SEQ ID NO: 15, and the second VH comprises the amino acid sequence shown in SEQ ID NO:

16. M is a linker site that is connected to the bispecific antibody or its antigen-binding fragment; L is a structural fragment connecting linker sites M and E; E is a structural fragment connecting L and D; Selected from the following structures: and ； D is a cytotoxic drug or a fragment thereof, said cytotoxic drug being selected from compounds of Formula I or II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of compounds of Formula I or II: 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; The cytotoxic drug and linker The corresponding fragment of the cytotoxic drug obtained after ligation is D in the general formula; and x is any integer from 1 to 10.

2. The antibody-drug conjugate of claim 1, 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.

3. The antibody-drug conjugate according to claim 1 or 2, wherein, Selected from the following structures: and 。 4. The antibody-drug conjugate of claim 1 or 2, wherein the cytotoxic drug is selected from the group consisting of compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of said compounds: and .

5. The antibody-drug conjugate of claim 1, wherein, Selected from the following structures: and ;and The cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, or prodrugs of the compounds: and .

6. The antibody-drug conjugate of claim 4, wherein, D represents the monovalent structure obtained by removing an H atom from the -OH or -NH2 group on the cytotoxic drug.

7. Antibody-drug conjugates, 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 in, Ab is a bispecific antibody or its antigen-binding fragment as defined in claim 1 or 2; This indicates the specific connection method between the thiol group of the cysteine ​​residue in the bispecific antibody or its antigen-binding fragment and the linker site in the antibody-drug conjugate that connects to the bispecific antibody or its antigen-binding fragment; and x represents the amount of drug load.

8. The antibody-drug conjugate of claim 7, wherein the thiol group in the bispecific antibody or its antigen-binding fragment is bonded to the linker site in the antibody-drug conjugate by a substitution reaction to form a thioether bond, thereby obtaining the antibody-drug conjugate.

9. 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.

10. A composition comprising one or more antibody-drug conjugates as described in any one of claims 1-9.

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

12. The composition of claim 11, wherein the DAR value of the composition is 3.0 to 4.0, 4.5 to 5.0, or 5.5 to 8.

0.

13. A pharmaceutical composition comprising one or more antibody-drug conjugates according to any one of claims 1-9 or compositions according to any one of claims 10-12, and a pharmaceutically acceptable carrier and / or excipient.

14. The pharmaceutical composition of claim 13, 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.

15. 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 1-9 or compositions according to any one of claims 10-12; 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.

16. Use of the antibody-drug conjugate of any one of claims 1-9, the composition of any one of claims 10-12, the pharmaceutical composition of claim 13 or 14, or the pharmaceutical combination of claim 15 in the preparation of a medicament, wherein the medicament is used in a subject for the prevention, treatment, and / or adjunctive treatment of cancers associated with c-MET and / or EGFR.

17. The use according to claim 16, 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.

18. The use according to claim 16 or 17, 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.

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