Antibody-drug conjugate targeting c-met and medical use thereof

Abstract

An antibody-drug conjugate targeting c-Met and the medical use thereof. The anti-c-Met antibody-drug conjugate has the structure as shown in formula (I): AB-(L-Y-D)n(I), wherein Ab is an anti-c-Met antibody, and L, Y and D are as defined in the description.

Description

Antibody-drug conjugate targeting c-Met and its medical use

[0001] This disclosure claims priority to patent application CN202310916920.7 filed on July 25, 2023. Technical Field

[0002] The present disclosure relates to anti-c-Met antibody-drug conjugates, methods for preparing the same, pharmaceutical compositions comprising the same, and uses thereof in preparing medicaments for treating c-Met-mediated diseases or conditions, particularly in preparing anti-cancer drugs. Background Art

[0003] Recent molecular biology and tumor pharmacology studies have demonstrated that cellular signaling pathways involving protein tyrosine kinases (PTKs) play a crucial role in tumor formation and progression. Over 50% of proto-oncogenes and oncogene products possess tyrosine kinase activity. The c-Met proto-oncogene belongs to the Ron subfamily of the PTK family. The encoded c-Met protein is a high-affinity receptor for hepatocyte growth factor / scatter factor (HGF / SF). The HGF / c-Met signaling pathway is closely associated with angiogenesis and tumor growth. Its sustained activation is a key factor in the carcinogenesis of tissue cells or the hyperproliferation of cancer cells. Inhibiting this pathway has become a new approach for targeted tumor therapy.

[0004] The c-Met proto-oncogene is located on the long arm of human chromosome 7 (7q31). It is over 120 kb in size and encodes a c-Met protein precursor with a molecular weight of approximately 150 kDa. This protein undergoes partial glycosylation to produce a 170 kDa glycoprotein, which is further cleaved into an α subunit (50 kDa) and a β subunit (140 kDa), linked by disulfide bonds to form the mature c-Met protein receptor. This heterodimer consists of two chains: the β chain has an extracellular region, a transmembrane region (also known as a membrane extension), and an intracellular region (containing the intracellular tyrosine kinase binding site). The α chain has only an extracellular portion, but it is heavily glycosylated and attached to the β chain via disulfide bonds. The extracellular regions of both subunits serve as the recognition sites for their respective ligands, while the intracellular regions possess tyrosine kinase activity.

[0005] There are three mechanisms of c-Met activation: HGF-dependent, HGF-independent, and other membrane-dependent pathways, such as CD44, a receptor on the hyaluronic acid membrane, adhesins, and RON signaling. The most common mechanism is HGF-dependent. HGF binds to c-Met at its N-terminus, promoting dimerization and autophosphorylation at Tyr1234 and Tyr1235 on the β chain. Phosphorylation of Tyr1349 and Tyr1356 near the C-terminus creates multiple binding sites for adaptor proteins. These adaptor proteins induce downstream signaling mediated by Pi3K / Akt, Ras / MAPK, c-Src, and STAT3 / 5, triggering diverse cellular responses, including cell survival and motility (closely linked to the Pi3K / Akt pathway), tumor metastasis, and cell proliferation (primarily mediated by Ras / MAPK). In addition, c-Met cross-talks with other membrane receptors, and it is now known that this cross-talk can promote tumor formation and metastasis. Since c-Met is the intersection of many pathways that lead to tumor formation and metastasis, targeting c-Met can relatively easily achieve simultaneous interference with many pathways, making c-Met a promising target for anti-tumor and anti-metastasis therapy.

[0006] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds. These ADCs leverage the antibody's specificity for tumor cell-specific or highly expressed antigens and the high efficacy of the cytotoxin, while avoiding toxic side effects on normal cells. This means that compared to traditional chemotherapy drugs, ADCs can precisely bind to tumor cells while minimizing the effects on normal cells.

[0007] Currently reported c-Met-targeted ADCs include Telisotuzumab Vedotin (ABBV-399, AbbVie, WO2017201204) and TR1801-ADC (Tanabe, Gymnopoulos, Marco, et al. "TR1801-ADC: a highly potent cMet antibody–drug conjugate with high activity in patient-derived xenograft models of solid tumors." Molecular Oncology 14.1(2020):54-68.). Telisotuzumab Vedotin is based on the anti-c-Met monoclonal antibody ABT-700 and the tubulin inhibitor MMAE connected by a cleavable linker. TR1801-ADC is an anti-c-Met antibody conjugated to a pyrrolobenzodiazepine dimer (PBD). Although there are some c-Met-targeted ADCs, none have been approved for marketing. Currently, only Telisotuzumab Vedotin is in Phase III clinical trials, while others are still in early clinical or preclinical stages, where their safety and efficacy are yet to be explored. Therefore, there is an urgent clinical need to develop c-Met-targeted ADCs with high safety and efficacy.

[0008] Summary of the Invention

[0009] The present disclosure provides an anti-c-Met antibody-drug conjugate or a pharmaceutically acceptable salt thereof and medical uses thereof.

[0010] Antibody-drug conjugates

[0011] The present disclosure provides an antibody-drug conjugate targeting c-Met or a pharmaceutically acceptable salt thereof, which has a structure shown in formula (I): Ab-(LYD)n Formula (I)

[0012] in:

[0013] Ab is an anti-c-Met antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence of any one of SEQ ID NOs: 10-12, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence of any one of SEQ ID NOs: 13-15;

[0014] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems;

[0015] -L- is a linker unit, which is -L 1 -L 2 -L 3 -L 4 -,

[0016] L 1 -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0017] L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20;

[0018] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0019] L 4 Selected from-NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6;

[0020] R 3 、R 4 and R 5are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0021] R 6 and R 7 are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

[0022] Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0023] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0024] R 1 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group or a heteroaryl group;

[0025] R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group or a heteroaryl group;

[0026] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0027] Or, R a and R2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0028] m is an integer from 0 to 4;

[0029] n is 1 to 10, and n is an integer or a decimal;

[0030] D is a camptothecin drug.

[0031] In some embodiments, D is exitecan or a derivative thereof.

[0032] In some embodiments, L 1 Connected to Ab, L 4 Connect with Y.

[0033] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof, wherein the anti-c-Met antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL); the VH and VL are selected from any one of the following groups:

[0034] (1) the VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1-3, and the VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 5 and 6, for example, the amino acid sequence shown in SEQ ID NO: 22 is RAX1KSVSTSTYNYLH, and X1 is selected from N or D;

[0035] Table 1. CDR sequences

[0036] (2) The VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of any one of SEQ ID NOs: 10-12, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of any one of SEQ ID NOs: 13-15.

[0037] In some embodiments, the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0038] In some embodiments, the anti-c-Met antibody comprises a VH and a VL, wherein any HCDR comprised in the VH has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned HCDRs; and / or any LCDR comprised in the VL has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned LCDRs. In some specific embodiments, the amino acid mutations are conservative substitutions, replacements, or modifications, and / or deletions or additions that do not affect function.

[0039] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody, its VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2, and 3, and its VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 7, 5 and 6.

[0040] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody, its VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2, and 3, and its VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 4, 5 and 6.

[0041] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.

[0042] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody comprises a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 9. The anti-c-Met antibody is a murine antibody.

[0043] Heavy chain variable region:

[0044] Light chain variable region:

[0045] The amino acid residues of the VH / VL CDRs of the anti-human c-Met antibody are determined and annotated according to the Kabat numbering system, with the CDRs in bold.

[0046] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the heavy chain variable region of the c-Met antibody further comprises a heavy chain FR region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof.

[0047] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the heavy chain FR region sequence on the heavy chain variable region of the c-Met antibody is derived from a human germline heavy chain sequence, for example, human germline heavy chain IGHV 3-33*01; a framework sequence comprising FR1, FR2, FR3 and FR4 regions of the human germline heavy chain IGHV 3-33*01, or a mutant sequence thereof, for example, the mutant sequence is a back mutation of 0-10 amino acids.

[0048] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the c-Met antibody, the light chain FR region sequence on its light chain variable region is selected from a human germline light chain sequence, for example, the human germline light chain IGKV085 or IGKV 4-1*01, comprising the framework sequence of FR1, FR2, FR3 and FR4 regions of the human germline light chain IGKV085 and IGKV 4-1*01 or a mutant sequence thereof, for example, the mutant sequence is a back mutation of 0-10 amino acids.

[0049] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody is a humanized antibody. In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody comprises a heavy chain variable region sequence selected from SEQ ID NOs: 10-12 and a light chain variable region sequence selected from SEQ ID NOs: 13-15.

[0050] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody comprises a combination of a heavy chain variable region sequence and a light chain variable region sequence selected from any one of a) to c):

[0051] a) a heavy chain variable region sequence of SEQ ID NO: 10, or a heavy chain variable region sequence having at least 90% sequence identity thereto, and a light chain variable region sequence of SEQ ID NO: 13, or a light chain variable region sequence having at least 90% sequence identity thereto;

[0052] b) a heavy chain variable region sequence of SEQ ID NO: 11, or a heavy chain variable region sequence having at least 90% sequence identity thereto, and a light chain variable region sequence of SEQ ID NO: 14, or a light chain variable region sequence having at least 90% sequence identity thereto;

[0053] Alternatively, c) the heavy chain variable region sequence of SEQ ID NO: 12, or a heavy chain variable region sequence having at least 90% sequence identity thereto, and the light chain variable region sequence of SEQ ID NO: 15, or a light chain variable region sequence having at least 90% sequence identity thereto.

[0054] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody comprises a heavy chain variable region sequence of SEQ ID NO: 11 and a light chain variable region sequence of SEQ ID NO: 14.

[0055] Heavy chain variable region:

[0056] Ab-9

[0057] Ab-10

[0058] Ab-11

[0059] Light chain variable region:

[0060] Ab-9

[0061] Ab-10

[0062] Ab-11

[0063] In some embodiments, the anti-c-Met antibody further comprises a light chain constant region, and / or a heavy chain constant region.

[0064] In some embodiments, the light chain constant region is derived from a kappa light chain, a lambda light chain, or a variant of any of the foregoing. For example, it is derived from a human kappa light chain, a human lambda light chain, or a variant of any of the foregoing. In some specific embodiments, the light chain constant region is derived from a human kappa light chain or a variant thereof.

[0065] In some embodiments, the heavy chain constant region is derived from IgG1, IgG2, IgG3, IgG4, or a variant of any of the foregoing, for example, from human IgG1, human IgG2, human IgG3, human IgG4, or a variant of any of the foregoing.

[0066] In some embodiments, the anti-c-Met antibody further comprises an Fc region. In some specific embodiments, the Fc region is derived from the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some specific embodiments, the Fc region is the Fc region of human IgG2.

[0067] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, the c-Met antibody is a humanized antibody, wherein the heavy chain constant region of the humanized antibody comprises a constant region derived from human IgG1 or a variant thereof, human IgG2 or a variant thereof, human IgG3 or a variant thereof, or human IgG4 or a variant thereof. In some embodiments, the heavy chain constant region of the humanized antibody comprises a constant region of human IgG1 or a variant thereof, or human IgG2 or a variant thereof, or human IgG4 or a variant thereof. In some embodiments, the heavy chain constant region of the humanized antibody comprises a constant region of human IgG2 or a variant thereof.

[0068] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody:

[0069] (1) the heavy chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 16, 18, and 20, or a sequence having at least 80% sequence identity thereto; and / or the light chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 17, 19, and 21, or a sequence having at least 80% sequence identity thereto;

[0070] (2) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 16, or an amino acid sequence having at least 80% sequence identity thereto; and / or the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 17, or an amino acid sequence having at least 80% sequence identity thereto;

[0071] (3) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence having at least 80% sequence identity thereto; and / or the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having at least 80% sequence identity thereto; or

[0072] (4) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 20, or a sequence having at least 80% sequence identity thereto; and / or the light chain comprises an amino acid sequence as shown in SEQ ID NO: 21, or a sequence having at least 80% sequence identity thereto.

[0073] In the context of this disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity; "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0074] Ab-9 humanized antibody:

[0075] Heavy chain:

[0076] Light chain:

[0077] Ab-10 humanized antibody:

[0078] Heavy chain:

[0079] Light chain:

[0080] Ab-11 humanized antibody:

[0081] Heavy chain:

[0082] Light chain:

[0083] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the anti-c-Met antibody comprises a heavy chain sequence as shown in SEQ ID NO: 18 and a light chain sequence as shown in SEQ ID NO: 19.

[0084] In some embodiments, the anti-c-Met antibodies encompass antigen-binding fragments. In some embodiments, the antigen-binding fragments include, but are not limited to, Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, and domain antibodies. For example, the antigen-binding fragment is an scFv, Fv, Fab, or Fab' fragment. In some embodiments, the anti-c-Met antibodies include monospecific antibodies, multispecific antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies). In some embodiments, the anti-c-Met antibodies include diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv, and the like.

[0085] In some embodiments, the anti-c-Met antibody in the antibody-drug conjugate binds to or competes for binding to the same c-Met epitope as any of the aforementioned anti-c-Met antibodies.

[0086] In some embodiments, the anti-c-Met antibody in the antibody-drug conjugate blocks the binding of any of the aforementioned anti-c-Met antibodies to c-Met.

[0087] In some embodiments, the binding of the anti-c-Met antibody in the antibody-drug conjugate to c-Met is blocked by any of the aforementioned anti-c-Met antibodies.

[0088] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items,

[0089] in:

[0090] Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0091] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen and an alkyl group;

[0092] R 1 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl;

[0093] R 2 selected from hydrogen atoms, halogenated alkyl groups and C 3-6 Cycloalkyl;

[0094] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0095] m is 0 or 1.

[0096] In some embodiments, the antibody-drug conjugate represented by formula (I) (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein Y is selected from:

[0097] The O end of Y is connected to the linker unit L.

[0098] In some embodiments, the antibody-drug conjugate represented by the general formula (I) (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0099] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O and S, wherein said C 1- 8 alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0100] L 2 Selected from-NR 4 (CH2CH2O)pCH2CH2C(O)-、-NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- or a chemical bond, wherein p is an integer from 1 to 20;

[0101] L 3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0102] L 4 Selected from-NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - and chemical bonds, wherein t is an integer from 1 to 6;

[0103] R 3 、R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0104] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0105] In some embodiments, the antibody-drug conjugate represented by the general formula (I) (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0106] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight chain heteroalkyl of 1 to 8 chain atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0107] L 2 Selected from-NR 4 (CH2CH2O)pCH2CH2C(O)-、-NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- or a chemical bond, wherein p is an integer from 1 to 20;

[0108] L 3A peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0109] L 4 Selected from-NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - and chemical bonds, wherein t is an integer from 1 to 6, non-limiting examples being 1, 2, 3, 4, 5, and 6;

[0110] R 3 、R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0111] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0112] In some embodiments, the antibody-drug conjugate represented by the general formula (I) (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0113] L 1 for s 1 is an integer from 2 to 8, non-limiting examples being 2, 3, 4, 5, 6, 7 and 8;

[0114] L 2 is a chemical bond;

[0115] L 3 is a tetrapeptide residue; for example, L 3 is the tetrapeptide residue of GGFG;

[0116] L 4 -NR 5 (CR 6 R7 )t-,R 5 、R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2;

[0117] The L 1 The end is connected to PC, L 4 Connect the Y end.

[0118] In some embodiments, the camptothecin drug is, for example, exitecan.

[0119] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items is an antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof:

[0120] in:

[0121] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0122] L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20;

[0123] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0124] R 1 is selected from hydrogen, halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0125] R 2is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0126] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0127] R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0128] R 6 and R 7 are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

[0129] m is an integer from 0 to 4;

[0130] n is 1 to 10, and n is an integer or a decimal;

[0131] Ab is as defined in general formula (I).

[0132] In some embodiments, the antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein n is 1 to 10, for example, 1 to 8, 2 to 8, 2 to 7, 2 to 4, 3 to 8, 3 to 7, 3 to 6, 4 to 7 or 4 to 6, and n is a decimal or an integer. In some embodiments, n is 1 to 8, and n is a decimal or an integer. In some embodiments, n is 3 to 7, and n is a decimal or an integer. In some embodiments, n is 4 to 6, and n is a decimal or an integer. In some embodiments, n is an average value of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10. In some embodiments, n is an average of about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.

[0133] In some embodiments, the antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein:

[0134] The -LY- is shown in the following structure:

[0135] s 1 is an integer from 2 to 8;

[0136] L 2 , L 3 、R1 、R 2 、R 5 、R 6 、R 7 and m are as defined above in the general formula (II).

[0137] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items is of formula (III) (Ab-L b -YD) shown in the antibody - drug conjugate or a pharmaceutically acceptable salt thereof:

[0138] in:

[0139] s 1 is an integer from 2 to 8;

[0140] Pc, R 1 、R 2 、R 5 、R 6 、R 7 , m and n are as defined in the general formula (II).

[0141] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein -L- is:

[0142] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein -LY- is selected from:

[0143] In some embodiments, wherein -LY- is selected from:

[0144] In some embodiments, wherein -LY- is

[0145] In some embodiments, wherein -LY- is:

[0146] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items is selected from:

[0147] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the antibody-drug conjugate is:

[0148] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items is selected from:

[0149] wherein Ab and n are as defined in the general formula (I).

[0150] In some embodiments, the antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the antibody-drug conjugate is:

[0151] in:

[0152] n is 1 to 8, n is a decimal or an integer, such as an integer or decimal of 3-7, or an integer or decimal of 4-6;

[0153] Ab is an anti-c-Met antibody comprising a heavy chain as shown in SEQ ID NO: 18 and a light chain as shown in SEQ ID NO: 19.

[0154] Preparation method

[0155] In some embodiments, the present disclosure provides a method for preparing an antibody-drug conjugate, comprising: reacting any of the aforementioned anti-c-Met antibodies with a drug to obtain the antibody-drug conjugate. Furthermore, the method further comprises: purifying the antibody-drug conjugate.

[0156] In some embodiments, a method for preparing an antibody-drug conjugate as shown in formula (I) comprises the following steps:

[0157] After Ab is reduced, it is coupled with the general formula (La-YD) to obtain a compound represented by the general formula (II);

[0158] Wherein, Ab is the anti-c-Met antibody disclosed herein; W, L 2 , L 3 、R 1 、R 2 、R 5 to R 7 , m and n are as defined in formula (I).

[0159] In some embodiments, the reducing agent is TCEP.

[0160] In some embodiments, the reducing agent reduces disulfide bonds on the antibody.

[0161] Pharmaceutical composition

[0162] The present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate according to any one of the preceding items or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0163] Medical uses and treatments

[0164] The present disclosure provides use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, or a pharmaceutical composition comprising the same as a drug.

[0165] The present disclosure provides the use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, as described in any of the preceding items for use in a medicament for treating a disease or condition mediated by c-Met. In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as described in any of the preceding items, is used for treating cancer. In some embodiments, the cancer is a cancer that expresses c-Met. In some embodiments, the cancer is a cancer that expresses low, medium, and / or high c-Met, for example, a cancer that expresses medium and / or high c-Met. In some embodiments, the cancer is gastric cancer, lung cancer, intestinal cancer, and / or liver cancer.

[0166] The antibody-drug conjugate provided by the present disclosure as described in any of the preceding items has tumor targeting, tumor cell toxicity, and bystander killing, and can be used to inhibit the activity of tumor cells in vitro or in vivo in a subject, kill tumor cells, and treat diseases related to c-Met mediation.

[0167] In some specific embodiments, the present disclosure provides methods for treating a subject having a cancer or tumor, or a subject at risk of developing cancer, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate or pharmaceutical composition. In some embodiments, the cancer is a c-Met-expressing cancer. In some embodiments, the cancer is gastric cancer, lung cancer, intestinal cancer, and / or liver cancer.

[0168] In some embodiments, the anti-c-Met antibody-drug conjugates or pharmaceutical compositions of the present disclosure can be administered by any suitable method known in the art, including but not limited to parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), intratumoral administration. The anti-c-Met antibody-drug conjugates or pharmaceutical compositions can be administered by any suitable route, such as by infusion or bolus injection. Administration can be systemic or local.

[0169] In some embodiments, the dosage regimen can be adjusted to obtain the best intended response (e.g., therapeutic or preventive response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the therapeutic situation.

[0170] definition

[0171] In order to make the present disclosure more easily understood, certain technologies and sciences are specifically defined below. Unless otherwise explicitly defined in the present disclosure, all other technologies and sciences used in the present disclosure have the meanings commonly understood by those skilled in the art in the art to which the present disclosure belongs.

[0172] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be construed to have an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0173] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0174] The term "c-Met" or "c-Met polypeptide" or "c-Met receptor" refers to a receptor tyrosine kinase that binds human growth factor (HGF). Unless otherwise specified herein, references to mouse c-Met (mc-Met) or monkey c-Met (cyno-c-Met) refer to human c-Met (hc-Met). Human, mouse, and cynomolgus monkey c-Met, as used herein, are encoded by nucleotide or polypeptide sequences provided by GenBank, such as the human polypeptide encoded by the nucleotide sequence provided in GenBank Accession No. NM_000245, or the human protein or its extracellular domain encoded by the polypeptide sequence provided in GenBank Accession No. NP_000236. The original single-chain precursor protein is post-translationally cleaved to produce α and β subunits, which are linked by disulfide bonds to form the mature receptor. The receptor tyrosine kinase c-Met participates in cellular processes including, for example, migration, invasion, and morphogenesis associated with tissue regeneration during embryogenesis.

[0175] The term "c-Met-related disorder or condition" refers to any disease, disorder, or condition that results from the adverse expression or lack of expression, adverse regulation or lack of regulation, or deleterious activity or lack of activity of c-Met, or that can be modulated, treated, or cured by modulating c-Met expression or activity. For example, activation of the HGF / c-Met pathway can be expected in a large proportion of cancer patients, or in patients whose disease is indeed driven by changes related to the c-Met pathway. For example, upregulation is attributed to various mechanisms, such as overexpression of HGF and / or c-Met, or constitutive activation through c-Met mutations. c-Met-related disorders or conditions include, but are not limited to, proliferative diseases, including, but not limited to, cancers, including, for example, gastric cancer, lung cancer, intestinal cancer, and / or liver cancer.

[0176] "Antibody" is used in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. An antibody may refer to an immunoglobulin, a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to the amino acid composition and arrangement order of their heavy chain constant regions. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class of Ig, subclasses can be further divided based on differences in the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0177] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (V region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region includes three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. The three hypervariable regions determine the specificity of the antibody and are also known as the complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0178] In the present disclosure, the antibody light chain may further comprise a light chain constant region. Exemplarily, the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

[0179] In the present disclosure, the antibody heavy chain may further comprise a heavy chain constant region. Exemplarily, the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4, or variants thereof.

[0180] The term "murine antibody," as used herein, refers to a monoclonal antibody against human c-Met produced in mice according to the knowledge and skill in the art. During preparation, a test subject is injected with a c-Met antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In one embodiment of the present disclosure, the murine c-Met antibody may further comprise a light chain constant region of a murine kappa or lambda chain, or variants thereof, or a heavy chain constant region of a murine IgG1, IgG2, IgG3, or IgG4, or variants thereof.

[0181] The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody. This can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies is first established. The variable region gene is then cloned from the mouse hybridoma cells and then cloned into the constant region gene of the human antibody for recombinant expression.

[0182] The term "humanized antibody", also referred to as CDR-grafted antibody humanization, refers to the antibody produced by transplanting the CDR sequence of a mouse to the antibody variable region framework of a person, i.e., different types of human germline antibody framework sequences. The strong antibody variable antibody response induced by carrying a large amount of mouse protein components can be overcome in chimeric antibodies. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. Germline DNA sequences of human heavy and light chain variable region genes can be obtained in the "VBase" human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The human antibody variable region framework is designed and selected, wherein the light chain FR region sequence on the antibody light chain variable region is selected from a human germline light chain sequence, for example, the human germline light chain IGKV085 or IGKV 4-1*01, comprising the FR1, FR2, FR3, and FR4 regions of the human germline light chain IGKV085 and IGKV 4-1*01; wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from a human germline heavy chain sequence, for example, the human germline heavy chain IGHV 3-33*01, comprising the FR1, FR2, FR3, and FR4 regions of the human germline heavy chain IGHV 3-33*01. To avoid a decrease in immunogenicity and a decrease in activity, the human antibody variable region can be subjected to minimal back mutations to maintain activity.

[0183] The term "antigen binding site" may be a discrete, three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present disclosure.

[0184] The term "epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids (non-adjacent amino acids are brought into close proximity in space by the tertiary folding of the protein). Epitopes formed by adjacent amino acids are typically retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are typically lost after treatment with a denaturing solvent. Epitopes typically exist in a unique spatial conformation that includes at least 3-15 amino acids. Methods for determining epitopes are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0185] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when human c-Met or its epitope is used as an analyte and an antibody is used as an antibody, the antibody binds to the epitope at a specific binding rate of less than 10 when measured by surface plasmon resonance (SPR) technology in an instrument. -7 M or even smaller equilibrium dissociation constant (K D ) binds to a predetermined antigen or an epitope thereof, and its affinity for binding to the predetermined antigen or an epitope thereof is at least twice as high as its affinity for binding to the predetermined antigen or an epitope thereof (or a nonspecific antigen other than a closely related antigen, such as BSA, etc.).

[0186] " Affinity " refers to the overall intensity of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding antibody (e.g., an antigen). Unless otherwise indicated, as used herein, " binding affinity " refers to internal binding affinity, which reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its antibody Y can generally be represented by an equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art (including those described herein). The term " kassoc " or " ka " refers to the on-rate of specific antibody-antigen interactions, and the term " kdis " or " kd " as used herein is intended to refer to the off-rate of specific antibody-antigen interactions. As used herein, the term " KD " refers to the equilibrium dissociation constant, which is derived from the ratio (i.e., kd / ka) of kd and ka and is expressed as molar concentration (M). The KD value of an antibody can be determined using methods known in the art, for example: methods for determining the KD of an antibody include measuring surface plasmon resonance using a biosensor system, such as a system, or measuring affinity in solution by solution equilibrium titration (SET). "Cross-reactivity" refers to the ability of an antibody (or fragment thereof) of the present disclosure to bind to c-Met from a different species. For example, an antibody of the present disclosure that binds to human c-Met may also bind to c-Met from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR or ELISA), or binding or functional interaction with cells that physiologically express c-Met. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.

[0187] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally speaking, comparison is made when the two sequences are aligned to obtain the maximum percentage homology. To determine the percentage of amino acid sequence identity, alignment can be achieved in a variety of ways within the skill of the art, for example, using computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences, such as BLAST.

[0188] The term "internalization" refers to the transport of a portion from the outside of a cell to the inside. The internalized portion can be located in an intracellular compartment. An "internalized" or "internalized" antigen or antibody refers to an antigen or antibody that is capable of being transported from the outside of a target cell to the inside. It is generally understood by those skilled in the art that the process of cellular internalization generally refers to the movement of cell surface molecules across the plasma membrane from the cell surface to the interior of the cell. After internalization, the endosome can be transported to the lysosome for degradation or recycled to the cell surface. The cellular internalization rate of a given cell surface molecule provides a measure of the kinetics of the movement of the molecule from the cell surface across the plasma membrane to the interior of the cell. Internalization activity or internalization rate of antigens and antibodies can be monitored and / or measured by various techniques known in the art, including acid dissociation (Li N. et al., Methods Mol. Biol., 457:305–17, 2008) and toxin killing assays (Pahara J. et al. Exp Cell Res., 316:2237–50, 2010; and Mazor et al., J. Immunol. Methods, 321:41–59, 2007). Many antibody labeling techniques, dyes, and kits for antibody labeling that can be used to quantify and monitor internalization are commercially available (e.g., pHrodo iFL antibody labeling methods, reagents, and kits sold by Thermo Fisher Scientific).

[0189] For example, antibody-drug conjugates (ADCs) bind to tumor cell surface antigens through the antibody in the ADC, which is then internalized into endosomes and converted from endosomes to lysosomes. Then, under the action of hydrolases in the lysosomes, the bioactive molecules (e.g., toxins or payloads) are dissociated from the ADC. The dissociated bioactive molecules enter the cytoplasm from the lysosomes and kill tumor cells. The bioactive molecules that escape from the killed tumor cells can further kill surrounding tumor cells that do not express or express low levels of the antigen (the so-called bystander effect).

[0190] "Mutant" or "variant" refers to a polypeptide containing at least one amino acid mutation (such as substitution, deletion or insertion) compared to the "parent" amino acid sequence, as long as the variant is still able to bind to the antigen or its fragment. Amino acid mutations (also known as amino acid modifications) include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of a binding molecule (e.g., an antibody). Any combination of deletions, insertions and substitutions can be introduced into the "parent" amino acid sequence to obtain the final variant. Amino acid mutations also include those that can change the post-translational processes of the binding molecule, such as changing the number or position of glycosylation sites. For example, depending on the length of the CDRs and FRs themselves, 1, 2, 3, 4, 5 or 6 amino acids can be inserted into or deleted from each CDR, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids can be inserted into or deleted from each FR. Furthermore, amino acid substitutions can be introduced into the CDR, VH or FR regions of the heavy and / or light chains, for example, conservative substitutions.

[0191] A "conservative substitution" refers to a replacement with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when replacing an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit a specific change in properties.

[0192] The term "camptothecin drugs" refers to cytotoxic camptothecin and its derivatives, including but not limited to 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, exitecan, irinotecan or 9-nitro-10-hydroxycamptothecin and its derivatives or pharmaceutically acceptable salts.

[0193] The term "antibody drug conjugate" (ADC) refers to an antibody linked to a drug via a linker (or connecting unit). In some embodiments, in the present disclosure, an "antibody drug conjugate" refers to an anti-C-met antibody linked to a toxic drug via a connecting unit.

[0194] The term "drug" refers to cytotoxic drugs, which are represented by D and are chemical molecules that have a strong ability to disrupt the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells in principle at sufficiently high concentrations, but due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. The term includes toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 and radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.

[0195] The term "Linker Unit" or "Linking Fragment" or "Linking Unit" refers to a chemical structure fragment or bond that is connected to an antibody at one end and to a drug at the other end. It can also be connected to other linkers before being connected to the drug. In a non-limiting embodiment, it is represented as L and L1 to L4, where the L1 end is connected to the antibody and the L4 end is connected to the structural unit Y and then to the drug (D).

[0196] The term "drug loading" refers to the average amount of cytotoxic drug loaded per ligand (anti-c-Met antibody) in the ADC, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading can range from 1 to 20, for example, 1 to 10, cytotoxic drugs (D) per antibody (Pc). In the embodiments of the present disclosure, the drug loading is expressed as n, and exemplary values ​​are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the average of any two values ​​therebetween. Thus, the drug loading can be an integer or a decimal. The average amount of drug per ADC molecule after the conjugation reaction can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, monoclonal antibody size variant assay (CE-SDS), and HPLC characterization.

[0197] The disclosed monoclonal antibody size variant determination method (CE-SDS) can employ capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) ultraviolet detection to quantitatively determine the purity of recombinant monoclonal antibody products based on molecular weight under reducing and non-reducing conditions according to capillary electrophoresis (Chinese Pharmacopoeia 2015 edition, 0542).

[0198] Although the drug to antibody ratio has an exact value for a particular conjugate molecule (e.g., n in Formula (I)), it will be understood that when used to describe a sample containing many molecules, the value will often be an average value due to some degree of heterogeneity typically associated with the conjugation step. The average loading of an immunoconjugate sample is referred to herein as the drug to antibody ratio or "DAR." In some embodiments, the DAR is between about 1 and about 10, e.g., 1-8, and is typically about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0. In some embodiments, at least 50% of the sample by weight is a compound having an average DAR plus or minus 2; in some specific embodiments, at least 50% of the sample is a conjugate containing an average DAR plus or minus 1. Examples include wherein the DAR is about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4. 0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.4, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0. In some embodiments, a DAR of 'about x' means that the measured value of DAR is within 20% of x.

[0199] The detection method of DAR, for example, is to extrapolate the DAR value from the LC-MS data of reduced and deglycosylated samples. LC / MS allows the quantification of the average number of payload (drug moiety) molecules connected to the antibody in the ADC. HPLC separates the antibody into light and heavy chains, and also separates the heavy chain (HC) and light chain (LC) according to the number of linker-payload groups of each chain. Mass spectrometry data can identify the types of components in the mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc. Based on the average loading amount of the LC and HC chains, the average DAR of the ADC can be calculated. The DAR of a given immunoconjugate sample represents the average number of drug (payload) molecules connected to a tetrameric antibody containing two light chains and two heavy chains. For example, the DAR detection method in WO2018142322.

[0200] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, such as an alkyl group containing 1 to 12 carbon atoms, an alkyl group containing 1 to 10 carbon atoms, or an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. For example, lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment. In non-limiting embodiments, the substituent is one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0201] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

[0202] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane group by removing two hydrogen atoms, and is a straight or branched group containing 1 to 20 carbon atoms, for example, an alkylene group containing 1 to 12 carbon atoms, or containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). Alkylene can be substituted or unsubstituted, and when substituted, the substituent can be substituted at any available point of attachment. In non-limiting embodiments, the substituent is independently optionally selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic radical, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0203] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl are as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituent can be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0204] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, for example, 3 to 12 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0205] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, but excluding the ring portion of -OO-, -OS- or -SS-, the remaining ring atoms are carbon. For example, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; or it contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups.

[0206] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group having 5 to 20 members (e.g., 6 to 14 members, or 7 to 10 members) and wherein the monocyclic rings share one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. Spiro heterocyclic groups are divided into single spiro heterocyclic groups, double spiro heterocyclic groups or multi-spiro heterocyclic groups according to the number of shared spiro atoms between the rings. For example, it is a 4-yuan / 4-yuan, 4-yuan / 5-yuan, 4-yuan / 6-yuan, 5-yuan / 5-yuan or 5-yuan / 6-yuan single spiro heterocyclic group. Non-limiting examples of spiro heterocyclic groups include:

[0207] The term "fused heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 20 members (e.g., 6 to 14 members, or 7 to 10 members), wherein each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. Depending on the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, for example, 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0208] The term "bridged heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 14 members (e.g., 6 to 14 members, or 7 to 10 members), wherein any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. Depending on the number of rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups. Non-limiting examples of bridged heterocyclic groups include:

[0209] The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:

[0210] wait.

[0211] The heterocyclyl group may be optionally substituted or unsubstituted. When substituted, the substituent may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0212] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system, for example, 6- to 10-membered, such as phenyl or naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0213] The aryl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0214] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. In non-limiting embodiments, the heteroaryl group is 5 to 10 members, or is 5 or 6 members, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0215] The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0216] The term "amino-protecting group" is intended to protect the amino group with an easily removable group in order to maintain the amino group intact while other parts of the molecule undergo reactions. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl. These groups may optionally be substituted with 1-3 substituents selected from halogen, alkoxy, or nitro. For example, the amino-protecting group is 9-fluorenylmethyloxycarbonyl.

[0217] The term "cycloalkylalkyl" refers to an alkyl group substituted with one or more cycloalkyl groups, wherein alkyl is as defined above, and wherein cycloalkyl is as defined above.

[0218] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0219] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0220] The term "hydroxy" refers to an -OH group.

[0221] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0222] The term "amino" refers to -NH2.

[0223] The term "nitro" refers to -NO2.

[0224] The term "amido" refers to -C(O)N(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0225] The term "carboxylate" refers to -C(O)O(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0226] The present disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds of formula (I) with reference to relevant literature. When preparing deuterated forms of compounds of formula (I), commercially available deuterated starting materials can be used, or they can use conventional techniques to synthesize deuterated reagents, which include but are not limited to deuterated borane, trideuteroborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane etc.

[0227] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and that the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0228] "Substituted" refers to one or more hydrogen atoms in a group, and in non-limiting embodiments, up to 5, for example, 1 to 3 hydrogen atoms are independently replaced by a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without paying too much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0229] "Administer," "apply," and "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "apply," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0230] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibody-drug conjugates of the present disclosure, to a subject who has, is suspected of having, or is prone to having one or more diseases or their symptoms, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although the embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, they should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as the Student's t-test, the chi-square test, the U test according to Mann and Whitney, the Kruskal-Wallis test (H test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0231] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0232] The term "drug carrier" as used in the context of the present invention refers to a system that can alter the way a drug enters the human body and its distribution within the body, control the rate of drug release, and deliver the drug to a targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, non-limiting examples of which include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while having good membrane permeability, making them excellent drug carriers.

[0233] The term "excipient" refers to any additive in a pharmaceutical preparation other than the main drug, also known as an adjuvant. Examples include binders, fillers, disintegrants, and lubricants in tablets; the base component of semisolid ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0234] The term "diluent," also known as filler, primarily increases the weight and volume of a tablet. The addition of a diluent not only maintains a certain volume, but also reduces dosage variations of the primary ingredient and improves the drug's compressibility. When the tablet contains an oily component, an absorbent is added to absorb the oil and maintain a "dry" state, facilitating tablet production.

[0235] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic drug). BRIEF DESCRIPTION OF THE DRAWINGS

[0236] FIG1 shows the bystander killing activity of ADC-1 against a mixture of EBC-1 cells and SW480 cells. DETAILED DESCRIPTION

[0237] Example

[0238] The following examples further illustrate the present disclosure, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0239] Example 1. Design and preparation of antibody-drug conjugates

[0240] 1.1 Anti-c-Met Antibodies

[0241] The following antibody Ab-10 was prepared according to conventional antibody methods, for example, vector construction, transfection into eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019), expression, isolation, and purification.

[0242] The following is the sequence of the Ab-10 humanized antibody:

[0243] Heavy chain:

[0244] Light chain:

[0245] 1.2. Preparation of Compound 1

[0246] The title compound 1 was synthesized according to the method provided in "Example 58 on page 163 of the specification of patent CN104755494A".

[0247] 1.3. Preparation of Compounds 9A and 9B

[0248] N-((2R,10S)-10-Benzyl-2-cyclopropyl-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]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

[0249] N-((2S,10S)-10-Benzyl-2-cyclopropyl-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]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

[0250] first step

[0251] Benzyl 2-cyclopropyl-2-hydroxyacetate 9a

[0252] 2a (1.3 g, 11.2 mmol; prepared using the method disclosed in patent application WO2013 / 106717) was dissolved in 50 mL of acetonitrile. Potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol), and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 hours, filtered through celite, and the filter cake was rinsed with ethyl acetate (10 mL). The combined filtrates were concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography using solvent System C to obtain the title product 9a (2 g, 86.9% yield).

[0253] Step 2

[0254] 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid benzyl ester 9b

[0255] 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol, prepared using the method disclosed in patent application "CN105829346A") were added to a reaction flask, and 4 mL of tetrahydrofuran was added. The atmosphere was replaced with argon three times, and the temperature was cooled to 0-5°C in an ice-water bath. Potassium tert-butoxide (109 mg, 0.98 mmol) was added. The ice bath was removed, and the mixture was warmed to room temperature and stirred for 40 minutes. 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL x 2) and chloroform (10 mL x 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 4 mL of dioxane, 2 mL of water was added, and sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added. The mixture was stirred at room temperature for 2 hours. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent System C to obtain the title product 9b (48 mg, yield: 19%).

[0256] MS m / z(ESI):515.0[M+1].

[0257] Step 3

[0258] 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid 9c

[0259] 9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixture of tetrahydrofuran and ethyl acetate (v:v = 2:1). Palladium on carbon (12 mg, 10% content, dry form) was added, and the atmosphere was replaced with hydrogen three times. The reaction was stirred at room temperature for 1 hour. The reaction solution was filtered through celite, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude title product 9c (13 mg). This product was directly used in the next reaction without purification.

[0260] MS m / z(ESI):424.9[M+1].

[0261] Step 4

[0262] (9H-fluoren-9-yl)methyl (2-(((1-cyclopropyl-2-(((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]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 9d

[0263] 1b (10 mg, 18.8 μmol) was added to a reaction flask, followed by 1 mL of N,N-dimethylformamide. The atmosphere was replaced with argon three times, and the temperature was cooled to 0-5°C in an ice-water bath. One drop of triethylamine was added dropwise, followed by the addition of crude product 9c (13 mg, 30.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol). The reaction was stirred in an ice-water bath for 40 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent System B to obtain the title product 9d (19 mg, 73.6% yield).

[0264] MS m / z(ESI):842.1[M+1].

[0265] Step 5

[0266] 2-((2-Aminoacetamido)methoxy)-2-cyclopropyl-N-((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]indolizino[1,2-b]quinolin-1-yl)acetamide 9e

[0267] 9d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 1 mL of toluene was added, and the mixture was concentrated under reduced pressure twice. 3 mL of n-hexane was added to the residue, and the supernatant was poured out after standing to retain the solid. The solid residue was concentrated under reduced pressure and pumped dry to obtain the crude title product 9e (17 mg), which was used directly in the next reaction without purification.

[0268] MS m / z(ESI):638.0[M+18].

[0269] Step 6

[0270] N-((2R,10S)-10-Benzyl-2-cyclopropyl-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]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

[0271] N-((2S,10S)-10-Benzyl-2-cyclopropyl-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]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

[0272] The crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, replaced with argon three times, and cooled to 0-5°C in an ice-water bath. 8 g (21.2 mg, 44.8 μmol, prepared by the method disclosed in patent application "EP2907824") in 0.3 mL of N,N-dimethylformamide solution was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol) was added. The mixture was stirred in an ice bath for 10 minutes. The ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour to produce compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4mg, 9-B: 1.7mg).

[0273] MS m / z(ESI):1074.4[M+1].

[0274] Single configuration compound 9-A (shorter retention time):

[0275] UPLC analysis: retention time 1.14 minutes, purity: 85% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0276] 1H NMR (400MHz, DMSO-d6): δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H) ,7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,1 H),4.54-4.40(m,2H),3.76-3.64(m,4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,1H),2.80-2.62(m,1H),2.45-2.30(m, 3H),2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H).

[0277] Single configuration compound 9-B (longer retention time):

[0278] UPLC analysis: retention time 1.16 minutes, purity: 89% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0279] 1H NMR (400MHz, DMSO-d6): δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H), 7.31(s,1H),7.26-7.13(m,3H),6.99(s,1H),6.55-6.48(m,1H),5.60- 5.50(m,1H),5.41(s,2H),5.35-5.15(m,2H),4.78-4.68(m,1H),4.60- 4.40(m,2H),3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H) ,3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13( m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52- 1.39(m,3H),1.34-1.12(m,4H),0.91-0.79(m,3H),0.53-0.34(m,4H).

[0280] 1.4. Preparation of Antibody-Drug Conjugate-1 (ADC-1)

[0281] The following structure of antibody-drug conjugate-1 (ADC-1) was prepared, wherein Ab in ADC-1 is Ab-10,

[0282] At 37°C, add an appropriate amount of histidine-histidine hydrochloride buffer solution (10 mM histidine-histidine hydrochloride, pH = 5.8) to the antibody Ab-10, followed by an appropriate amount of 200 mM EDTA (pH 6.3) to bring the antibody concentration to 15 g / L. Then, add a prepared aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (10 mM, 14 eq), and incubate at 25°C with shaking for 22-24 hours, then stop the reaction.

[0283] Compound 9-A (11 eq) was dissolved in dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 2 hours to stop the reaction. The reaction solution was desalted and purified using a Capto S Impact cationic chromatography column (eluent: 50 mM HAc-NaAc buffer, pH 5.5, containing 390 mM NaCl) to obtain an exemplary product of the general formula ADC-1 (ADC-1) (HAc-NaAc buffer) and stored refrigerated at 4°C.

[0284] The target DAR value was n = 5.3. The average drug loading was calculated by RP-HPLC, and the actual DAR values ​​were n = 4.7-5.9.

[0285] 1.5. Preparation of Antibody-Drug Conjugate-2 (ADC-2)

[0286] Ab-10 and compound 1 were prepared according to the preparation method described in step 1.4 above to obtain antibody-drug conjugate-2 (ADC-2) with the following structure, wherein Ab in ADC-2 is Ab-10, the target DAR value is 6,

[0287] Example 2. Binding activity of antibody-drug conjugates to c-Met protein

[0288] Biacore technology was used to test the affinity of the antibody-drug conjugate ADC-1 for c-Met proteins from different species (human and cynomolgus macaque). A Protein A sensor chip was embedded in a Biacore 8K instrument using HBS-EP+ as the assay buffer. Each cycle consisted of ligand capture, analyte injection, and regeneration. The instrument temperature was set at 25°C. ADC-1 and Ab-10 were prepared in HBS-EP+ buffer and captured in the detection channel (Fc2) using Protein A at a flow rate of 10 μL / min and a capture time of 30 s. Analytes, including human, cynomolgus macaque, and mouse c-Met proteins, were prepared at varying concentrations in HBS-EP+ buffer and injected into the reference and detection channels (Fc1 & 2) of the chip at a flow rate of 30 μL / min. Analytes were serially diluted two-fold, and experimental parameters were set. Finally, the chip was regenerated using Glycine 1.5 for 30 s at a flow rate of 30 μL / min. Biacore 8K Control Software collected and saved the SPR signal, and then used Biacore 8K Evaluation analysis software for data processing. The signal value of the detection channel (Fc2) was subtracted from the signal value of the corresponding reference channel (Fc1) to obtain the corrected signal curve. The affinity evaluation used the double subtraction method. A zero concentration cycle (Sample 0) with the same parameter settings was added before the sample cycle. The sample cycle was subtracted from the zero concentration cycle (Sample–Sample 0) to obtain the secondary corrected signal curve. The affinity kinetic curve was fitted according to the 1:1 Langmuir binding model, and K was calculated. D The affinity results of the antibody or antibody-drug conjugate to the protein are shown in Table 2.

[0289] The experimental results showed that ADC-1 and its naked antibody Ab-10 had strong affinity for both human and cynomolgus macaque c-Met proteins.

[0290] Table 2. Affinity of ADC-1 for human or cynomolgus monkey c-Met

[0291] Example 3. Evaluation of the ability of antibody-drug conjugates to inhibit tumor cell proliferation

[0292] The CellTiter Glo assay was used to detect the effects of antibody-drug conjugates on the proliferation of different tumor cell lines in vitro.

[0293] MKN45 cells, NCI-H1993 cells, NCI-H441 cells, and NCI-N87 cells were cultured in RPMI1640 complete medium containing 10% FBS, EBC-1 cells were cultured in MEM complete medium containing 10% FBS, and SW480 cells were cultured in L-15 medium containing 10% FBS.

[0294] For the experiment, cells were trypsinized with 0.25% EDTA, centrifuged at 400 g for 5 minutes, and the supernatant discarded. Each cell line was resuspended in its respective culture medium. Cell density was adjusted using its respective culture medium; the serum concentration for NCI-H1299 cells in the assay system was 2%, and the serum concentration for the remaining cells was 5%. In a 96-well plate, 100 μL of cell suspension was added to each well. NCI-H1993 cells, EBC-1 cells, NCI-H441 cells, MKN-45 cells, NCI-N87 cells, and SW480 cells were seeded at 1000 cells / well; NCI-H1299 cells and HCT116 cells were seeded at 500 cells / well; and Hs746T cells were seeded at 1500 cells / well. The cell plates were incubated overnight at 37°C in a 5% CO2 incubator. ADC-1, its naked anti-Ab-10, and its toxin fraction were serially diluted and added to the cell culture medium. After 6 days of cell culture, the cells were cultured. The assay was performed using the Cell Counting-Lite 2.0 Luminescent Cell Viability Assay Kit (VAZYME, DD1101-02) according to the manufacturer's instructions. Chemiluminescence was detected using a microplate reader (BMG, CLARIOstar plus), and data were analyzed using GraphPad Prism (version 9) software.

[0295] The experimental results shown in Table 3 show that the ADC-1 disclosed herein has strong inhibitory activity against gastric cancer cells MKN-45 (high expression of c-Met), which is superior to naked antibodies and toxins. The results show that the ADC-1 disclosed herein has strong tumor proliferation inhibition activity.

[0296] Among the lung cancer cells shown in Table 3, NCI-H1993 and EBC-1 showed high expression of c-Met, NCI-H441 showed moderate expression, and NCI-H1299 showed low expression; among gastric cancer cells, Hs 746T and MKN-45 showed high expression of c-Met, and NCI-N87 showed low expression; and colon cancer cells HTC116 and SW480 both showed low expression of c-Met.

[0297] As shown in Table 3, ADC-1 also exhibited strong anti-proliferation activity against other c-Met-high-expressing tumor cell lines (NCI-H1993, EBC-1, and Hs 746T), with EBC-1 lung cancer cells being particularly sensitive to ADC-1. While naked anti-c-Met antibodies showed minimal activity against the moderately expressing lung cancer cell line NCI-H441, toxin-loaded ADC-1 exhibited strong anti-proliferation activity. However, the toxin-loaded ADC-1 exhibited less potent inhibitory activity against cell lines with low c-Met expression (NCI-H1299, NCI-N87, HCT116, and SW480), despite the toxin's ability to significantly inhibit these cell lines.

[0298] The above results demonstrate that the disclosed ADC-1 has specific targeting properties, exhibiting strong inhibitory activity against the lung cancer cell lines NCI-H1993 and EBC-1, which both highly express c-Met, and the gastric cancer cell line Hs746T. Furthermore, ADC-1 exhibits strong inhibitory activity against the lung cancer cell line NCI-H441, despite minimal inhibitory activity from anti-c-Met antibodies. Furthermore, ADC-1 exhibits significantly less toxicity against nonspecific cells (e.g., normal cells) with low c-Met expression.

[0299] Table 3. IC values ​​of ADC-1 for inhibition of proliferation of various tumor cell lines 50 Value (nM)

[0300] Example 4. Endocytosis ability of antibody-drug conjugates

[0301] To examine the time-dependent endocytosis of antibody-drug conjugates after binding to c-Met-positive cells at a single concentration, human lung cancer cells (EBC-1) were evaluated using ADC-1, naked antibody Ab-10, Teli-VcMMAE, and naked antibody ABT-700 groups. Teli-VcMMAE (i.e., ABBV-399, with MMAE as the toxin) and its naked antibody ABT-700 were prepared in-house based on the structure of telisotuzumab vedotin disclosed in WHO Drug Information, Vol. 30, No. 2, 2016, p. 319.

[0302] EBC-1 cells were cultured in MEM medium containing 10% FBS, washed once with PBS, and then stained with TrypLE TM The cells were digested into a single cell suspension using Express Enzyme (Gibco, 12605-010) and centrifuged at 400 g for 5 min. The cell density was adjusted to 2 × 10 cells / mL using FACS buffer (PBS 1x, pH 7.2, and 2% FBS). 6 / mL, 50μL was added to each well of a 96-well round-bottom plate, and the groups were set as: endocytosis 0h, endocytosis 0.5h, endocytosis 2h, endocytosis 4h, and endocytosis 24h. 50μL of 40nM (2× concentration) diluted antibody or antibody-drug conjugate was added to each well. The detection group included ADC-1, naked antibody Ab-10, Teli-VcMMAE, and naked antibody ABT-700, so that the final concentration was 20nM. Incubate at 4°C for 60min. After incubation, centrifuge at 400g for 5min, discard the supernatant, and wash twice with 200μL FACS Buffer per well. The cells were resuspended in culture medium and placed in an incubator at 37°C and 5% CO2 for 0h, 0.5h, 2h, 4h, or 24h of endocytosis. After different endocytosis times, TrypLE TM Express Enzyme digested cells to single cell suspension, washed once with 200 μL FACS buffer, and added secondary antibody (Alexa Fluor 5000) diluted in FACS buffer. 647AffiniPure Mouse Anti-Human IgG (H+L), 1:500, Jackson, 209-605-008) was incubated at 4°C in the dark for 30 minutes. Centrifuge at 400g for 5 minutes, discard the supernatant, and wash each well twice with 200μL FACS buffer. Resuspend the cells in 50μL FACS buffer and fix them with 50μL 4% paraformaldehyde. Mix thoroughly and store at 4°C in the dark. After the samples are collected, analyze the Alexa Fluor 500-μL FACS buffer using a flow cytometer. 647 fluorescence signal. Flowjo software was used to analyze the data and calculate the gMFI value: endocytosis rate = (endocytosis 0h gMFI - endocytosis nh gMFI) / endocytosis 0h gMFI. The results are shown in Table 4.

[0303] Table 4. Evaluation of cell endocytosis performance

[0304] The results showed that the internalization rates of ADC-1 and its naked antibody Ab-10 by EBC-1 cells gradually increased with prolonged incubation, showing a time-dependent pattern. Furthermore, at 4 hours, both ADC-1 and its naked antibody (approximately 80% internalization rate) exhibited higher internalization rates than Teli-VcMMAE and its naked antibody ABT-700 (approximately 55% internalization rate), indicating that ADC-1 and its naked antibody can be rapidly internalized by target cells.

[0305] Example 5. Bystander Killing Effect of Antibody-Drug Conjugates

[0306] EBC-1 cells (c-Met high expression) and SW480 cells (c-Met low expression) were cultured in MEM medium containing 10% FBS and L-15 medium containing 10% FBS, respectively. They were trypsinized to a single cell suspension and centrifuged at 400 g for 5 min. The cells were washed once with PBS and then adjusted to a cell density of 1×10 6 / mL, and use CellTrace TM Violet (Thermo, C34557) labeled EBC-1, CellTrace TM SW480 cells were labeled with CFSE (Thermo, C34554A) in PBS at 37°C for 15 minutes. After labeling, staining was terminated with complete medium, centrifuged at 400 g for 5 minutes, and washed twice with PBS. EBC-1 cells and SW480 cells were resuspended in a 1:1 mixture of MEM medium containing 5% FBS and L15 medium and seeded into 24-well plates. 9 × 10 cells were plated per well in the mixed culture group. 4 EBC-1 and 1.5×10 4 SW480 cells per well, 1.5×10 cells per well in the SW480 single culture group 4 Cells were cultured in 500 μL of culture medium and allowed to adhere overnight in a 5% CO2, 37°C incubator. The next day, 2× the concentration of ADC-1 was added to a 24-well plate, resulting in final concentrations of 0.5 nM, 5 nM, and 50 nM, respectively. An equal volume of fresh culture medium was added to the control group. The cells were incubated in a cell culture incubator for 72 hours. After 72 hours, the cells were washed once with PBS, digested with trypsin to a single-cell suspension, and counted using a cell counter. The ratio of CTV-positive and CFSE-positive cells was determined using a flow cytometer (BD Celesta Flow cytometer), and the number of EBC-1 and SW480 cells in each well was calculated. GraphPad Prism 9 software was used for plotting, and the data were statistically analyzed using Tukey's multiple comparisons test.

[0307] The experimental results are shown in Figure 1. When SW480 cells with low c-Met expression were cultured alone, the cell number was largely unaffected by different concentrations of ADC-1. However, when co-cultured with EBC-1 cells with high c-Met expression, the SW480 cell number decreased with increasing ADC-1 doses.

[0308] The results showed that the ADC-1 disclosed herein had a clear bystander killing effect. The ADC did not kill SW480 cells with low c-Met expression cultured alone. However, after mixing EBC-1 cells with high c-Met expression with SW480 cells with low c-Met expression, the ADC also had a killing effect on the low c-Met expressing cells.

[0309] Example 6. In vivo efficacy evaluation of human lung cancer NCI-H441 cell mouse transplant tumor model

[0310] Balb / c nude mice were subcutaneously inoculated with NCI-H441 cells (5×10 6 ), and wait until the tumor volume grows to an average of 140 mm 3 Afterwards, the mice were randomly divided into 4 groups according to tumor size and body weight, including a vehicle control group and three ADC-1 dosage groups (1, 3, and 10 mg / kg), with 6 mice in each group. The grouping day was designated as D0.

[0311] The mice were intraperitoneally injected twice (D0, D7) with vehicle (0.9% NaCl) or drug ADC-1 (1, 3, 10 mg / kg). After grouping, the tumor volume was measured twice a week and the mouse body weight was recorded for 21 days.

[0312] Data were recorded using Excel statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT (number of animals in each group); graphs were drawn using GraphPad Prism software, and data were statistically analyzed using Two-way ANOVA or One-way ANOVA.

[0313] Tumor volume was calculated as follows: Tumor volume (V) = (length × width 2 ) / 2.

[0314] Relative tumor volume (RTV) = V t / V0×100(%), where V0 is the tumor volume measured during group administration, V t is the tumor volume at each measurement.

[0315] Relative tumor growth rate T / C (%) = (T RTV / C RTV) × 100%, where T and C are the tumor volumes of the treatment and control groups at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. Tumor inhibition rate TGI (%) = 1-T / C (%).

[0316] The experimental results are shown in Table 5. Compared with the vehicle control group, ADC-1 at 1, 3, and 10 mg / kg doses all exhibited significant tumor inhibition, with TGI% of 92.91%, 93.70%, and 95.71%, respectively. Furthermore, no significant weight loss was observed in the experimental animals during dosing. This demonstrates that the test drug has a strong therapeutic effect against tumors, exhibiting a 92.91% tumor inhibition rate even at a 1 mg / kg dose. Furthermore, the test drug did not cause significant toxic side effects in the experimental animals, demonstrating good tolerability and safety.

[0317] Table 5. Inhibitory effect on the growth of human lung cancer NCI-H441 subcutaneous transplanted tumor model in nude mice Note: All groups were compared with the vehicle control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0318] Example 7. In vivo efficacy evaluation of human gastric cancer MKN-45 nude mouse transplant tumor model

[0319] Female Balb / c Nude mice were subcutaneously inoculated with 5×10 6 MKN-45 cells were used to establish a subcutaneous tumor model. When the tumor grew to approximately 117 mm 3 The mice were then randomly divided into seven groups, each consisting of six mice, based on tumor size and body weight. These included a vehicle control group, a naked antibody Ab-10 group (10 mg / kg), three ADC-1 dose groups (1, 3, and 10 mg / kg), a reference drug Teli-VcMMAE group (3 mg / kg), and an IgG2-drug control group (3 mg / kg, IgG2 antibody conjugated to the same linker and toxic drug as ADC-1, DAR 5.3). Each group received a single intraperitoneal injection (D0). Animal body weight and tumor size were measured twice weekly during the experiment.

[0320] Tumor volume was calculated as follows: Tumor volume = (length × width 2 ) / 2. Relative tumor change rate ΔT / ΔC (%) = (tumor volume at the end of the dosing group - tumor volume at the beginning of the dosing group) / (tumor volume at the end of the vehicle group - tumor volume at the beginning of the vehicle group) × 100%. Tumor growth inhibition rate TGI% = (1-ΔT / ΔC) × 100%.

[0321] Data were recorded using Excel statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT (number of animals in each group); graphs were drawn using GraphPad Prism software, and data were statistically analyzed using Two-way ANOVA or One-way ANOVA.

[0322] The experimental results are shown in Table 6. Compared with the vehicle control group, ADC-1 3 mg / kg and ADC-1 10 mg / kg both significantly inhibited the growth of MKN-45 tumors in vivo, with TGI% of 94.72% and 103.56%, respectively. However, the reference drug Teli-VcMMAE (3 mg / kg) at the same dose did not show significant anti-tumor activity, with a TGI% of 40.28%. Similarly, even at a dose of 10 mg / kg, naked anti-Ab-10 and IgG2-toxin did not show significant anti-tumor activity, with TGI% of 48.34% and 23.42%, respectively. This shows that the test drug ADC-1 has a good anti-tumor effect. At the same dose, the test drug ADC-1 is significantly more effective than the reference drug Teli-VcMMAE and naked anti-Ab-10, and does not produce obvious toxic side effects in the experimental animals, with good tolerability and safety. Interestingly, even at a low dose of 3 mg / kg, ADC-1 achieved the same tumor inhibitory effect as 10 mg / kg, and both doses were well tolerated by tumor-bearing mice, suggesting a wide therapeutic window for ADC-1.

[0323] Table 6. Inhibitory effect on the growth of human gastric cancer MKN-45 subcutaneous transplanted tumor model in nude mice Note: All groups were compared with the vehicle control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0324] Example 8. In vivo efficacy evaluation of human gastric cancer GA6831PDX nude mouse transplant tumor model

[0325] BALB / c nude mice were subcutaneously inoculated with human gastric cancer GA6831 tumors (from Gastric cancer xenograft model GA6831 tumor-bearing mice were harvested for tumor tissue and cut into tumor masses with a diameter of 2-3 mm to establish a human gastric cancer subcutaneous xenograft tumor model. When the tumor grew to an average of about 151 mm 3 The mice were randomly divided into 4 groups according to tumor size and body weight, including a vehicle control group and three ADC-1 dosage groups (1, 3, and 10 mg / kg), with 7 mice in each group. The grouping day was designated as D0.

[0326] The mice were intraperitoneally injected with vehicle (0.9% NaCl) or drug ADC-1 (1, 3, 10 mg / kg) on ​​D0, D7, and D14, respectively. The tumor volume and mouse body weight were measured twice a week after grouping.

[0327] Data were recorded using Excel statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT (number of animals in each group); graphs were drawn using GraphPad Prism software, and data were statistically analyzed using Two-way ANOVA or One-way ANOVA.

[0328] Tumor volume was calculated as follows: Tumor volume (V) = (length × width 2 ) / 2. Relative tumor growth rate ΔT / ΔC (%) = (tumor volume at the end of the dosing group - tumor volume at the start of the dosing group) / (tumor volume at the end of the vehicle group - tumor volume at the start of the vehicle group) × 100%. Tumor growth inhibition rate TGI% = (1-ΔT / ΔC) × 100%.

[0329] The experimental results are shown in Table 7. Compared with the solvent control group, 1mg / kg, 3mg / kg and 10mg / kg ADC-1 significantly inhibited the In the in vivo growth model of GA6831 gastric cancer subcutaneous xenografts, the TGI% were 65.65%, 73.56%, and 91.70%, respectively. The animals showed no significant weight loss during dosing. This indicates that the drug has a good therapeutic effect on tumors and has no significant toxic side effects in the animals, demonstrating good tolerability and safety.

[0330] Table 7. Inhibitory effect on the growth of human gastric cancer GA6831 PDX nude mouse subcutaneous transplant tumor model Note: All groups were compared with the vehicle control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0331] Example 9. In vivo efficacy evaluation of human colorectal cancer CR3150PDX nude mouse transplant tumor model

[0332] BALB / c nude mice were subcutaneously inoculated with human colorectal cancer CR3150 tumors (from Colorectal cancer xenograft model Tumor tissue was collected from CR3150 tumor-bearing mice and cut into tumor pieces with a diameter of 2-3 mm to establish a human colorectal cancer subcutaneous xenograft tumor model. When the tumor grew to an average of about 156 mm 3 The mice were randomly divided into three groups according to tumor size and body weight, namely, a vehicle control group and two ADC-1 dosage groups (3 and 10 mg / kg), with 7 mice in each group. The day of grouping was designated as Day 0.

[0333] The mice were intraperitoneally injected with a vehicle (0.9% NaCl) and ADC-1 (3 and 10 mg / kg) on ​​D0 and D7, respectively. The tumor volume and body weight of the mice were measured twice a week after grouping.

[0334] Data were recorded using Excel statistical software: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT (number of animals in each group); graphs were drawn using GraphPad Prism software, and data were statistically analyzed using Two-way ANOVA or One-way ANOVA.

[0335] Tumor volume was calculated as follows: Tumor volume (V) = (length × width 2 ) / 2. Relative tumor growth rate ΔT / ΔC (%) = (tumor volume at the end of the dosing group - tumor volume at the start of the dosing group) / (tumor volume at the end of the vehicle group - tumor volume at the start of the vehicle group) × 100%. Tumor growth inhibition rate TGI% = (1-ΔT / ΔC) × 100%.

[0336] The experimental results are shown in Table 8. Compared with the vehicle control group, 3 and 10 mg / kg ADC-1 significantly inhibited the In the in vivo colorectal cancer CR3150 subcutaneous xenograft model, the TGI% was 70.58% and 106.95%, respectively. Furthermore, no significant weight loss or unexpected death was observed in the experimental animals during dosing. This demonstrates that the test drug has a good therapeutic effect on tumors and has no significant toxic side effects in the experimental animals, demonstrating good tolerability and safety.

[0337] Table 8. Inhibitory effect on the growth of human colorectal cancer CR3150PDX nude mouse subcutaneous transplant tumor model Note: All groups were compared with the vehicle control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0338] Example 10. In vivo safety evaluation in cynomolgus monkeys

[0339] 1) Safety of ADC-1

[0340] ADC-1 was injected intravenously into cynomolgus monkeys at 5, 15, and 40 mg / kg, with 10 monkeys in each group, half male and half female. The doses were administered every 2 weeks for 13 consecutive weeks (7 doses in total), followed by a 4-week recovery period.

[0341] The results showed that no animals were found dead or dying throughout the study. The main toxic target organs were the gastrointestinal tract and the lymphohematopoietic system; decreases in red blood cell (RBC), hemoglobin, hematocrit (HCT), and reticulocyte (RET) counts, as well as decreased albumin (ALB) levels, were observed in the 40 mg / kg ADC-1-treated cynomolgus monkeys. After a 4-week recovery period, no lesions were observed. Based on these findings, the highest non-severely toxic dose (HNSTD) of ADC-1 in cynomolgus monkeys was 40 mg / kg.

[0342] 2) Comparison of the safety of ADC-3

[0343] ADC-12 was synthesized by referring to the method provided in Example 24 on page 61 of the specification of patent CN106687480B.

[0344] ADC-12 was injected intravenously into cynomolgus monkeys at 1, 3, and 9 mg / kg doses every 2 weeks for a total of 6 doses.

[0345] Results showed that pathological findings in the 1mg / kg ADC-12 group were limited to decreased lymphocytes in the spleen and thymus, with no other significant abnormalities. Pathological findings in the 3mg / kg ADC-12 group included lesions in the immune and hematopoietic systems, digestive system (liver, pancreas, gastrointestinal tract), heart, kidneys, and adrenal glands. In the 9mg / kg ADC-12 group, most animals were near death or had died, indicating that HNSTD was only at 1mg / kg.

[0346] Although specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof having a structure shown in formula (I): Ab-(LYD)n Formula (I) in: Ab is an anti-c-Met antibody, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of any one of SEQ ID NOs: 10-12, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of any one of SEQ ID NOs: 13-15; The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; -L- is a linker unit, which is -L 1 -L 2 -L 3 -L 4 -, L 1 -(succinimidyl-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6; R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 1 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; m is an integer from 0 to 4; n is 1 to 10, and n is an integer or a decimal; D is a camptothecin drug, preferably exitecan or its derivatives.

2. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the VH of the anti-c-Met antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1-3, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 5 and 6, respectively; Preferably, 1) the anti-c-Met antibody comprises VH of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2 and 3, respectively, and VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 7, 5 and 6, respectively, or 2) the anti-c-Met antibody has a VH comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1, 2, and 3, respectively, and a VL comprising a LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively; The CDRs are defined according to the Kabat system.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the anti-c-Met antibody is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody, preferably a humanized antibody.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, wherein the anti-c-Met antibody comprises any one of the following: (1) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 11, or having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 14, or having at least 90% sequence identity thereto; (2) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 10, or having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 13, or having at least 90% sequence identity thereto; (3) The VH comprises an amino acid sequence as shown in SEQ ID NO: 12, or a sequence having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 15, or a sequence having at least 90% sequence identity thereto.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the anti-c-Met antibody further comprises an Fc region; Preferably, the Fc region is derived from the Fc region of human IgG1, IgG2, IgG3 or IgG4; more preferably Fc region of human IgG2.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the anti-c-Met antibody comprises a combination of light chains and heavy chains selected from the group consisting of: The heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence having at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having at least 90% sequence identity thereto; The heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 16, or having at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 17, or having at least 90% sequence identity thereto; or, The heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 20, or having at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 21, or having at least 90% sequence identity thereto.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein n is 1 to 8, and n is a decimal or an integer, Preferably, n is an integer or decimal from 3 to 7, More preferably, n is an integer or decimal from 4 to 6.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, which is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by formula II: in: W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; R 1 is selected from a hydrogen atom, a halogen, a cycloalkylalkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; m is an integer from 0 to 4; n is 1 to 10, and n is an integer or a decimal; Ab is as defined in any one of claims 1-6.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein: The -LY- is shown in the following structure: s 1 is an integer from 2 to 8; L 2 , L 3 , R 1 , R 2 , R 5 , R 6 , R 7 and m as defined in claim 8.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, which is an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof represented by general formula (III): in: s 1 is an integer from 2 to 8; preferably 5; Ab, R 1 , R 2 , R 5 , R 6 , R 7 , m and n are as defined in claim 8.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein -L- is:

12. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein -LY- is selected from: Preferably 13. A method for preparing the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, comprising: Ab and LYD are coupled to react to obtain the antibody-drug conjugate; Optionally, the method further comprises: purifying the antibody-drug conjugate.

14. A pharmaceutical composition comprising: The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, and One or more pharmaceutically acceptable excipients, diluents or carriers.

15. Use of the antibody-drug conjugate according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 14 in the preparation of a medicament for treating a disease or condition, wherein: Preferably, the disease or condition is cancer, or a c-Met-mediated disease or condition; More preferably, the cancer is a cancer expressing c-Met; Most preferably, the cancer is gastric cancer, lung cancer, intestinal cancer or liver cancer.

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