Anti-claudin antibody-drug conjugate and its medical use

Abstract

The present invention relates to an anti-claudin antibody-drug conjugate and its pharmaceutical use. Specifically, the present invention relates to a ligand-drug conjugate represented by the general formula (Pc-LYD), where Pc is an anti-Claudin18.2 antibody or an antigen-binding fragment thereof, and L, Y, and n are as defined in the specification. JPEG2023505708000060.jpg4248

Description

[Technical Field]

[0001] This application claims priority to a Chinese patent application filed on December 12, 2019 (application number CN201911273041.7) and a Chinese patent application filed on September 30, 2020 (application number CN202011060513.3).

[0002] The present disclosure relates to anti-claudin antibody-drug conjugates, particularly anti-Claudin18.2 antibody-exatecan analog conjugates, methods for preparing the same, drug compositions containing the antibody-drug conjugates, and their use in preparing drugs for treating Claudin18.2-mediated diseases or conditions, particularly in preparing anti-cancer drugs. [Background technology]

[0003] Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0004] Claudin-18 (CLDN18), a protein encoded by the Claudin18 gene in humans, belongs to the tight junction protein family and regulates intercellular molecular flow. The Claudin protein structure contains four transmembrane domains and two extracellular loops, with its N- and C-termini located in the cytoplasm. Claudin-18 has two splice variants, Claudin18.1 and Claudin18.2, which differ by eight amino acids in the first extracellular loop alone. The expression of Claudin18.1 and Claudin18.2 differs. Claudin18.1 is selectively expressed in normal lung cells, whereas Claudin18.2 is severely restricted in normal cells but frequently allosterically activated and overexpressed in several tumor types (e.g., gastric, lung, and pancreatic cancers). Claudin18.2 is considered a potential therapeutic target for gastric cancer and other cancer types, and the discovery of this target also provides a new option for the treatment of gastric cancer.

[0005] Antibody drug conjugates (ADCs) combine monoclonal antibodies or antibody fragments with biologically active cytotoxins via stable chemical linkers, taking advantage of the specificity of antibodies for binding to surface antigens on normal and tumor cells and the high efficiency of cytotoxic substances, while avoiding the drawbacks of the former, such as the relatively low therapeutic efficacy, and the latter, such as the excessive toxicity and side effects. Compared with conventional chemotherapy drugs, antibody drug conjugates can bind more precisely to tumor cells and reduce their impact on normal cells.

[0006] Currently, patents relating to antibodies and ADC drugs targeting Claudin18.2 have been reported, such as WO2020200196A1, WO2016166122 and WO2016165762. However, there is still a need to develop more effective and safer anti-Claudin18.2 antibody-drug conjugates for better use in treating tumors related to Claudin18.2. Summary of the Invention

[0007] The present disclosure relates to an ADC of an anti-Claudin18.2 antibody and uses thereof, and provides an ADC drug in which an anti-Claudin18.2 antibody or an antigen-binding fragment is conjugated with an exatecan analog, which is a cytotoxic substance.

[0008] Accordingly, an object of the present disclosure is to provide a ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof: [ka] however, Y is -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)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R bare the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group and a heterocyclyl group, or R a and R b together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, R 1 is selected from halogen, halogenated alkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups; R 2 is selected from a hydrogen atom, a halogen, a halogenated alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group, or R 1 and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, Or R a and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, m is an integer from 0 to 4, n is 1 to 10, and n is a decimal or an integer; L is a linker unit, Pc is an anti-Claudin18.2 antibody or an antigen-binding fragment thereof.

[0009] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by any one of the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, the anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the same sequences as those of the heavy chain variable region represented by the sequence SEQ ID NO:3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the same sequences as those of the light chain variable region represented by the sequence SEQ ID NO:4, or ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the same sequences as those of the heavy chain variable region represented by the sequence SEQ ID NO:5, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the same sequences as those of the light chain variable region represented by the sequence SEQ ID NO:6.

[0010] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by any one of the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, the anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the sequences of which are set forth in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the sequences of which are set forth in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively; or iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, whose sequences are represented by SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, whose sequences are represented by SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, respectively.

[0011] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by any one of the general formula (Pc-LYD) described above or a pharmaceutically acceptable salt thereof, the anti-Claudin18.2 antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.

[0012] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by any one of the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, the anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, (1) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO:3 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO:4 or has at least 90% identity thereto; (2) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 24 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO: 21 or has at least 90% identity thereto; (3) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO:5 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO:6 or has at least 90% identity thereto; or (4) The heavy chain variable region has an amino acid sequence represented by SEQ ID NO:31 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO:28 or has at least 90% identity thereto.

[0013] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt thereof, the anti-Claudin18.2 antibody is a humanized antibody, and the humanized antibody comprises a framework region derived from a human antibody or a framework region mutant thereof, and the framework region mutant is a back mutation having at most 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) in the light chain framework region and / or the heavy chain framework region of the human antibody, respectively; Preferably, the framework region variants comprise a mutation selected from the following (a) or (b): (a) the light chain variable region optionally comprises one or more amino acid backmutations selected from 22S, 85I, and 87H, and / or the heavy chain variable region optionally comprises one or more amino acid backmutations selected from 48I, 82T, and 69M; or (b) the light chain variable region optionally comprises one or more amino acid backmutations selected from 4L or 22S, and / or the heavy chain variable region optionally comprises one or more amino acid backmutations selected from 38K, 40R, 48I, 66K, 67A, 69L, 71L, and 73K; Preferably, the framework region variants include mutations selected from: (a-1) the light chain variable region contains the amino acid backmutations 22S, 85I, and 87H, and the heavy chain variable region contains the amino acid backmutations 48I and 82T; or (b-1) The light chain variable region contains an amino acid backmutation selected from 4L.

[0014] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt thereof, the anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises the following heavy chain variable region and light chain variable region, i.e., (vii) the heavy chain variable region has the sequence represented by SEQ ID NO:3, and the light chain variable region has the sequence represented by SEQ ID NO:4; (viii) the heavy chain variable region has the sequence represented by SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, and the light chain variable region has the sequence represented by SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23; (ix) the heavy chain variable region has the sequence represented by SEQ ID NO:5 and the light chain variable region has the sequence represented by SEQ ID NO:6; or (x) the heavy chain variable region has the sequence represented by SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, and the light chain variable region has the sequence represented by SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30; Preferably, the anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises the heavy chain variable region and light chain variable region shown below, i.e. (xi) the heavy chain variable region has the sequence represented by SEQ ID NO:31 and the light chain variable region has the sequence represented by SEQ ID NO:29; or (xii) the heavy chain variable region has the sequence represented by SEQ ID NO:26, and the light chain variable region has the sequence represented by SEQ ID NO:23.

[0015] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt thereof, the anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region and a light chain constant region, preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions and common variants thereof, and the light chain constant region is selected from human antibody kappa and lambda chain constant regions and common variants thereof, more preferably, the antibody comprises a heavy chain constant region whose sequence is represented by SEQ ID NO:7 and a light chain constant region whose sequence is represented by SEQ ID NO:8, most preferably, the antibody comprises a heavy chain having at least 90% identity with a heavy chain having the amino acid sequence represented by SEQ ID NO:35 or SEQ ID NO:42, and a light chain having at least 90% identity with a light chain having the amino acid sequence represented by SEQ ID NO:36 or SEQ ID NO:39, The antibody comprises a heavy chain having at least 90% identity with a heavy chain having an amino acid sequence represented by SEQ ID NO:37 or SEQ ID NO:49, and a light chain having at least 90% identity with a light chain having an amino acid sequence represented by SEQ ID NO:38 or SEQ ID NO:46.

[0016] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by any one of the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, the anti-Claudin18.2 antibody or antigen-binding fragment thereof is (c) a heavy chain whose sequence is represented by SEQ ID NO:35 and a light chain whose sequence is represented by SEQ ID NO:36; (d) a heavy chain having the sequence represented by SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45 and a light chain having the sequence represented by SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41; (e) a heavy chain having the sequence set forth in SEQ ID NO:37 and a light chain having the sequence set forth in SEQ ID NO:38; or (f) a heavy chain having the sequence represented by SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52 and a light chain having the sequence represented by SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48.

[0017] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by any one of the above general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, the anti-Claudin18.2 antibody is h1901-11, which comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:44 and a light chain having the sequence set forth in SEQ ID NO:41; or h1902-5, which comprises a heavy chain having an amino acid sequence represented by SEQ ID NO:49 and a light chain having an amino acid sequence represented by SEQ ID NO:47.

[0018] In some embodiments of the present disclosure, the antigen-binding fragment is selected from a Fab, a Fab', a F(ab')2, a single-chain antibody (scFv), a dimerized V region (diabody), and a disulfide-bond stabilized V region (dsFv).

[0019] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above aspects or a pharmaceutically acceptable salt thereof, n may be an integer or decimal number between 1 and 10, and n may be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. n is a decimal number or integer of 2 to 8, preferably a decimal number or integer of 3 to 8, more preferably a decimal number or integer of 5 to 9, or preferably a decimal number or integer of 2 to 7. In some embodiments, n is a decimal number or integer of 3.5 to 4.5.

[0020] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt thereof, however, Y is -O-(CR a Rb ) 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 atom, or an alkyl group; R 1 is a halogenated alkyl group or C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, a halogenated alkyl group, or C 3-6 cycloalkyl groups, Or R 1 and R 2 C, along with the carbon atoms attached to them. 3-6 forming a cycloalkyl group, m is 0 or 1.

[0021] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt thereof, Y is [ka] Selected from However, the O-end of Y is connected to the linker unit L.

[0022] In some embodiments of the present disclosure, in the provided Ligand-Drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt or solvate thereof, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -It is.

[0023] In some embodiments, L 1 -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, where W is selected from C 1-8Alkyl group, C 1-8 Alkyl group-C 3-6 a cycloalkyl group or a straight chain heteroalkyl group of 1 to 8 chain atoms, said heteroalkyl group containing 1 to 3 heteroatoms selected from N, O or S, said C 1-8 Alkyl group, C 1-8 Alkyl group-C 3-6 The cycloalkyl groups or straight chain heteroalkyl groups of 1 to 8 chain atoms are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl groups.

[0024] In some embodiments, L 2 is -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, provided that p 1 is an integer between 1 and 20. In some embodiments, L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are selected from amino acid residues formed by the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group.

[0025] In some embodiments, L 4 is -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH2) t - and a chemical bond, where t is an integer of 1 to 6.

[0026] In some embodiments, 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.

[0027] In some embodiments, 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.

[0028] In some embodiments of the present disclosure, in the Ligand-Drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt or solvate thereof, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, where W is selected from C 1-8 Alkyl group, C 1-8 alkyl-cycloalkyl groups or straight chain heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl groups contain 1 to 3 heteroatoms selected from N, O or S, and the C 1-8 the alkyl group, the cycloalkyl group, and the straight-chain heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl groups; L 2 is -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, provided that p 1 is an integer between 1 and 20, L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are selected from amino acid residues formed by the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and are optionally further substituted with one or more substituents selected from halogen, hydroxyl group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group, and cycloalkyl group; L 4 is -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH2) t - or a chemical bond, where 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.

[0029] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt thereof, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - and L 1 teeth [ka] and s 1 is an integer between 2 and 8, L 2 is a chemical bond, L 3 is a tetrapeptide residue, preferably L 3 is the tetrapeptide residue of GGFG (SEQ ID No: 55), L 4 is -NR 5 (CR 6 R 7 )t- and R 5 , R 6 or R 7 are the same or different and each independently represents a hydrogen atom or an alkyl group; t is 1 or 2; However, the above L 1 The end is connected to Pc, and L 4 The end is connected to a Y.

[0030] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt thereof, -L- is [ka] is.

[0031] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt thereof, -LY- is optionally [ka] Selected from.

[0032] In some embodiments of the present disclosure, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof described in any one of the above is represented by the general formula (Pc-L a-YD) or a pharmaceutically acceptable salt thereof, [ka] however, W, L 2 , L 3 , R 5 , R 6 , R 7 is as defined above for the linker unit -L-; Pc, n, R 1 , R 2 , m is as defined in the general formula (Pc-LYD).

[0033] In some embodiments of the present disclosure, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof described in any one of the above is represented by the general formula (Pc-L b -YD) or a pharmaceutically acceptable salt thereof, [ka] however, s 1 is an integer from 2 to 8, Pc, R 1 , R 2 , R 5 ~R 7 , m and n are represented by the general formula (Pc-L a -YD).

[0034] In some embodiments of the present disclosure, in the ligand-drug conjugate represented by the general formula (Pc-LYD) described in any one of the above or a pharmaceutically acceptable salt thereof, the ligand-drug conjugate is [ka] Selected from Here, Pc and n are as defined in the general formula (Pc-LYD).

[0035] In some embodiments of the present disclosure, there is provided a ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate is [ka] Selected from Here, n is as defined in the general formula (Pc-LYD), and antibodies h1902-5 and h1901-11 are as defined above.

[0036] The present disclosure provides compounds of the general formula (Pc-L a

[0013] The present invention further provides a method for preparing a ligand-drug conjugate represented by formula (I) or a pharmaceutically acceptable salt thereof, the method comprising: [ka] Pc' is expressed by the general formula (L a -YD), and the compound represented by the general formula (Pc-L a -YD), however, Pc is the anti-Claudin18.2 antibody or an antigen-binding fragment thereof as described above, and Pc' is obtained by reducing Pc; W, L 2 , L 3 , R 1 , R 2 , R 5 ~R 7 , m and n are represented by the general formula (Pc-L a -YD).

[0037] The present disclosure further provides a method for preparing an antibody-drug conjugate represented by the general formula (Pc-L'-D), which comprises: [ka] and then coupling with a compound of general formula (L'-D) to obtain a compound, Pc is the anti-Claudin18.2 antibody or antigen-binding fragment thereof as described above, n is as defined in the general formula (Pc-LYD).

[0038] In another aspect, the present disclosure provides a drug composition comprising the ligand-drug conjugate or a pharmaceutically acceptable salt thereof described in any one of the above and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0039] In another aspect, the present disclosure provides a use of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof described in any one of the above, or a drug composition comprising the same, as a drug. In some embodiments, the drug is used to treat a Claudin18.2-mediated disease or condition, and the Claudin18.2-mediated disease or condition is preferably a Claudin18.2-highly expressing cancer. In some embodiments, the drug is used to treat cancer. In some embodiments, the cancer is selected from the group consisting of squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krueckenberg's tumor, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's tumor, and thyroid cancer. Sarcoma, systemic light-chain amyloidosis, and Merkel cell carcinoma are preferred, and more preferably, the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer is selected from non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

[0040] In another aspect, the present disclosure provides a use of any one of the ligand-drug conjugates or pharmaceutically acceptable salts thereof, or a pharmaceutical composition comprising the same, in preparing a medicament for treating a Claudin18.2-mediated disease or condition, wherein the Claudin18.2-mediated disease or condition is a Claudin18.2-highly expressed cancer. In some embodiments, the disease is selected from the group consisting of squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krueckenberg's tumor, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's tumor, and thyroid cancer. Sarcoma, systemic light-chain amyloidosis, and Merkel cell carcinoma are preferred, and more preferably, the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma, the lung cancer is selected from non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

[0041] In another aspect, the present disclosure provides a use of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of the above claims, or a drug composition comprising the same, in the preparation of a drug for treating or preventing a tumor, wherein the tumor and cancer are head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelomatous myeloma, thyroid cancer ... Preferably, the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; more preferably, the lung cancer is selected from non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

[0042] In another aspect, the present disclosure further relates to a method for treating and / or preventing a tumor, the method comprising administering to a subject in need thereof a therapeutically effective amount or a prophylactically effective amount of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof described in any one of the above, or a pharmaceutical composition containing the same, and preferably, the tumor is a cancer associated with high expression of Claudin18.2.

[0043] In another aspect, the present disclosure further relates to a method for treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of the above claims, or a drug composition comprising the same, wherein the tumors and cancers include head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, renal cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone marrow ... thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, thyroid cancer, Preferred are cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Kruckenberg's tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light-chain amyloidosis, and Merkel cell carcinoma; more preferably, the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia.

[0044] The active compound (e.g., a ligand-drug conjugate or pharmaceutically acceptable salt thereof described herein) can be prepared in a form suitable for administration by any suitable route, and preferably the active compound is in a unit dosage form or in a form that allows a subject to self-administer a single dose. The unit dosage forms of the present disclosure may be troches, capsules, cachets, bottled drug solutions, drug powders, granules, tablets, suppositories, reconstituted powders, or liquid formulations.

[0045] The dosage of the active compound or composition used in the therapeutic methods of the present disclosure generally varies depending on the severity of the disease, the weight of the subject, and the potency of the active compound, but as a general guide, a suitable unit dose may be from 0.1 mg to 1000 mg.

[0046] The pharmaceutical composition of the present disclosure may contain one or more additives in addition to the active compound, and the additives may be selected from components such as fillers, diluents, binders, wetting agents, disintegrants, or excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0047] The Claudin18.2 antibody and antibody-drug conjugate according to the present disclosure have good affinity with cell surface antigens, good cell endocytosis efficiency and strong tumor inhibition efficiency, as well as broader drug application potential and are suitable for clinical drug application. [Brief explanation of the drawings]

[0048] [Figure 1] This shows the results of FACS detection of the binding of a humanized antibody to human Claudin 18.2 at the cellular level. [Figure 2] NUGC4 cell endocytosis experiment of humanized antibody. [Figure 3A-3C] Figure 3A shows the ADCC effect of antibodies in NUGC4 cells with different Claudin18.2 expression levels. Figure 3B shows the ADCC effect of antibodies in NUGC4 cells with moderate Claudin18.2 expression, and Figure 3C shows the ADCC effect of antibodies in NUGC4 cells with high Claudin18.2 expression. [Figure 4] 1 shows the results of a tumor inhibition experiment using ADC-1 of the present disclosure. [Figure 5] 1 shows the results of a tumor inhibition experiment using ADC-2 of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0049] 1. Terminology Unless otherwise limited, all technical and scientific terms used herein are consistent with those commonly understood by one of ordinary skill in the art. Although the present disclosure can be practiced or tested using any methods and materials similar or equivalent to those described herein, the preferred methods and materials are described herein. In describing and claiming the present disclosure, the following terminology will be used in accordance with the following definitions.

[0050] When trade names are used in this disclosure, they are intended to include formulations of the trade name product, generic drugs and active drug portions of the trade name product.

[0051] Unless stated to the contrary, terms used in the specification and claims have the following meanings.

[0052] The term "drug" refers to a chemical substance that can alter or investigate the physiological functions and pathological conditions of the body and can be applied to the prevention, diagnosis, and treatment of diseases. Drugs include cytotoxic drugs. There is no strict distinction between drugs and poisons. A poison is a chemical substance that has a toxic effect on the body and harms human health at a small dose, while any drug can cause a toxic reaction at an excessively large dose. A cytotoxic drug is a substance that inhibits or prevents cellular function and / or causes cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficient concentrations, but due to a lack of specificity, they can also induce apoptosis of normal cells while killing tumor cells, resulting in severe side effects. Cytotoxic drugs include toxins such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants, and animals, radioactive isotopes (e.g., 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.

[0053] The term "linker unit," "linker," or "connecting fragment" refers to a chemical structural fragment or bond that is connected at one end to a ligand (e.g., an antibody or antigen-binding fragment thereof) and at the other end to a drug, and may be connected to another linker and then to the drug.

[0054] The linker may comprise one or more linker elements. Exemplary linker elements include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid ester ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). The linker may comprise an extender, a spacer, and an amino acid unit and can be synthesized by methods known in the art, for example, as described in US 2005-0238649 A1. The linker may be a "cleavable linker" that facilitates drug release in cells. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0055] Abbreviation Linker units include, but are not limited to: MC=6-maleimidocaproyl, which has the following structure: [ka] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker); Citrulline = 2-amino-5-ureidopentanoic acid, PAB = p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker element); Me-Val-Cit = N-methyl-valine-citrulline (but the linker peptide bond is modified so that it is not cleaved by cathepsin B); MC(PEG)6-OH = maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines); SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate, SPDP = N-succinimidyl 3-(2-pyridyldithio)propionate, SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid ester, and IT = iminothiolane.

[0056] The term "ligand-drug conjugate" refers to a ligand connected to a biologically active drug via a linking unit. The "ligand-drug conjugate" of the present disclosure is preferably an antibody-drug conjugate (ADC), in which a monoclonal antibody or antibody fragment is connected to a biologically active toxic drug via a linking unit. The antibody may be conjugated to the drug directly or via a linker. The average number of drug modules (average drug load or drug loading, which may be expressed as an n value) of each antibody may range, for example, from about 0 to about 20 drug modules; in some embodiments, each antibody is conjugated to 1 to about 10 drug modules; and in some embodiments, each antibody is conjugated to 1 to about 8 drug modules.

[0057] The term "average drug loading" or "drug loading amount" refers to the average number of cytotoxic drugs loaded onto each ligand in a ligand-drug conjugate molecule. This may be expressed as the ratio of the drug amount to the antibody amount. The drug loading range is such that each ligand (Pc) may be linked to 0 to 12 cytotoxic drugs, preferably 1 to 10 cytotoxic drugs. In an embodiment of the present disclosure, the drug loading amount is represented by n, which may be referred to as the drug-antibody ratio (DAR) value. n may be a non-zero integer or decimal number between 0 and 12, preferably an integer or decimal number between 1 and 10, more preferably between 2 and 8, and may be either an integer or decimal number, and most preferably between 3 and 8. Exemplary values are the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average drug amount in each ADC molecule after the coupling reaction can be characterized by conventional methods, such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC.

[0058] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0059] Claudin 18 (CLD18) molecules (GenBank accession numbers: splice variant 1 (CLD18A1): NP_057453, NM016369 and splice variant 2 (CLD18A2 or Claudin18.2): NM_001002026, NP_001002026) are integral transmembrane proteins located in tight junctions between epithelia and endothelia. Occludin and claudins are the major transmembrane protein components of tight junctions. The strong intercellular adhesion properties of claudins create a primary barrier that prevents and regulates paracellular transport of solutes and maintains cell polarity by restricting the lateral diffusion of membrane lipids and proteins. Proteins at tight junctions are involved in the organization of epithelia. Reports suggest that these proteins are largely inaccessible to antibodies in well-structured epithelia, but are exposed in tumor cells.

[0060] The term "antibody" refers to an immunoglobulin, which has a tetrapeptide chain structure consisting of two heavy chains and two light chains connected by interchain disulfide bonds. Immunoglobulins are divided into five types based on the amino acid composition and sequence order of the immunoglobulin heavy chain constant region, or may be referred to as immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds; for example, IgG may be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ or λ chains based on the constant region. Each of the five types of Ig may have either κ or λ chains.

[0061] In full-length antibody heavy and light chains, approximately 110 amino acids near the N-terminus are highly variable and form the variable region (Fv region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region. The variable region contains three highly variable regions (HVRs) and four framework regions (FRs) whose sequences are relatively conserved. The three highly variable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

[0062] The terms "fully humanized antibody," "fully human antibody," or "fully human antibody" are also referred to as "fully human monoclonal antibodies," in which both the variable and constant regions of the antibody are of human origin. The development of monoclonal antibodies has gone through four stages: mouse monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The technologies related to the preparation of fully human antibodies mainly include human hybridoma technology, EBV-mediated B lymphocyte transformation technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0063] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. The binding fragments included in the term "antigen-binding fragment" are selected from antigen-binding fragments of Fab, Fab', F(ab')2, single-chain antibodies (scFv), dimerized V regions (diabodies), disulfide-stabilized V regions (dsFv), and peptides containing CDRs, and examples include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single antibody arm; (v) a single domain or dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR) or (vii) a combination of two or more isolated CDRs optionally connected by a synthetic linker. Alternatively, the two domains of an Fv fragment, VL and VH, are encoded by separate genes, but can be recombinantly linked with a synthetic linker to generate a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Such antibody fragments can be obtained by conventional techniques known to those skilled in the art, and, like intact antibodies, fragments are selected for their functionality. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of different isotypes, such as, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0064] Typically, Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating an IgG antibody molecule with the protease papain (e.g., cleaving the heavy chain at amino acid residue 224), in which the N-terminal portion of the heavy chain and the light chain are linked together by a disulfide bond.

[0065] F(ab')2 is typically obtained by digesting the lower disulfide bond in the hinge region of IgG with the enzyme pepsin. It has a molecular weight of approximately 100,000 and is an antibody fragment containing two Fab regions connected by a hinge, retaining antigen-binding activity.

[0066] Generally, Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, which is obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.

[0067] Alternatively, Fab' can be produced by inserting DNA encoding the Fab' fragment into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0068] The terms "single-chain antibody," "single-chain Fv," or "scFv" refer to a molecule comprising an antibody heavy chain variable domain (or VH) and an antibody light chain variable domain (or VL) connected by a linker. Such scFv molecules may have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example, 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers for use in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0069] The term "CDR" refers to one of the six highly variable regions in an antibody variable domain that primarily mediates antigen binding. Typically, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The amino acid sequence boundaries of a CDR can be determined by any one of a variety of well-known methods. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 of the light chain variable domain and CDR2 and CDR3 of the heavy chain variable domain. Further numbering conventions include the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc MP, Dev. Comp. Immunol., 27, 55-77(2003)).

[0070] The term "antibody framework" refers to the part of a variable domain, VL or VH, that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain. In essence, it is a variable domain without the CDRs.

[0071] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an immunoglobulin or antibody. An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0072] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, an antibody binds to an antigen at a specific site within a range of about 10 -7 Less than m, e.g., about 10 -8 M, 10 -9 M or 10 -10 It binds with an affinity (KD) less than or equal to M.

[0073] The term "KD" refers to the dissociation equilibrium constant of an antibody-antigen interaction. Typically, antibodies (or antigen-binding fragments) of the present disclosure have a dissociation equilibrium constant of about 10 -7 Less than m, e.g., about 10 -8 M or 10 -9 It binds to Claudin18.2 (or its epitope) with a dissociation equilibrium constant (KD) of less than M. For example, in the present disclosure, the affinity between an antibody and a cell surface antigen is measured as a KD value by the FACS method.

[0074] The term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. A nucleic acid molecule may be single-stranded or double-stranded, with double-stranded DNA being preferred. A nucleic acid is "operatively connected" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively connected to a coding sequence if it affects the transcription of the coding sequence.

[0075] "Identity" of amino acid sequences refers to the percentage of amino acid residues in a first sequence that are similar to those in a second sequence, with gaps introduced, if necessary, during the process of aligning amino acid sequences to maximize the percentage of sequence identity, and with any conservative substitutions not considered part of the sequence identity. To measure the percentage of amino acid sequence identity, alignment can be achieved by several methods within the skill of the art, such as publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters to be applied to measure alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0076] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments can be linked. In another embodiment, the vector is a viral vector into which other DNA segments can be linked to the viral genome. The vectors disclosed herein can either autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), or can integrate into the genome of a host cell after introduction and thereby replicate along with the host genome (e.g., non-episomal mammalian vectors).

[0077] Methods for producing and purifying antibodies and antigen-binding fragments well known in the art are described, for example, in Chapters 5 to 8 and 15 of Reisenko's Antibody Laboratory Techniques Manual. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments described in the invention have one or more human FR regions added to non-human CDR regions by genetic engineering techniques. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) homepage, http: / / imgt.cines.fr, by aligning the IMGT human antibody variable region germline gene database with MOE software, or from the immunoglobulin journal Lefranc, G., The Immunoglobulin Facts Book, Academic Press, 2001, ISBN 012441351.

[0078] The term "host cell" refers to a cell into which an expression vector has already been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Bacteria amenable to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli and Salmonella, Bacillaceae, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells.

[0079] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into host cells. In one preferred conventional technique, a mammalian expression system results in antibody glycosylation, particularly at the N-terminal end of the Fc region. Positive clones are expanded in a bioreactor culture to produce antibodies. The culture medium secreting the antibodies can be purified by conventional techniques, for example, through a Sepharose A or G FF column. Nonspecifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and the antibody fragments are detected and collected by SDS-PAGE. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and multimers can be removed by conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, for example, at -70°C, or lyophilized.

[0080] The term "peptide" refers to a compound fragment intermediate between amino acids and proteins, consisting of two or more amino acid molecules connected to each other by peptide bonds, and is a structural and functional fragment of a protein.

[0081] The term "sugar" refers to a biopolymer consisting of the three elements C, H, and O, and may be divided into monosaccharides, disaccharides, polysaccharides, and the like.

[0082] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group, a straight or branched chain group containing 1 to 20 carbon atoms, with alkyl groups containing 1 to 12 carbon atoms being preferred, alkyl groups containing 1 to 10 carbon atoms being more preferred, and alkyl groups containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 or 6 carbon atoms) being most preferred. 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 groups, 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.More preferred are 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, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available attachment site, and the substituents are preferably independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0083] The term "heteroalkyl group" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein the alkyl group is as defined above.

[0084] The term "alkylene group" refers to a saturated, straight-chain or branched-chain aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms of an alkane parent, and is a straight-chain or branched-chain group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms (1, 2, 3, 4, 5, or 6 carbon atoms). Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH-), 1,1-ethylidene (-CH(CH)-), 1,2-ethylidene (-CHCH)-, 1,1-propylidene (-CH(CHCH)-), 1,2-propylidene (-CHCH(CH)-), 1,3-propylidene (-CHCHCHCH-), 1,4-butylidene (-CHCHCHCHCH-), and 1,5-butylidene (-CHCHCHCHCHCH-). The alkylene group may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available attachment site, and the substituents are preferably independently optionally substituted with one or more substituents selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0085] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), where alkyl group and cycloalkyl group are defined above. Non-limiting examples of alkoxy groups include methoxy group, ethoxy group, propoxy group, butoxy group, cyclopropoxy group, cyclobutoxy group, cyclopentyloxy group, and cyclohexyloxy group. An alkoxy group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably independently one or more groups selected from alkyl group, alkenyl group, alkynyl group, alkoxy group, alkylthio group, alkylamino group, halogen, mercapto group, hydroxy group, nitro group, cyano group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, and heterocycloalkylthio group.

[0086] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl group ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms (including 3, 4, 5, 6, 7, or 8 carbon atoms). Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups, and polycyclic cycloalkyl groups include spirocyclic, fused, and bridged cycloalkyl groups.

[0087] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which may be nitrogen, oxygen, or S(O). m(where m is an integer of 0, 1, or 2), but does not include the -OO-, -OS-, or -SS- ring moiety, with the remaining ring atoms being carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms (1, 2, 3, or 4 heteroatoms), and more preferably the cycloalkyl ring contains 3 to 10 ring atoms (3, 4, 5, 6, 7, 8, 9, or 10 ring atoms). Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclyl groups include spirocyclic, fused-ring, and bridged-ring heterocyclyl groups.

[0088] The term "spiroheterocyclyl group" refers to a polycyclic heterocyclyl group having 5- to 20-membered rings sharing one atom (called a spiro atom) between the rings, in which one or more ring atoms is nitrogen, oxygen, or S(O). m (where m is an integer of 0 to 2), with the remaining ring atoms being carbon. It may contain one or more double bonds, but none of the rings has a completely conjugated π-electron system. It is preferably 6 to 14-membered, more preferably 7 to 10-membered. Depending on the number of spiro atoms shared between the rings, spiroheterocyclyl groups are classified as monospiroheterocyclyl groups, bisspiroheterocyclyl groups, or polyspiroheterocyclyl groups, and preferred are monospiroheterocyclyl groups and bisspiroheterocyclyl groups. More preferred are 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocyclyl groups. Non-limiting examples of spiroheterocyclyl groups include: [ka] Includes.

[0089] The term "fused heterocyclyl group" refers to a 5- to 20-membered polycyclic heterocyclyl group in which each ring in the system shares an adjacent pair of atoms with another ring in the system, one or more rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system, and one or more ring atoms is nitrogen, oxygen, or S(O) m (where m is an integer of 0, 1, or 2), and the remaining ring atoms are carbon. A fused heterocyclyl group is preferably 6 to 14-membered, and more preferably 7 to 10-membered (7, 8, 9, or 10-membered). Depending on the number of constituent rings, it may be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclyl group, preferably a bicyclic or tricyclic, and more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclyl group. Non-limiting examples of fused heterocyclyl groups are: [ka] Includes.

[0090] The term "bridged heterocyclyl group" refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings share two non-directly connected atoms, and may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system, and in which one or more ring atoms is nitrogen, oxygen, or S(O) m (where m is an integer of 0, 1, or 2), and the remaining ring atoms are carbon. 6 to 14-membered rings are preferred, and 7 to 10-membered rings (7, 8, 9, or 10-membered rings) are more preferred. Depending on the number of constituent rings, bridged heterocyclyl groups may be classified as bicyclic, tricyclic, tetracyclic, or polycyclic, and are preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclyl groups are: [ka] Includes.

[0091] The heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, in which the ring connected to the parent structure is a heterocyclyl ring, non-limiting examples of which include: [ka] Includes:

[0092] The heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogen atoms, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0093] The term "aryl group" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π-electron system, preferably 6- to 10-membered (6, 7, 8, 9, or 10-membered) group, such as a phenyl group and a naphthyl group, preferably a phenyl group. The aryl group ring may be fused to a heteroaryl group, heterocyclyl group, or cycloalkyl group ring, in which the ring connected to the parent structure is an aryl group ring, non-limiting examples of which are: [ka] Includes.

[0094] The aryl group may be substituted or unsubstituted. When the aryl group is substituted, the substituents are preferably independently one or more groups selected from an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylthio group, an alkylamino group, a halogen atom, a mercapto group, a hydroxy group, a nitro group, a cyano group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a cycloalkoxy group, a heterocycloalkoxy group, a cycloalkylthio group, and a heterocycloalkylthio group.

[0095] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms (1, 2, 3, or 4 heteroatoms) and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered (5-, 6-, 7-, 8-, 9-, or 10-membered heteroaryl group), more preferably 5- or 6-membered, such as furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, and tetrazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, where the ring connected to the parent structure is the heteroaryl ring, non-limiting examples of which include: [ka] Includes.

[0096] The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogen atoms, mercapto groups, hydroxy groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0097] The term "amino-protecting group" refers to a group that protects an amino group with an easily removable group so that the amino group is not altered when other parts of the molecule react. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, t-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl. These groups may be optionally substituted with 1 to 3 substituents (1, 2, or 3 substituents) selected from halogen, alkoxy, or nitro groups. The amino-protecting group is preferably 9-fluorenylmethyloxycarbonyl.

[0098] The term "halogenated alkyl group" refers to an alkyl group in which one or more hydrogens have been replaced with halogens, wherein the alkyl group is as defined above.

[0099] The term "deuterated alkyl group" refers to an alkyl group in which one or more deuterium atoms have replaced a hydrogen atom on the alkyl group, wherein the alkyl group is as defined above.

[0100] The term "hydroxyalkyl group" refers to an alkyl group in which a hydrogen is replaced by one or more hydroxy groups, wherein the alkyl group is as defined above.

[0101] The term "hydroxy group" refers to an --OH group.

[0102] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0103] The term "amino group" refers to -NH2.

[0104] The term "nitro group" refers to -NO2.

[0105] The term "cyano" refers to -CN.

[0106] The term "amide group" refers to -C(O)N(alkyl group) or (cycloalkyl group), wherein alkyl group and cycloalkyl group are as defined above.

[0107] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the phrase includes both cases where the event or circumstance occurs and cases where it does not occur. For example, "a heterocyclyl group optionally substituted with an alkyl group" means that an alkyl group may, but need not, be present, and the phrase includes both cases where the heterocyclyl group is substituted with an alkyl group and cases where the heterocyclyl group is not substituted with an alkyl group.

[0108] "Substituted" means that one or more hydrogen atoms in a group, preferably at most five, more preferably one, two or three hydrogen atoms, are independently replaced with a substituent. Substituents are located only at their chemically feasible positions, and those skilled in the art can determine possible or impossible substitutions (by experiment or theory) without much effort. For example, an amino group or hydroxy group having free hydrogen may be unstable if it is bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0109] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof with other chemical components, and other components such as physiologically / pharmaceutically acceptable carriers and excipients, to facilitate administration to the body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0110] The term "pharmaceutically acceptable salt" or "pharmaceutically available salt" refers to a salt of a ligand-drug conjugate of the present disclosure or a salt of an active compound described herein, which salt is safe and effective when administered to a subject and possesses the desired biological activity. The ligand-drug conjugates of the present disclosure contain at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically available salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0111] In one embodiment of the present disclosure, the cytotoxic drug is conjugated to a sulfhydryl group of the antibody by a linking unit. (1) controlling the molar ratio of the coupling reagent to the monoclonal antibody; (2) controlling the reaction time and temperature; (3) selecting different reaction reagents; The loading of the ligand-cytotoxic drug conjugate can be controlled by a number of methods, including but not limited to: The preparation of common pharmaceutical compositions is shown in the Chinese Pharmacopoeia.

[0112] The term "pharmaceutically acceptable carrier," as used in the present disclosure, refers to a system that can modify the drug's entry into a subject and its distribution in the body, control the drug's release rate, and transport the drug to a target organ. The drug carrier's release and targeting system can reduce drug degradation and loss, lower side effects, and improve bioavailability. For example, polymer surfactants used as carriers can self-assemble to form various aggregates due to their unique amphiphilic structure, including micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules and have good membrane permeability, making them good drug carriers.

[0113] The term "excipient" refers to additives other than the active compound in a pharmaceutical composition, which may also be referred to as auxiliary materials. For example, binders, fillers, disintegrants, and lubricants in tablets, base materials in semi-solid preparations such as ointments and creams, and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure adjusters, and coloring agents in liquid preparations may all be referred to as excipients.

[0114] The term "diluent," also known as a filler, is primarily used to increase the weight and volume of tablets. Adding a diluent not only ensures a consistent volume, but also reduces the dosage deviation of the active ingredient and improves the compressibility of the drug. When a tablet contains an oily component, an absorbent must be added to absorb the oily component to maintain a "dry" state and facilitate tablet formation. Examples include starch, lactose, calcium inorganic salts, and microcrystalline cellulose.

[0115] The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Acceptable solvents and vehicles used include water, Ringer's solution, and isotonic sodium chloride solution. Sterile injectable preparations may also be sterile injectable oil-in-water microemulsions in which the active ingredient is dissolved in the oil phase. For example, the active ingredient may be dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin and processed to form a microemulsion. The injectable solutions or microemulsions can be infused into the subject's bloodstream via local bolus injection. Alternatively, solutions and microemulsions are preferably administered in a manner that maintains a constant, cyclical concentration of the compounds of the present disclosure. To maintain such a constant concentration, a continuous intravenous administration device can be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous pump.

[0116] The pharmaceutical compositions may be in the form of sterile injectable aqueous or oleaginous suspensions for intramuscular and subcutaneous administration. Such suspensions can be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents, as described above. Sterile injectable preparations may also be sterile injectable solutions or suspensions prepared in non-toxic, gastrointestinal-acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Sterile fixed oils are also conveniently used as solvents or suspending media. For this purpose, any suitable fixed oil, including synthetic monoglycerides or diglycerides, can be used. Fatty acids, such as oleic acids, can also be used to prepare injectables.

[0117] 2. Synthesis method To achieve the synthesis objectives, the following synthesis technology scheme is adopted: General formula (Pc-L a -YD), which is a process for preparing a compound of the formula [ka] After reducing Pc, the general formula (L a -YD) and the general formula (Pc-La -YD), wherein TCEP is preferred as the reducing agent, and disulfide bonds on reduced antibodies are particularly preferred; However, Pc, W, L 2 , L 3 , R 1 , R 2 , R 5 ~R 7 , m and n are represented by the general formula (Pc-L a -YD).

[0118] The foregoing specification provides details of one or more embodiments of the present disclosure. Although the present disclosure can be practiced or tested using any methods and materials similar or equivalent to those described herein, the preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully illustrate preferred embodiments of the present disclosure. These examples should not be construed in any way as limiting the scope of the present disclosure, which is limited only by the claims. [Example]

[0119] 1. Preparation of antibodies Example 1-1: Construction of a cell line with high expression of claudin18.2 The pCDH-hClaudin18.2 lentiviral expression vector plasmid and Lipofectamine 3000 transfection reagent for pVSV-G and pCMV-dR8.91 lentiviral packaging vectors were transfected into 293T virus packaging cells. The virus-containing culture supernatant was collected, filtered, and centrifuged at ultrahigh speed. The concentrated virus was then used to infect the human gastric signet ring cell carcinoma cell line NUGC4. The cells were then selected with puromycin for 2-3 weeks, followed by FACS single-cell sorting. Claudin18.2 expression levels were determined based on tumor IHC scores. Cells with a tumor IHC score of 3 were considered high Claudin18.2 expressing cells, and cells with a tumor IHC score of 2 were considered moderate Claudin18.2 expressing cells. NUGC4 / hClaudin18.2 monoclonal cell lines with high Claudin18.2 expression were selected by detecting Claudin18.2 expression on the surface of lentivirus-infected NUGC4 cells using FACS. At the same time, NUGC4 clonal cell lines with moderate Claudin18.2 expression were selected by detecting Claudin18.2 expression on the surface of wild-type NUGC4 cells using FACS, whereas wild-type NUGC4 cells were considered low Claudin18.2 expressing cells. The selected monoclonal cell lines were expanded and frozen in a refrigerator for subsequent experiments.

[0120] Claudin18.2 sequence Genbank: NP_001002026: (SEQ ID NO: 1) MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV;

[0121] Claudin18.2 DNA sequence: (SEQ ID NO:2) 1 AGAATTGCGC TGTCCACTTG TCGTGTGGCT CTGTGTCGAC ACTGTGCGCC ACCATGGCCG 61 TGACTGCCTG TCAGGGCTTG GGGTTCGTGG TTTCACTGAT TGGGATTGCG GGCATCATTG 121 CTGCCACCTG CATGGACCAG TGGAGCACCC AAGACTTGTA CAACAACCCC GTAACAGCTG 181 TTTTCAACTA CCAGGGGCTG TGGCGCTCCT GTGTCCGAGA GAGCTCTGGC TTCACCGAGT 241 GCCGGGGCTA CTTCACCCTG CTGGGGCTGC CAGCCATGCT GCAGGCAGTG CGAGCCCTGA 301 TGATCGTAGG CATCGTCCTG GGTGCCATTG GCCTCCTGGT ATCCATCTTT GCCCTGAAAT 361 GCATCCGCAT TGGCAGCATG GAGGACTCTG CCAAAGCCAA CATGACACTG ACCTCCGGGGA 421 TCATGTTCAT TGTCTCAGGT CTTTGTGCAA TTGCTGGAGT GTCTGTGTTT GCCAACATGC 481 TGGTGACTAA CTTCTGGATG TCCACAGCTA ACATGTACAC CGGCATGGGT GGGATGGTGC 541 AGACTGTTCA GACCAGGTAC ACATTTGGTG CGGCTCTGTT CGTGGGCTGG GTCGCTGGAG 601 GCCTCACACT AATTGGGGGT GTGATGATGT GCATCGCCTG CCGGGGCCTG GCACCAGAAG 661 AAACCAACTA CAAAGCCGTT TCTTATCATG CCTCAGGCCA CAGTGTTGCC TACAAGCCTG 721 GAGGCTTCAA GGCCAGCACT GGCTTTGGGT CCAACACCAA AAACAAGAAG ATATACGATG 781 GAGGTGCCCG CACAGAGGAC GAGGTACAAT CTTATCCTTC CAAGCACGAC TATGTGTAAT 841 GCTCTAAGAC CTCTCAGCAC GGGCGGAAGA AACTCCCGGA GAGCTCACCC AAAAAACAAG 901 GAGATCCCAT CTAGATTTCT TCTTGCTTTT GACTCACAGC TGGAAGTTAG AAAAGCCTCG 961 ATTTCATCTT TGGAGAGGCC AAATGGTCTT AGCCTCAGTC TCTGTCTCTA AATATTCCAC 1021 CATAAAACAG CTGAGTTATT TATGAATTAG AGGCTATAGC TCACATTTTC AATCCTCTAT 1081 TTCTTTTTTT AAATATAACT TTCTACTCTG ATGAGAGAAT GTGGTTTTAA TCTCTCTCTC 1141 ACATTTTGAT GATTTAGACA GACTCCCCCT CTTCCTCCTA GTCAATAAAC CCATTGATGA 1201 TCTATTTCCC AGCTTATCCC CAAGAAAACT TTTGAAAGGA AAGAGTAGAC CCAAAGATGT 1261 TATTTTCTGC TGTTTGAATT TTGTCTCCCC ACCCCCAACT TGGCTAGTAA TAAACACTTA 1321 CTGAAGAAGA AGCAATAAGA GAAAGATATT TGTAATCTCT CCAGCCCATG ATTCCGGTTT 1381 TCTTACACTG TGATCTTAAA AGTTACCAAA CCAAAGTCAT TTTCAGTTTG AGGCAACCAA 1441 ACCTTTCTAC TGCTGTTGAC ATCTTCTTAT TACAGCAACA CCATTCTAGG AGTTTCCTGA 1501 GCTCTCCACT GGAGTCCTCT TTCTGTCGCG GGTCAGAAAT TGTCCCTAGA TGAATGAGAA 1561 AATTATTTTTT TTTAATTTAA GTCCTAAATA TAGTTAAAAT AAATAATGTT TTAGTAAAAT 1621 GATACACTAT CTCTGTGAAA TAGCCTCACC CCTACATGTG GATAGAAGGA AATGAAAAAA 1681 TAATTGCTTT GACATTGTCT ATATGGTACT TTGTAAAGTC ATGCTTAAGT ACAAATTCCA 1741 TGAAAAGCTC ACTGATCCTA ATTCTTTCCC TTTGAGGTCT CTATGGCTCT GATTGTACAT 1801 GATAGTAAGT GTAAGCCATG TAAAAGTAA ATAATGTCTG GGCACAGTGG CTCACGCCTG 1861 TAATCCTAGC ACTTTGGGAG GCTGAGGAGG AAGGATCACT TGAGCCCAGA AGTTCGAGAC 1921 TAGCCTGGGC AACATGGAGA AGCCCTGTCT CTACAAAATA CAGAGAGAAA AAATCAGCCA 1981 GTCATGGTGG CCTACACCTG TAGTCCCAGC ATTCCGGGAG GCTGAGGTGG GAGGATCACT 2041 TGAGCCCAGG GAGGTTGGGG CTGCAGTGAG CCATGATCAC ACCACTGCAC TCCAGCCAGG 2101 TGACATAGCG AGATCCTGTC TAAAAAAATA AAAAATAAAT AATGGAACAC AGCAAGTCCT 2161 AGGAAGTAGG TTAAAACTAA TTCTTTAAAA AAAAAAAAAA GTTGAGCCTG AATTAAATGT 2221 AATGTTTCCA AGTGACAGGT ATCCACATTT GCATGGTTAC AAGCCACTGC CAGTTAGCAG 2281 TAGCACTTTC CTGGCACTGT GGTCGGTTTT GTTTTGTTTT GCTTTGTTTA GAGACGGGGT 2341 CTCACTTTCC AGGCTGGCCT CAAACTCCTG CACTCAAGCA ATTCTTCTAC CCTGGCCTCC 2401 CAAGTAGCTG GAATTACAGG TGTGCGCCAT CACAACTAGC TGGTGGTCAG TTTTGTTACT 2461 CTGAGAGCTG TTCACTTCTC TGAATTCACC TAGAGTGGTT GGACCATCAG ATGTTTGGGC 2521 AAAACTGAAA GCTCTTTGCA ACCACACACC TTCCCTGAGC TTACATCACT GCCCTTTTGA 2581 GCAGAAAGTC TAAATTCCTT CCAAGACAGT AGAATTCCAT CCCAGTACCA AAGCCAGATA 2641 GGCCCCCTAG GAAACTGAGG TAAGAGCAGT CTCTAAAAAC TACCCACAGC AGCATTGGTG 2701 CAGGGGAACT TGGCCATTAG GTTATTATTT GAGAGGAAG TCCTCACATC AATAGTACAT 2761 ATGAAAGTGA CCTCCAAGGG GATTGGTGAA TACTCATAAG GATCTTCAGG CTGAACAGAC 2821 TATGTCTGGG GAAAGAACGG ATTATGCCCC ATTAAATAAC AAGTTGTGTT CAAGAGTCAG 2881 AGCAGTGAGC TCAGAGGCCC TTCTCACTGA GACAGCAACA TTTAAACCAA ACCAGAGGAA 2941 GTATTTGTGG AACTCACTGC CTCAGTTTGG GTAAAGGATG AGCAGACAAG TCAACTAAAG 3001 AAAAAAGAAA AGCAAGGAGG AGGGTTGAGC AATCTAGAGC ATGGAGTTTG TTAAGTGCTC 3061 TCTGGATTTG AGTTGAAGAG CATCCATTTG AGTTGAAGGC CACAGGGCAC AATGAGCTCT 3121 CCCTTCTACC ACCAGAAAGT CCCTGGTCAG GTCTCAGGTA GTGCGGTGTG GCTCAGCTGG 3181 GTTTTTAATT AGCGCATTCT CTATCCCAACA TTTAATTGTT TGAAAGCCTC CATATAGTTA 3241 GATTGTGCTT TGTAATTTTG TTGTTGTTGC TCTATCTTAT TGTATATGCA TTGAGTATTA 3301 ACCTGAATGT TTTGTTACTT AAATATTAAA AACACTGTTA TCCTACAGTT。

[0122] Example 1-2: Production of Anti-Human Claudin 18.2 Monoclonal Antibody 1 Immunization The anti-human Claudin 18.2 monoclonal antibody was produced by immunized mice. The experimental SJL white mice were female, 6 - 8 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd., Animal Production License Number: SCXK (Beijing) 2012 - 0001). Breeding environment: SPF level. After purchasing the mice, they were bred in the laboratory environment for 1 week, with the light / dark cycle adjusted at 12 / 12 hours, the temperature at 20 - 25 °C, and the humidity at 40 - 60%. The mice acclimated to the environment were immunized according to the following protocol. The immunization antigen was huClaudin18.2-HEK293 cells (HEK-293 stable transfection cell line transfected with the human Claudin18.2 plasmid). Immunization protocol: Before the first immunization of cells, TiterMax (R) Gold Adjuvant (Sigma Cat No. T2684) was injected intraperitoneally (IP) into the mice at 0.1 mL / mouse. 30 minutes later, 0.1 mL of cell suspension diluted to a concentration of 1×10 8 / mL with physiological saline was injected intraperitoneally (IP) into each mouse. After the cells were evenly dispersed, inoculation was performed on days 0, 14, 28, 42, and 56. Blood was collected on days 21, 35, 49, and 63, and the antibody titer in the mouse serum was determined by the ELISA method. After the 4th - 5th immunizations, mice with high and stable antibody titers in the serum were selected for spleen cell fusion. Three days before spleen cell fusion, additional immunization was performed, and 1×10 7 cells were injected intraperitoneally (IP).

[0123] 2 Spleen Cell Fusion By an optimized PEG-mediated fusion process, spleen lymphocytes and myeloma cells Sp2 / 0 cells (ATCC (R) CRL-8287 TM ) were fused to obtain hybridoma cells. The hybridoma cells obtained by fusion were 0.5 - 1×10 6The cells were resuspended in complete medium (IMDM medium containing 20% FBS, 1x HAT, and 1x OPI) at a density of 1 / mL and seeded at 100 μL / well into a 96-well plate. After incubation at 37°C and 5% CO2 for 3-4 days, 100 μL / well of HAT complete medium was added and cultured for 3-4 days until needle-like clones were obtained. The supernatant was removed, and 200 μL / well of HT complete medium (IMDM medium containing 20% FBS, 1x HT, and 1x OPI) was added. After incubation at 37°C and 5% CO2 for 3 days, ELISA detection was performed.

[0124] 3. Hybridoma cell selection Depending on the density of hybridoma cell growth, hybridoma culture supernatants were detected using a binding ELISA method. Cells that strongly bound to huClaudin18.2-HEK293 cells but did not bind to HEK293 cells were selected, amplified, cryopreserved, and subcloned 2-3 times until single-cell clones were obtained. Each time the cells were subcloned, detection was also required in a cell binding experiment. Hybridoma clones were obtained by selection through the above experiments, and antibodies were further prepared using serum-free cell culture methods. The antibodies were purified according to the purification example and prepared for use in the detection example.

[0125] Examples 1-3: Humanization of mouse antibodies Monoclonal hybridoma cell lines mAb1901 and mAb1902 with high in vitro activity were selected, and the monoclonal antibody sequences were cloned, humanized, recombinantly expressed, and their activity evaluated. The process for cloning sequences from hybridomas is as follows: Hybridoma cells in the logarithmic growth phase were harvested, and RNA was extracted with Trizol (Invitrogen, 15596-018) (following the instructions in the reagent kit manual) and reverse transcribed (PrimeScript TMThe cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig primer set (Novagen, TB326 Rev.B 0503) and then sent to Sequencing for sequencing. The amino acid sequences corresponding to the hybridoma cell DNA sequences obtained are shown in SEQ ID NOs: 3 to 6.

[0126] mAb1901 Mouse Heavy Chain Variable Region (SEQ ID NO:3) EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQGTSVTVSS;

[0127] mAb1901 Mouse Light Chain Variable Region (SEQ ID NO:4) DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKLELK;

[0128] mAb1902 mouse heavy chain variable region (SEQ ID NO:5) EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGTTLTVSS;

[0129] mAb1902 mouse light chain variable region (SEQ ID NO:6) DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKLEIK;

[0130] The above mouse heavy chain variable region and light chain variable region were connected to the heavy chain constant region of a human IgG1 antibody and the human κ light chain constant region, respectively, to form chimeric antibodies ch1901 and ch1902, which will be described later. The constant region is selected from the following sequences:

[0131] Heavy chain constant region of human IgG1 antibody: (SEQ ID NO:7) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0132] Human kappa light chain constant region: (SEQ ID NO:8) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0133] The humanization of mouse monoclonal antibodies was carried out according to the methods disclosed in many publications in this field. That is, human constant domains were used instead of the parent (mouse antibody) constant domains, and human germline sequences were selected based on the homology between mouse and human antibodies, followed by CDR grafting. In the present invention, candidate molecules with good activity were selected and humanized, and the results are as follows:

[0134] 1. Mouse antibody CDR region In Table 1, the amino acid residues of the VH / VL CDRs are determined and annotated according to the Kabat numbering system. The CDR sequences of the mouse antibodies are shown in Table 1. [Table 1]

[0135] 2. Selection of human germline FR region sequences Based on the obtained mouse antibody VH / VL CDR typical structures, the heavy and light chain variable region sequences were compared with the antibody Germline database to obtain highly homologous human germline templates, among which the human germline light chain framework regions were derived from the human kappa light chain gene.

[0136] 2.1 Design of humanized modifications and back mutations of mAb1901 An appropriate human antibody germline was selected, and the mAb1901 mouse antibody was humanized. The CDR regions of the mouse antibody mAb1901 were grafted onto the selected humanized template, and the humanized variable regions were replaced and recombined with IgG constant regions to form a complete antibody. At the same time, the FR regions in the V regions of the humanized antibody were backmutated. Exemplary backmutation patterns and combinations are as follows: [Table 2] [Table 3]

[0137] In the above table, the heavy chain variable region is connected to a human IgG1 heavy chain constant region represented by SEQ ID NO:7 to form the heavy chain of the full-length antibody, while the light chain variable region is connected to a human κ light chain constant region represented by SEQ ID NO:8 to form the light chain of the full-length antibody. In other embodiments, the heavy chain variable region and the light chain variable region may be connected to other heavy chain constant regions and light chain constant regions, respectively, to form a full-length antibody.

[0138] 2.2 Humanization and back-mutation design of mAb1902 An appropriate human antibody germline was selected, and the mAb1902 mouse antibody was humanized. The CDR regions of the mouse antibody mAb1902 were grafted onto the selected humanized template, and the humanized variable regions were replaced and recombined with IgG constant regions to form a complete antibody. At the same time, the FR regions in the V regions of the humanized antibody were backmutated. Exemplary backmutation patterns and combinations are as follows: [Table 4] [Table 5]

[0139] In the above table, the heavy chain variable region is connected to the human IgG1 heavy chain constant region represented by SEQ ID NO:7 to form the heavy chain of the full-length antibody, and the light chain variable region is connected to the human κ light chain constant region represented by SEQ ID NO:8 to form the light chain of the full-length antibody.

[0140] Chimeric antibody ch1901 ch1901 heavy chain: (SEQ ID NO:35) EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; ch1901 light chain (SEQ ID NO:36) DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0141] Chimeric antibody ch1902 ch1902 heavy chain (SEQ ID NO:37) EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGT TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; ch1902 light chain (SEQ ID NO:38) DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0142] Table 6 shows the humanized antibodies of mAb1901. [Table 6] [Table 7]

[0143] Table 8 shows the humanized antibodies of mAb1902. [Table 8]

[0144] The light and heavy chain sequences of the humanized antibody mAb1902 are shown in Table 9 below. [Table 9]

[0145] The positive control antibody of the present disclosure is IMAB-362 (according to WO2016166122) JPEG0007720305000029.jpg101121

[0146] Each of the above antibodies was cloned, expressed, and purified using conventional gene cloning and recombinant expression methods.

[0147] II. Preparation of Compounds

[0148] Experimental methods in the examples of this disclosure for which specific conditions are not specified generally follow common conditions or conditions recommended by the manufacturers of raw materials or products. Reagents for which specific sources are not specified are commonly available commercially.

[0149] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d), deuterated chloroform (CDCl), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). The chemical shifts were 10 -6 Expressed in ppm.

[0150] MS measurements were performed using a FINNIGAN LCQAd(ESI) mass spectrometer (manufacturer: Thermo, model number: Finnigan LCQ advantage MAX). UPLC measurements were performed using a Waters Acquity UPLC SQD liquid chromatograph mass spectrometer. HPLC measurements were performed using an Agilent 1200DAD high performance liquid chromatograph (Sunfire C18 150 x 4.6 mm column) and a Waters 2695-2996 high performance liquid chromatograph (Gimini C18 150 x 4.6 mm column). UV-HPLC measurements were performed using a Thermo nanodrop 2000 ultraviolet spectrophotometer.

[0151] Growth inhibition rate and IC 50 The values were measured using a PHERA starFS microplate reader (BMG, Germany).

[0152] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used as silica gel plates for thin layer chromatography (TLC). The silica gel plate specifications for thin layer chromatography (TLC) are 0.15 mm to 0.2 mm, and the silica gel plate specifications for separating and purifying products by thin layer chromatography are 0.4 mm to 0.5 mm.

[0153] Column chromatography generally used Yantai Huanghai 200–300 mesh silica gel as the carrier.

[0154] Known starting materials according to the present disclosure may be synthesized by adopting or following methods known in the art, or may be purchased commercially from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), and Darui Chemical.

[0155] In the examples, all reactions were carried out in an argon or nitrogen atmosphere unless otherwise specified. An argon or nitrogen atmosphere refers to an argon or nitrogen balloon with a volume of approximately 1 L connected to the reaction flask.

[0156] A hydrogen atmosphere refers to a hydrogen balloon with a volume of approximately 1 L attached to the reaction flask.

[0157] The pressurized hydrogenation reaction was carried out using a Parr 3916EKX hydrogenation apparatus and a Seiran QL-500 hydrogen generator or an HC2-SS hydrogenation apparatus. The hydrogenation reaction was usually carried out after three cycles of evacuation and hydrogen filling.

[0158] The microwave reaction was carried out using a CEM Discover-S 908860 microwave reactor.

[0159] In the examples, unless otherwise specified, the solutions used in the reactions refer to aqueous solutions. Unless otherwise specified in the examples, the reaction temperature is room temperature. Room temperature is the optimum reaction temperature, with the temperature range being 20°C to 30°C.

[0160] Preparation of PBS buffer solution with pH 6.5 in the example: 8.5 g of KH2PO4, 8.56 g of K2HPO4.3H2O, 5.85 g of NaCl, and 1.5 g of EDTA were taken and placed in a flask, and the volume was adjusted to 2 L. The solution was completely dissolved by ultrasonication and then shaken to obtain the solution.

[0161] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used to purify the compound include A: dichloromethane and isopropyl alcohol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system, and the volume ratio of the solvents may be adjusted depending on the polarity of the compound, or may be adjusted by adding a small amount of triethylamine and an acidic or basic reagent, etc.

[0162] Some of the compounds of the present disclosure have been characterized by Q-TOF LC / MS, which utilized an Agilent 6530 accurate mass quadrupole-time of flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1 × 75 mm column).

[0163] For the YD drug moiety of the antibody-drug conjugates of the present disclosure, reference is made to PCT / CN2019 / 107873, and the synthesis and testing of related compounds are cited in this patent, the synthesis of non-limiting examples of which are cited as follows:

[0164] Example 1 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)-1-hydroxycyclopropane-1-formamide 1 [ka] Exatecan mesylate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application EP0737686A1) was added to 1 mL of N,N-dimethylformamide and cooled to 0-5°C in an ice-water bath. One drop of triethylamine was added and the reaction mixture was stirred until the solution became clear. 1-Hydroxycyclopropylformate 1a (1.4 mg, 3.7 μmol, prepared by the known method disclosed in Tetrahedron Letters, 25(12), 1269-72, 1984) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.8 mg, 13.7 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 1 (1.6 mg, yield 82.1%). MS m / z (ESI): 520.2 [M+1] 1 H NMR (400 MHz, CDCl3): δ 7.90-7.84 (m, 1H), 7.80-7.68(m, 1H), 5.80-5.70 (m, 1H), 5.62-5.54(m, 2H), 5.44-5.32 (m, 2H), 5.28-5.10(m, 2H), 3.40-3.15 (m, 3H), 2.44 (s, 3H), 2.23(t, 1H), 2.06-1.75 (m, 2H), 1.68-1.56 (m, 1H), 1.22-1.18 (m, 2H), 1.04-0.98 (m, 2H), 0.89 (t, 3H).

[0165] Example 2 (S)-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)-2-hydroxyacetamide 2-A (R)-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)-2-hydroxyacetamide 2-B [ka] To 1b (4 mg, 7.53 μmol), 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide were added, the atmosphere was purged with argon three times, and the mixture was cooled to 0-5°C in an ice-water bath. 0.3 mL of N-methylmorpholine was added dropwise and stirred until the reaction mixture became clear. 2-Cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared according to the method disclosed in patent application WO2013106717), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 1 hour. The ice-water bath was removed, and the mixture was heated to 30°C and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product, compound 2, was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A - water (10 mmol NHOAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title products (2-A: 1.5 mg, 2-B: 1.5 mg). MS m / z (ESI): 534.0 [M+1]. Single-configuration compound 2-B (relatively short retention time) UPLC analysis: retention time: 1.06 minutes, purity: 88% (chromatogram: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetyl triol). 1 H NMR (400 MHz, DMSO-d6): δ 8.37 (d, 1H), 7.76 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.58-5.56 (m, 1H), 5.48 (d, 1H), 5.41 (s, 2H), 5.32-5.29 (m, 2H), 3.60 (t, 1H), 3.19-3.13 (m, 1H), 2.38 (s, 3H), 2.20-2.14 (m, 1H), 1.98 (q, 2H), 1.87-1.83 (m, 1H), 1.50-1.40 (m, 1H), 1.34-1.28 (m, 1H), 0.86 (t, 3H), 0.50-0.39 (m, 4H). Single configuration compound 2-A (relatively long retention time) UPLC analysis: retention time: 1.10 minutes, purity: 86% (chromatogram: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetyl triol). 1 H NMR (400 MHz, DMSO-d6): δ 8.35 (d, 1H), 7.78 (d, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58-5.53 (m, 1H), 5.42 (s, 2H), 5.37 (d, 1H), 5.32 (t, 1H), 3.62 (t, 1H), 3.20-3.15 (m, 2H), 2.40 (s, 3H), 2.25-2.16 (m, 1H), 1.98 (q, 2H), 1.87-1.82 (m, 1H), 1.50-1.40 (m, 1H), 1.21-1.14 (m, 1H), 0.87 (t, 3H), 0.47-0.35 (m, 4H).

[0166] Example 3 (S)-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)-3,3,3-trifluoro-2-hydroxypropanamide 3-A (R)-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)-3,3,3-trifluoro-2-hydroxypropanamide 3-B [ka] 1b (5.0 mg, 9.41 μmol) was added to 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, cooled to 0-5°C in an ice-water bath, and 0.3 mL of N-methylmorpholine was added dropwise and stirred until the reaction mixture became clear. 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplier: Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 10 minutes. The ice-water bath was removed, and the mixture was heated to 30°C and stirred for 8 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product, compound 3, was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A - water (10 mmol NHOAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min). The corresponding components were collected and concentrated under reduced pressure to give the title products (3-A: 1.5 mg, 3-B: 1.5 mg). MS m / z (ESI): 561.9 [M+1]. Single-configuration compounds (relatively short retention times) UPLC analysis: retention time: 1.11 minutes, purity: 88% (chromatogram: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetyl triol). 1 H NMR (400 MHz, DMSO-d6): δ 8.94 (d, 1H), 7.80 (d, 1H), 7.32 (s, 1H), 7.20 (d, 1H), 6.53 (s, 1H), 5.61-5.55 (m, 1H), 5.45-5.23 (m, 3H), 5.15-5.06 (m, 1H), 4.66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.26-2.20 (m, 1H), 2.16-2.08 (m, 1H), 2.02-1.94 (m, 1H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H). Single configuration compound (relatively long retention time) UPLC analysis: retention time: 1.19 minutes, purity: 90% (chromatogram: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetyl triol). 1 H NMR (400 MHz, DMSO-d6): δ 8.97 (d, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.16 (d, 1H), 6.53 (s, 1H), 5.63-5.55 (m, 1H), 5.45-5.20 (m, 3H), 5.16-5.07 (m, 1H), 4.66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.22-2.14 (m, 1H), 2.04-1.95 (m, 2H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

[0167] Example 4 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)-1-hydroxycyclopentane-1-formamide 4 [ka] 1 mL of N,N-dimethylformamide was added to 1b (3.0 mg, 5.64 μmol), cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added and stirred until the reaction mixture became clear. 1-Hydroxycyclopentaneformic acid 4a (2.2 mg, 16.9 μmol, prepared according to the method disclosed in patent application WO2013106717) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were added to the reaction mixture in that order. After addition, the mixture was stirred at 0-5°C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 4 (2.5 mg, yield 80.9%). MS m / z (ESI): 548.0 [M+1]. 1 H NMR (400 MHz, CDCl3): δ 7.73-7.62 (m, 2H), 5.75-5.62 (m, 1H), 5.46-5.32 (m, 2H), 5.26-5.10 (m, 1H), 3.30-3.10 (m, 1H), 2.43 (s, 3H), 2.28-2.20 (m, 2H), 2.08-1.84 (m, 8H), 1.69-1.58 (m, 2H), 1.04-1.00 (m, 2H), 0.89 (t, 3H).

[0168] Example 5 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)-1-(hydroxymethyl)cyclopropane-1-formamide 5 [ka] 1 mL of N,N-dimethylformamide was added to 1b (2.0 mg, 3.76 μmol), cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added and stirred until the reaction mixture became clear. 1-(Hydroxymethyl)-cyclopentaneformic acid 5a (0.87 mg, 7.5 μmol, prepared according to the method disclosed in patent application WO201396771) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 5 (1.0 mg, yield 50%). MS m / z (ESI): 533.9 [M+1]. 1 H NMR (400 MHz, CDCl3): δ 8.07 (s, 1H), 7.23-7.18 (m, 2H), 6.71-6.64 (m, 1H), 6.55-6.51 (m, 1H), 5.36-5.27 (m, 2H), 4.67-4.61 (m, 2H), 3.53-3.48 (m, 1H), 3.30-3.22 (m, 2H), 3.18-3.13 (m, 1H), 2.71-2.61 (m, 2H), 2.35-2.28 (m, 1H), 2.04-1.91 (m, 4H), 1.53-1.40 (m, 3H), 0.91-0.75 (m, 4H).

[0169] Example 6 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)-1-(hydroxymethyl)cyclobutane-1-formamide 6 [ka] 1 mL of N,N-dimethylformamide was added to 1b (3.0 mg, 5.64 μmol), cooled to 0-5°C in an ice-water bath, and one drop of triethylamine was added. The reaction mixture was stirred until clear. 1-(hydroxymethyl)cyclobutane-1-formic acid 6a (2.2 mg, 16.9 μmol, prepared by a known method disclosed in "Journal of the American Chemical Society, 2014, vol. 136, #22, pp. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction mixture, and the reaction mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 6 (2.1 mg, yield 67.9%). MS m / z (ESI): 548.0 [M+1]. 1H NMR (400 MHz, DMSO-d6): δ 7.85-7.62 (m, 1H), 6.88 (br,1H), 5.87-5.48 (m,2H), 5.47-5.33 (m,1H), 5.31-5.06 (m,1H), 4.25-3.91 (m, 2H), 3.25 (br, 1H), 2.60-2.32 (m, 3H), 2.23 (t, 1H), 2.15-1.95 (m, 3H), 1.70-1.56 (m, 2H), 1.41-1.17 (m, 9H), 1.03 (s, 1H), 0.95-0.80 (m, 2H).

[0170] Example 7 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)-1-hydroxycyclobutane-1-formamide 7 [ka] 1b (3.0 mg, 5.64 μmol) was added to 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, cooled to 0-5°C in an ice-water bath, and 0.3 mL of N-methylmorpholine was added dropwise. The reaction mixture was stirred until clear. 1-Hydroxycyclobutaneformic acid 7a (2.0 mg, 17.22 μmol, supplied by Yakuseki), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) were added sequentially to the reaction mixture. After addition, the mixture was stirred at 0-5°C for 10 minutes. The ice-water bath was removed, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by thin-layer chromatography using developing solvent system B to obtain the title product 7 (2.5 mg, 83.1% yield). MS m / z (ESI): 534.0 [M+1]. 1H NMR (400 MHz, DMSO-d6): δ 8.28 (d, 1H), 7.75 (d, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 6.12 (s, 1H), 5.59-5.51 (m, 1H), 5.41 (s, 2H), 5.20-5.01 (m, 2H), 3.27-3.17 (m, 1H), 3.15-3.05 (m, 1H), 2.71-2.63 (m, 1H), 2.37 (s, 3H), 2.12-2.05 (m, 1H), 2.03-1.94 (m, 2H), 1.92-1.78 (m, 4H), 1.50-1.42 (m, 1H), 0.90-0.83 (m, 4H).

[0171] Example 8 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaicosyl)oxy)-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)cyclopropane-1-formamide 8 [ka]

[0172] Step 1 Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylate 8c Benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104 mg, 0.54 mmol, prepared by the method disclosed in patent application US2005 / 20645) and (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate 8b (100 mg, 0.27 mmol, prepared by the method disclosed in patent application CN105829346A) were added to a reaction flask, 5 mL of tetrahydrofuran was added, the mixture was purged with argon three times, and the temperature was lowered to 0-5°C in an ice-water bath. Potassium tert-butoxide (61 mg, 0.54 mmol) was added, the ice-water bath was removed, the mixture was warmed to room temperature, and the mixture was stirred for 10 min. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The combined organic phase was concentrated. The resulting residue was dissolved in 3 mL of 1,4-dioxane, 0.6 mL of water was added, and sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (70 mg, 0.27 mmol) were added. The mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 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 B to give the title product 8c (100 mg, 73.6% yield). MS m / z (ESI): 501.0 [M+1].

[0173] Step 2 1-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid 8d 8c (50 mg, 0.10 mmol) was dissolved in 3 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (25 mg, 10% content) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to give the title product 8d (41 mg, 100% yield). MS m / z (ESI): 411.0 [M+1].

[0174] Step 3 (9H-Fluoren-9-yl)methyl (2-(((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)aminocarbonyl)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 8e 1b (7 mg, 0.013 mmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, the atmosphere was purged with argon three times, and the mixture was cooled to 0-5 °C in an ice-water bath. One drop of triethylamine was added, and a solution of 8d (7 mg, 0.017 mmol) in 0.5 mL of N,N-dimethylformamide was added. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (7 mg, 0.026 mmol) was added, and the mixture was stirred in an ice bath for 35 min. 10 mL of water was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phase was washed with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent system B to give the title product 8e (8.5 mg, 78.0% yield). MS m / z (ESI): 828.0 [M+1].

[0175] Step 4 1-((2-Aminoacetamido)methoxy)-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)cyclopropane-1-formamide 8f 8e (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, 0.1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, 2 mL of toluene was added, and the mixture was concentrated under reduced pressure. This procedure was repeated twice, 3 mL of n-hexane was added, and the upper layer of n-hexane was poured off. This procedure was repeated three times, and the mixture was concentrated under reduced pressure to give the crude product 8f (2.9 mg). This product was used directly in the next reaction without further purification. MS m / z (ESI): 606.0 [M+1].

[0176] Step 5 1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolyl-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazaicosyl)-oxy)-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)cyclopropane-1-formamide 8 Crude product 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged with argon three times, and cooled to 0-5°C in an ice-water bath. A 0.3 mL N,N-dimethylformamide solution of (S)-2-(-2-(-2-(6-(-2,5-dioxo-1H-pyrrolyl-1-yl)hexaneamino)acetamino)acetamino)-3-phenylpropionic acid 8g (2.7 mg, 5.80 μmol, prepared by the method disclosed in patent application "EP2907824") was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The mixture was reacted in an ice bath for 30 minutes, the ice bath was removed, the mixture was warmed to room temperature, and the mixture was stirred for 15 minutes. The reaction mixture was purified by high-performance liquid chromatography (column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A-water (10 mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding components were collected and concentrated under reduced pressure to give the title product 8 (2 mg, yield 39.0%). MS m / z (ESI): 1060.0 [M+1]. 1H NMR (400 MHz, DMSO-d6): δ 9.01 (d, 1H), 8.77 (t, 1H), 8.21 (t, 1H), 8.08-7.92 (m, 2H), 7.73 (d, 1H), 7.28 (s, 1H), 7.24-7.07 (m, 4H), 6.98 (s, 1H), 6.50 (s, 1H), 5.61 (q, 1H), 5.40 (s, 2H), 5.32 (t, 1H), 5.12 (q, 2H), 4.62 (t, 1H), 4.52 (t, 1H), 4.40-4.32 (m, 1H), 3.73-3.47 (m, 8H), 3.16-3.04 (m, 2H), 2.89 (dd, 1H), 2.69-2.55 (m, 2H), 2.37-2.23 (m, 4H), 2.12-1.93 (m, 4H), 1.90-1.74 (m, 2H), 1.52-1.38 (m, 4H), 1.33-1.11 (m, 5H), 0.91-0.81 (m, 4H).

[0177] Example 9 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)caproamide 9-A 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)caproamide 9-B [ka]

[0178] Step 1 2-Cyclopropyl-2-hydroxybenzyl acetate 9a 2a (1.3 g, 11.2 mmol, prepared according to the method disclosed in patent application WO2013 / 106717) was dissolved in 50 mL of acetonitrile, and 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 diatomaceous earth, and the filter cake was rinsed with ethyl acetate (10 mL). The combined filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using eluent system C to give the title product 9a (2 g, 86.9% yield).

[0179] Step 2 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate benzyl ester 9b 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to a reaction flask, 4 mL of tetrahydrofuran was added, and the mixture was purged with argon three times. The mixture was cooled to 0-5 °C in an ice-water bath, potassium tert-butoxide (109 mg, 0.98 mmol) was added, the ice-water bath was removed, the mixture was warmed to room temperature, and stirred for 40 min. 10 mL of ice-water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The combined organic phase was concentrated. The resulting residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol), and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 h. After adding 20 mL of water, the mixture was extracted with ethyl acetate (10 mL × 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 give the title product 9b (48 mg, yield 19%). MS m / z (ESI): 515.0 [M+1].

[0180] Step 3 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 9c 9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (12 mg, 10% content, dry) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to give the crude title product 9c (13 mg), which was used directly in the next reaction without further purification. MS m / z (ESI): 424.9 [M+1].

[0181] Step 4 (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 1b (10 mg, 18.8 μmol) was added to a reaction flask, 1 mL of N,N-dimethylformamide was added, and the mixture was purged with argon three times. The mixture was cooled to 0-5 °C in an ice-water bath, and one drop of triethylamine was added. 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) were added, and the mixture was stirred in an ice bath for 40 min. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was 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). MS m / z (ESI): 842.1[M+1].

[0182] Step 5 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 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 mixture was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure. This procedure was repeated twice. The residue was pulped with 3 mL of n-hexane, and after standing, the supernatant was poured off and the solid was retained. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude title product 9e (17 mg), which was used directly in the next reaction without further purification. MS m / z (ESI): 638.0[M+18].

[0183] Step 6 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)caproamide 9-A 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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)caproamide 9-B Crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged with argon three times, and cooled to 0-5°C in an ice-water bath. 8 g (21.2 mg, 44.8 μmol) of N,N-dimethylformamide in 0.3 mL was added, followed by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol). The mixture was stirred in an ice bath for 10 minutes, removed, and warmed to room temperature. Compound 9 was produced by stirring for 1 hour. The reaction mixture was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19 × 250 mm, mobile phase: A - water (10 mmol NH4OAc), B - acetonitrile, gradient elution, flow rate: 18 mL / min), and the corresponding components were collected and concentrated under reduced pressure to give the title products (9-A: 2.4 mg, 9-B: 1.7 mg). MS m / z (ESI): 1074.4 [M+1]. Single-configuration compound 9-A (relatively short retention time): UPLC analysis: Retention time: 1.14 min, Purity: 85% (Column: ACQUITY UPLC BEHC18 1.7 μm 2.1 × 50 mm, Mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile). 1H NMR (400 MHz, DMSO-d6): δ 8.60 (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, 1H), 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). Compound 9-B in a single configuration (comparatively long retention time): UPLC analysis: retention time: 1.16 minutes, purity: 89% (Kurarum: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm, mobile phase: A-water (5 mmol NH4OAc), B-アセトニトリル). 1H NMR (400 MHz, DMSO-d6): δ 8.68-8.60 (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).

[0184] 3. Preparation of anti-Claudin18.2 antibody ADC conjugate Analysis of drug loading in ADC bulk drug 1. UV-HPLC method The DAR value n of some ADC examples of the present disclosure was calculated using UV-HPLC, specifically as follows. 1. Measurement method: A cuvette containing sodium succinate buffer was placed in the reference absorption cell and the sample measurement absorption cell, respectively. After the solvent blank was subtracted, the cuvette containing the test solution was placed in the sample measurement absorption cell, and the absorbance at 280 nm and 370 nm was measured. 2. Calculation of results: The loading amount of the ADC solution was measured by ultraviolet spectrophotometry (instrument used: Thermo Nanodrop 2000 ultraviolet spectrophotometer). The principle is that the total absorbance value of the ADC solution at a certain wavelength is equal to the cumulative absorbance value of the drug and monoclonal antibody at that wavelength, i.e., (1)A 280 nm =ε mab-280 bC mab +ε Drug-280 bC Drug ε Drug-280 : The drug has an average molar extinction coefficient of 5100 at 280 nm, C Drug : drug concentration, ε mab-280 The average molar adsorption coefficient of the monoclonal antibody stock solution at 280 nm is 214600, C mab: Concentration of monoclonal antibody stock solution, b: The optical path length is 1 cm. Similarly, the total absorbance equation for the sample at 370 nm can be obtained, i.e., (2)A 370 nm =ε mab-370 bC mab +ε Drug-370 bC Drug ε Drug-370 : The average molar extinction coefficient of the drug at 370 nm is 19000, C Drug : drug concentration, ε mab-370 : The extinction coefficient of the monoclonal antibody stock solution at 370 nm is 0, C mab: Concentration of monoclonal antibody stock solution, b: The optical path length is 1 cm. The drug loading can be calculated using the two equations (1) and (2) in combination with the extinction coefficients and concentration data of the monoclonal antibody and drug at the two detection wavelengths. Drug loading = C Drug / C mab .

[0185] 2. RP-HPLC method The DAR values of some ADC Examples of the present disclosure were calculated by RP-HPLC (reverse-phase high-performance liquid chromatography), specifically as follows. 1,Method of measurement: The naked antibody (unconjugated antibody) and the test ADC sample (concentration 1 mg / mL) were reduced with 4 μL of DDT (Sigma) and then placed in a water bath at 37°C for 1 hour. The samples were then removed and placed in an inner tube. Detection was performed using an Agilent 1200 high-efficiency liquid chromatograph. The column used was an Agilent PLRP-S 1000A 8 μm 4.6 x 250 mm column. The column temperature was 80°C, the DAD detector wavelength was 280 nm, the flow rate was 1 mL / min, and the injection volume was 40 μL. The light and heavy chains were then identified by spectral comparison between the sample and the naked antibody. The DAR value n was calculated by integrating the spectrum of the detection sample. 2. Preparation of solutions 1) 0.25 M DTT solution: Preparation example: 5.78 mg of DTT was taken and thoroughly dissolved by adding 150 μL of purified water to obtain a 0.25 M DTT solution, which was then stored at -20°C. 2) Mobile phase A (0.1% TFA in water): Preparation example: 1000 mL of purified water was measured out in a measuring cylinder, 1 mL of TFA (Sigma) was added, and the mixture was thoroughly mixed uniformly before use. The mixture was then stored at 2-8°C for 14 days. 3) Mobile phase B (0.1% TFA in acetonitrile): Preparation example: 1000 mL of acetonitrile was measured out using a measuring cylinder, 1 mL of TFA was added, and the mixture was thoroughly mixed uniformly before use. The mixture was then stored at 2 to 8°C for 14 days. 3. Data analysis By comparing the spectra of the sample with the naked antibody, the positions of the light and heavy chains were distinguished, and the DAR value (n) was calculated by integrating the spectrum of the detected sample. The calculation formula is as follows: JPEG0007720305000039.jpg59150 Sum of LC peak areas = LC peak area + LC+1 peak area Sum of HC peak areas = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area LC DAR = Σ (number of connected drugs × peak area percentage) / sum of LC peak areas HC DAR = Σ (number of connected drugs × peak area percentage) / sum of HC peak areas DAR = LC DAR + HC DAR.

[0186] Example of Preparation of Claudin18.2 Antibody-Drug Conjugate Examples 3-1 and 3-2: ADC-1 and ADC-2 [ka] At 37°C, a PBS buffer solution containing antibody h1902-5 (0.05 M PBS buffer solution, pH 6.5, 10.0 mg / mL, 320.0 mL, 21.62 μmol) was added to a tri(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 11.03 mL, 110.3 μmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours to terminate the reaction. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (350 mg, 303 μmol) was dissolved in 13.2 mL of acetonitrile and 6.6 mL of DMSO, added to the reaction solution cooled to 25°C, placed in a water bath oscillator, and allowed to oscillate at 25°C for 3 hours, after which the reaction was stopped. The resulting reaction mixture was purified by ultrafiltration using 5 L of PBS buffer (50 mM, pH 6.5, 4% acetonitrile, 2% DMSO) and 5 L of succinate buffer (10 mM, pH 5.3) to remove small molecules. Sucrose was added to 60 mg / mL, and Tween-20 was added to 0.2 mg / mL. Finally, the exemplary product ADC-1 (10 mM succinate, pH 5.3, 10 mg / mL, 2.626 g) of the general formula of antibody-drug conjugate h1902-5-9-A was prepared. Yield: 81.81%. Calculate the mean value by UV-HPLC: n=6.8. Using the above method, antibody h1901-11 was used instead of antibody h1902-5, and combined with compound 9-A to prepare an exemplary product ADC-2 of the general formula antibody-drug conjugate h1901-11-9-A, with a DAR value of n=7.1.

[0187] Example 3-3 ADC-3 At 37°C, an aqueous solution of antibody h1901-11 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 1 mL, 67.5 nmol) was added to the prepared aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 10.1 μL, 101 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (0.58 mg, 540 nmol) was dissolved in 34 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and allowed to oscillate at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the exemplary product ADC-3 of the antibody-drug conjugate h1901-11-9-A in PBS buffer (0.72 mg / mL, 11.2 mL) and stored at 4 °C. Average values calculated by RP-HPLC: n = 2.51.

[0188] Example 3-4 ADC-4 At 37°C, an aqueous solution of antibody h1901-11 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 1 mL, 67.5 nmol) was added to the prepared aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 16.9 μL, 169 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (0.73 mg, 680 nmol) was dissolved in 43 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and allowed to oscillate at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-4 (0.62 mg / mL, 12.5 mL) as an exemplary product of antibody-drug conjugate h1901-11-9-A in PBS buffer and stored at 4 °C. Average values calculated by RP-HPLC: n = 4.06.

[0189] Example 3-5 ADC-5 At 37°C, an aqueous solution of antibody h1901-11 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 1 mL, 67.5 nmol) was added to the prepared aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 35.8 μL, 358 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (1.09 mg, 1015 nmol) was dissolved in 64 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and allowed to oscillate at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-5 (0.54 mg / mL, 12.5 mL), an exemplary product of antibody-drug conjugate h1901-11-9-A, in PBS buffer. The product was stored at 4 °C. Average values calculated by RP-HPLC: n = 6.8.

[0190] Example 3-6 ADC-6 At 37°C, an aqueous solution of antibody h1902-5 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 1.08 mL, 72.9 nmol) was added to the prepared aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 10.9 μL, 109 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (0.63 mg, 587 nmol) was dissolved in 40 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and allowed to oscillate at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the exemplary product ADC-6 of h1902-5-9-A in PBS buffer (0.7 mg / mL, 13.0 mL) and stored at 4 °C. Average values calculated by RP-HPLC: n = 2.69.

[0191] Example 3-7 ADC-7 At 37°C, an aqueous solution of antibody h1902-5 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 1.08 mL, 72.9 nmol) was added to the prepared aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.3 μL, 183 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (0.79 mg, 736 nmol) was dissolved in 50 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and allowed to oscillate at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the exemplary product ADC-7 of h1902-5-9-A in PBS buffer (0.6 mg / mL, 14.0 mL) and stored at 4 °C. Average values calculated by RP-HPLC: n = 4.25.

[0192] Example 3-8 ADC-8 At 37°C, an aqueous solution of antibody h1902-5 in PBS buffer (0.05 M PBS buffer, pH 6.5, 10.0 mg / mL, 1.08 mL, 72.9 nmol) was added to the prepared aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 38.7 μL, 387 nmol), placed in a water bath oscillator, and allowed to oscillate at 37°C for 3 hours before quenching. The reaction solution was then cooled to 25°C in a water bath. Compound 9-A (1.18 mg, 1099 nmol) was dissolved in 70 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath oscillator and allowed to oscillate at 25 °C for 3 hours to terminate the reaction. The reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain the exemplary product ADC-8 of h1902-5-9-A in PBS buffer (0.56 mg / mL, 14.2 mL) and stored at 4 °C. Average values calculated by RP-HPLC: n = 7.01.

[0193] Example 3-9 ADC-9 The prepared TCEP histidine buffer (10 mM histidine buffer, 1.717 mM, 1.16 L, 1.99 mmol) was added to histidine-acetate-Tris / EDTA buffer (10 mM histidine-acetate-Tris buffer, pH 7.2, 20.6 g / L, 6.49 L, 0.91 mmol) containing antibody h1902-5 at 12°C, and the mixture was placed in a thermostatic water bath and stirred at 12°C for 2 hours to terminate the reaction, yielding intermediate I solution. Compound 9-A (4.72 g, 4.39 mmol) was dissolved in 0.38 L of DMSO to prepare a DMSO solution of compound 9-A. 0.38 L of DMSO was added to the solution of intermediate I, and then the DMSO solution of compound 9-A was added. The mixture was placed in a thermostatic water bath and stirred at 12°C for 1 hour to terminate the reaction. The reaction mixture was purified using a Capto S Impact cation chromatography column. It was washed with nine column volumes of 0.05 M acetate buffer (pH 5.0) containing 10% (v / v) DMSO and six column volumes of 0.05 M acetate buffer (pH 5.0), followed by elution with 0.05 M acetate, 0.30 M sodium chloride buffer (pH 5.5) to remove free toxins and residual solvents. Ultrafiltration was performed at 22°C using a 7-fold volume of the cationic eluent (using a 30 KD polycellulose membrane package) to obtain the exemplary product ADC-9 of h1902-5-9-A. The average value calculated by RP-HPLC was 4.1.

[0194] The drug loading obtained in this example is a non-limiting example, and those skilled in the art can obtain conjugates with different DAR values (1 to 10, preferably 1 to 8, more preferably 2 to 8, 2 to 7) by adjusting the reaction conditions and reagents.

[0195] Biological evaluation Test Example 1: Cell-level ELISA binding experiment Cell-based ELISA experiments were used to detect the binding properties of Claudin18.2 antibodies. NUGC4 cells stably transfected with Claudin18.2 and grown to 90% confluency were cultured in 96-well cell plates. Cells were fixed with 4% paraformaldehyde for 1 hour. The plates were washed three times with PBST buffer (PBS, pH 7.4, containing 0.05% Tween-20). Then, 200 μL of 5% nonfat milk (Kwongming nonfat dry milk) sealing solution diluted with PBS was added per well. The plates were then incubated for 2.5 hours in a 37°C incubator or overnight (16–18 hours) at 4°C for sealing. After sealing, the sealing solution was discarded, and the plates were washed three times with PBST buffer. Different concentrations of the antibody to be tested, diluted in 50 μL of sample diluent (PBS, pH 7.4, containing 1% nonfat dry milk), were added per well. The plates were then incubated for 2 hours in a 37°C incubator. After incubation, the plate was washed five times with PBST, and 100 μL / well of HRP-conjugated sheep anti-human secondary antibody (Jackson Immuno Research, 109-035-003) diluted in sample diluent was added and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 50 μL / well of TMB chromogenic substrate (KPL, 52-00-03) was added and incubated at room temperature for 10-15 minutes. The reaction was stopped by adding 50 μL / well of 1 M H2SO4, and the absorbance at 450 nm was read using an MD Versa Max Tm microplate reader to determine the EC2 binding of the Claudin18.2 antibody to Claudin18.2. 50 The value was calculated. [Table 10] [Table 11] [Table 12]

[0196] Test Example 2: Antibody binding experiment at the cellular level NUGC4 cells stably transfected to express Claudin18.2 were cultured at 1 × 10 in FACS buffer (PBS (Sigma, P4417-100TAB) with 2% fetal bovine serum (Gibco, 10099141) pH 7.4). 6 A cell suspension was prepared at 100 μL / well in a 96-well round-bottom plate (Corning, 3795). After centrifuging and removing the supernatant, different concentrations of the Claudin18.2 antibody to be tested, diluted with FACS buffer (50 μL / well), were added and incubated in a refrigerator at 4°C for 1 hour, protected from light. After washing three times with FACS buffer at 300 g, a working concentration of Alexa Fluor 488 sheep anti-human IgG (H+L) (Invitrogen, A-11013) was added and incubated in a refrigerator at 4°C for 40 minutes, protected from light. After washing three times with FACS buffer at 300 g, the geometric mean fluorescence intensity was detected using a BD FACS Canto II flow cytometer to determine the binding EC of the Claudin18.2 antibody to NUGC4 cells stably transfected with Claudin18.2. 50 The values were calculated and the results are shown in FIG.

[0197] Test Example 3: Antibody endocytosis experiment The Claudin 18.2 antibody to be tested, pre-labeled with DyLight 488 NHS Ester (Thermofisher, 46403), was added to a final concentration of 5 μg / mL at 1 × 10 61 mL of Claudin18.2 was added to stably transfected NUGC4 cells and incubated on ice for 1 hour, protected from light. The cells were then washed three times with pre-chilled FACS buffer (PBS, pH 7.4, 2% fetal bovine serum) by centrifugation. The supernatant was removed and the cells were added to pre-warmed complete medium and placed in a 37°C, 5% CO2 incubator. After 0, 0.5, 1, 2, and 4 hours, the cells were removed and stored on ice, protected from light. After collecting all samples, the supernatant was removed by low-temperature centrifugation at 300 g. Elution buffer (0.05 M glycine, 0.1 M sodium chloride, pH 1.7) was added, followed by incubation at room temperature for 7 minutes. The cells were then washed once with FACS buffer at 300 g. Geometric mean fluorescence intensity (GME) was measured using a BD FACS Canto II flow cytometer to calculate the endocytosis efficiency of the Claudin18.2 antibody into stably transfected NUGC4 cells. The results (see Figure 2) show that the humanized antibody has good cell endocytosis efficiency.

[0198] Test Example 4: Measurement of antibody affinity based on flow cytometry On the day of the experiment, HEK293 / hClaudin18.2 cells were harvested into a U-bottom 96-well plate, with 1–2 × 10 cells per well. 5 Cells were added. Human Claudin18.2 antibody was added at an initial concentration of 5 μg / mL, diluted 2x (12 concentration points) in a 2x gradient and incubated at 4°C for 1 hour. IMAB362 was used as a positive control, and a negative control without antibody was used. After centrifugation, the antibody was removed and 100 μL / well of FITC anti-human IgG Fc antibody (200x) was added. The cells were incubated at 4°C in the dark for 30 minutes. After washing twice with PBS + 2% FBS, the cells were prepared for flow cytometry analysis. After starting the BD FACS Canto II and preheating, a new experiment was established in the BD FACSDiva software. The HEK293 / hClaudin18.2 negative control sample was detected, and the FSC and SSC voltages were adjusted to the appropriate values and saved. Quantum TMAccording to the FITC-5 MESF Kit instruction manual, blank sample B and calibration curve 1 were detected, and the FITC voltage was adjusted to the appropriate value and saved. The samples in the U-bottom 96-well plate were detected at the saved voltage, and the data were recorded. The experimental data was analyzed using Flowjo software to obtain the Geo average value, and the Quantum TM The MESF-Geo Mean calibration curve was fitted according to the FITC-5 MESF Kit instruction manual, and the molar concentration and free antibody concentration of human Claudin18.2 antibody bound to HEK293 / hClaudin18.2 cells were calculated based on the fluorescence concentration of the FITC anti-human IgG Fc antibody. The Bmax and dissociation constant KD of the antibody were calculated using the Scatchard plot method. The results are shown in Table 13. [Table 13]

[0199] Test Example 5: Evaluation of ADCC effect of antibodies Various NUGC4 cells (high / moderate / low Claudin18.2 expression) were digested and centrifuged at 1000 rpm, then resuspended and counted. 3 × 10 cells were used. 5 Cells were resuspended at a density of 1000 cells / mL in phenol red-free RPMI 1640 (Gibco, 11835-030) supplemented with 10% FBS (New Zealand Ultra-Low IgG Fetal Bovine Serum, Gibco, 1921005PJ). 25 μL of cells (7500 cells / well) were added to each well of a 96-well plate (Corning, 3903). Antibodies were diluted in the above phenol red-free medium to prepare a 3x antibody dilution solution, and 25 μL of antibody was added to the cell plate at each well. The plate was incubated at 37°C in a 5% CO2 incubator for 0.5 hours. Effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected and centrifuged at 1000 rpm, then resuspended and counted. Cells were collected at 3 × 10 6Resuspend the cells in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum) at a density of 7.5 × 10 cells / mL and place 25 μL of cells (7.5 × 10 cells) per well in experimental plates. 4 Cells (cells / well) were added and incubated at 37°C in a 5% CO2 incubator for 6 hours. Bright-Glo (Promega, E2610) was added to each well of the experimental plate at 75 μL / well, and chemiluminescence was detected using a microplate reader (PerkinElmer, VITOR3). The results (see Table 14 and Figures 3A to 3C) show that antibodies h1901-11 and h1902-5 both exhibit very strong ADCC activity in NUGC4 cells with varying levels of Claudin18.2 expression, ranging from low (Figure 3A) to moderate (Figure 3B) to high (Figure 3C). [Table 14]

[0200] Test Example 6: Inhibition test of in vitro tumor cell growth by compounds 1. Purpose of the test The purpose of this experiment was to detect the inhibitory activity of the drug compounds disclosed herein on the in vitro proliferation of U87MG cells (glioma cells, Chinese Academy of Sciences Cell Bank, Catalog # TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Product No. HTB-30). The cells were treated in vitro with different concentrations of the compounds and cultured for 6 days, after which the cells were analyzed by CTG (CellTiter-Glo). (R) Cell proliferation was detected using the Luminescent Cell Viability Assay (Promega, Product Number: G7573) reagent, and IC 50 The in vitro activity of the compound was evaluated based on the value.

[0201] 2. Experimental Method Hereinafter, the test method for the inhibitory activity of the compounds of the present disclosure on tumor cell proliferation in vitro will be described as an example, taking the test method for inhibiting the in vitro proliferation of U87MG cells as an example. This method can also be applied to, but is not limited to, the test of the inhibitory activity of the compounds of the present disclosure on tumor cell proliferation in vitro. 1. Cell culture: U87MG and SK-BR-3 cells were cultured in EMEM medium (GE, product number: SH30024.01) containing 10% FBS and McCoy's 5A medium (Gibco, product number 16600-108) containing 10% FBS, respectively. 2. Cell preparation: Logarithmic growth phase U87MG and SK-BR-3 cells were taken and washed once with PBS (phosphate buffer solution, Shanghai Yuanpei Biotechnology Co., Ltd.). After that, 2-3 mL of trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) was added and digested for 2-3 minutes. After the cells were completely digested, 10-15 mL of cell culture medium was added to elute the digested cells. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and 10-20 mL of cell culture medium was added to resuspend the cells to prepare a single-cell suspension. 3. Seeding cells onto plates: U87MG and SK-BR-3 single cell suspensions were mixed evenly and cultured in cell culture medium until the viable cell density reached 2.75 x 10 3 cells / mL and 8.25 × 10 3 The density-adjusted cell suspensions were adjusted to 180 μL / well and mixed evenly. 180 μL of medium was added to each well of a 96-well cell culture plate. 200 μL of medium alone was added to the outer wells of the 96-well plate. The culture plate was then incubated in an incubator at 37°C and 5% CO for 24 hours. 4. Preparation of compounds: Compounds were dissolved in DMSO (dimethyl sulfoxide, Shanghai Taitan Technology Co., Ltd.) to prepare stock solutions with an initial concentration of 10 mM. The small molecule compounds had an initial concentration of 500 nM and were formulated as follows: 30 μL of each test sample was added to the first column of a 96-well U-bottom formulation plate, resulting in a sample concentration of 100 μM. 20 μL of DMSO was added to each well of columns 2 to 11. 10 μL of the sample from column 1 was added to 20 μL of DMSO in column 2, mixed evenly, and 10 μL was added to column 3, and so on up to column 10. 5 μL of the drug in the formulation plate was added to each well in 95 μL of EMEM medium, mixed evenly, and then prepared for use. The initial concentration of ADC was 10 nM or 500 nM, and the formulation method was as follows: 100 μL of each test sample was added to the first column of a 96-well plate, with the sample concentration set to 100 nM or 5 μM. 100 μL of PBS was added to each well of columns 2 to 11. 50 μL of the sample from column 1 was added to 100 μL of PBS in column 2, mixed evenly, and 50 μL was added to column 3. In this way, 3-fold dilutions were made up to column 10. 5. Sample addition procedure: 20 μL of the prepared samples to be tested at different concentrations were added to the culture plate, and each sample was added to two parallel wells. The culture plate was incubated in an incubator (37°C, 5% CO2) for 6 days. 6. Color development procedure: A 96-well cell culture plate was taken out, and 90 μL of CTG solution was added to each well, followed by incubation at room temperature for 10 minutes. 7. Plate reading procedure: The 96-well cell culture plate was taken out and placed on a microplate reader (BMG labtech, PHERAstar FS), and the chemiluminescence was measured by the microplate reader.

[0202] 3. Data analysis The five pairs of data were processed and analyzed using Microsoft Excel and Graphpad Prism. See the table below for the results of the experiment. [Table 15]

[0203] Test Example 7: Cellular activity experiment of ADC molecules In this experiment, the killing activity of ADC molecules on human gastric cancer cell lines was detected in vitro using the CellTiter-Glo Luminescence Cell Viability Assay. On day 1, NUGC4-claudin18.2 low-expressing, NUGC4-claudin18.2 moderate-expressing, and NUGC4-claudin18.2 high-expressing cells were collected and plated at a density of 2.5 × 10 4 The solution was adjusted to 1 mL / mL and added at 90 μL per well to a 96-well white clear-bottom plate, with each well containing approximately 2500 cells. The cells were incubated overnight at 37°C in a 5% CO2 incubator. On day 2, the samples were diluted in a U-bottom 96-well plate, with an initial concentration of 5 μM and 4x gradient dilutions for nine concentration points. 10 μL of the diluted samples were added to the cell plate at 10 μL per well. The plate was incubated at 37°C in a 5% CO2 incubator for 6 days. On day 8, the cell culture plate was removed, 50 μL of Cell Titer-Glo Reagent was added per well, and the plate was incubated at room temperature for 2-3 minutes. Fluorescence readings were then read using a PHERAstar FS plate reader. Data analysis was performed using GraphPad Prism software. The results are shown in Table 16. [Table 16]

[0204] Biological evaluation of in vivo activity Test Example 8: Evaluation of the in vivo efficacy of ADC molecules Balb / c nude mice were subcutaneously injected with human gastric cancer NUGC4 (moderate Claudin 18.2 expression) cells (5 × 10 6 The mice were inoculated with 50% Matrigel (containing Matrigel per mouse) and divided into groups on day 0, with 8 mice per group, for a total of 5 groups. The mean tumor volume was approximately 84.41 mm. 3 It was. ADC was injected intraperitoneally, and administered three times in total, with each animal receiving 10 g / 0.1 mL injection according to body weight, on days 0, 4, and 11, respectively. From the day of grouping, ADC was intraperitoneally injected, a total of four times, administered every five days, with each animal receiving 10 g / 0.1 mL injection according to body weight. Tumor volume and body weight were measured twice a week and the data were recorded. Using Excel 2003 statistical software, the mean value was calculated using avg, the SD value was calculated using STDEV, the SEM value was calculated using STDEV / SQRT, and the P value for intergroup differences was calculated using TTEST. Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 Relative volume (RTV) = VT / V0 Tumor inhibition rate (%) = (CRTV-TRTV) / CRTV (%) where V0 and VT are the tumor volumes at the start of the experiment (the day of the first administration is day 0) and at the time of measurement and counting, respectively. CRTV and TRTV are the relative tumor volumes of the blank control group and the experimental group at the end of the experiment, respectively. The results are shown in Table 17 and Figures 4 and 5. [Table 17] Furthermore, the present invention includes the following aspects. [Aspect 1] A ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, [ka] however, Y is -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)- and -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 halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group and a heterocyclyl group, or R a and R b together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, R 1 is selected from halogen, halogenated alkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups; R 2 is selected from a hydrogen atom, a halogen, a halogenated alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group, or R 1 and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, Or R a and R 2 together with the carbon atoms to which they are attached form a cycloalkyl or heterocyclyl group, m is an integer from 0 to 4, n is 1 to 10, and n is a decimal or an integer; L is a linker unit, Pc is an anti-Claudin18.2 antibody or an antigen-binding fragment thereof; A ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof: [Aspect 2] The anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the same sequences as those of the heavy chain variable region represented by the sequence SEQ ID NO:3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the same sequences as those of the light chain variable region represented by the sequence SEQ ID NO:4, or ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the same sequences as those of the heavy chain variable region represented by the sequence SEQ ID NO:5, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the same sequences as those of the light chain variable region represented by the sequence SEQ ID NO:6; A ligand-drug conjugate represented by the general formula (Pc-LYD) according to Aspect 1, or a pharmaceutically acceptable salt thereof. [Aspect 3] The anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the sequences of which are set forth in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the sequences of which are set forth in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively; or iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the sequences of which are represented by SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the sequences of which are represented by SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, respectively; A ligand-drug conjugate represented by the general formula (Pc-LYD) according to Aspect 1 or 2, or a pharmaceutically acceptable salt thereof. [Aspect 4] The ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of Aspects 1 to 3, wherein the anti-Claudin18.2 antibody is a mouse antibody, a chimeric antibody, or a humanized antibody. [Aspect 5] The anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, (1) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO:3 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO:4 or has at least 90% identity thereto; (2) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 24 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO: 21 or has at least 90% identity thereto; (3) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO:5 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO:6 or has at least 90% identity thereto; or (4) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 31 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO: 28 or has at least 90% identity thereto; A ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of Aspects 1 to 4, or a pharmaceutically acceptable salt thereof. [Aspect 6] the anti-Claudin18.2 antibody is a humanized antibody, the humanized antibody comprises a framework region derived from a human antibody or a variant of the framework region thereof, and the variant of the framework region is a back mutation having at most 10 amino acids in the light chain framework region and / or the heavy chain framework region of the human antibody, respectively; Preferably, the framework region variants comprise a mutation selected from the following (a) or (b): (a) the light chain variable region optionally comprises one or more amino acid backmutations selected from 22S, 85I, and 87H, and / or the heavy chain variable region optionally comprises one or more amino acid backmutations selected from 48I, 82T, and 69M; or (b) the light chain variable region optionally comprises one or more amino acid backmutations selected from 4L and 22S, and / or the heavy chain variable region optionally comprises one or more amino acid backmutations selected from 38K, 40R, 48I, 66K, 67A, 69L, 71L, and 73K; Preferably, the framework region variants include mutations selected from: (a-1) the light chain variable region contains the amino acid backmutations 22S, 85I, and 87H, and the heavy chain variable region contains the amino acid backmutations 48I and 82T; or (b-1) the light chain variable region contains 4L amino acid backmutations; A ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of Aspects 1 to 5, or a pharmaceutically acceptable salt thereof. [Aspect 7] The anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises the following heavy chain variable region and light chain variable region: (vii) the heavy chain variable region has the sequence represented by SEQ ID NO:3, and the light chain variable region has the sequence represented by SEQ ID NO:4; (viii) the heavy chain variable region has the sequence represented by SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, and the light chain variable region has the sequence represented by SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23; (ix) the heavy chain variable region has the sequence represented by SEQ ID NO:5 and the light chain variable region has the sequence represented by SEQ ID NO:6; or (x) the heavy chain variable region has the sequence represented by SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, and the light chain variable region has the sequence represented by SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30; Preferably, the anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises the heavy chain variable region and light chain variable region shown below, i.e. (xi) the heavy chain variable region has the sequence represented by SEQ ID NO:31 and the light chain variable region has the sequence represented by SEQ ID NO:29; or (xii) the heavy chain variable region has the sequence represented by SEQ ID NO:26, and the light chain variable region has the sequence represented by SEQ ID NO:23; A ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of Aspects 1 to 6, or a pharmaceutically acceptable salt thereof. [Aspect 8] The anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region and a light chain constant region, Preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions and common variants thereof, and the light chain constant region is selected from human antibody κ and λ chain constant regions and common variants thereof; More preferably, the anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region whose sequence is represented by SEQ ID NO:7 and a light chain constant region whose sequence is represented by SEQ ID NO:8; Most preferably, the anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain having at least 90% sequence identity with the heavy chain represented by SEQ ID NO:35 or SEQ ID NO:42 and a light chain having at least 90% sequence identity with the light chain represented by SEQ ID NO:36 or SEQ ID NO:39; or a heavy chain having at least 90% sequence identity with the heavy chain represented by SEQ ID NO: 37 or SEQ ID NO: 49, and a light chain having at least 90% sequence identity with the light chain represented by SEQ ID NO: 38 or SEQ ID NO: 46; A ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of Aspects 1 to 7, or a pharmaceutically acceptable salt thereof. [Aspect 9] The anti-Claudin18.2 antibody or antigen-binding fragment thereof is (c) a heavy chain whose sequence is represented by SEQ ID NO:35 and a light chain whose sequence is represented by SEQ ID NO:36; (d) a heavy chain having the sequence represented by SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45 and a light chain having the sequence represented by SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41; (e) a heavy chain having the sequence set forth in SEQ ID NO:37 and a light chain having the sequence set forth in SEQ ID NO:38; or (f) a heavy chain having the sequence represented by SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52 and a light chain having the sequence represented by SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48; A ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of Aspects 1 to 8, or a pharmaceutically acceptable salt thereof. [Aspect 10] The anti-Claudin18.2 antibody h1901-11, which comprises a heavy chain having the amino acid sequence represented by SEQ ID NO: 44 and a light chain having the amino acid sequence represented by SEQ ID NO: 41; or h1902-5, which comprises a heavy chain represented by SEQ ID NO: 49 and a light chain represented by SEQ ID NO: 47; A ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of Aspects 1 to 9, or a pharmaceutically acceptable salt thereof. [Aspect 11] A ligand-drug conjugate or a pharmaceutically acceptable salt thereof represented by the general formula (Pc-LYD) according to any one of Aspects 1 to 10, wherein n is a decimal or integer of 2 to 8, and preferably a decimal or integer of 3.5 to 4.5. [Aspect 12] Y is -O-(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 atom, or an alkyl group; R 1 is a halogenated alkyl group or C 3-6 is a cycloalkyl group, R 2 is a hydrogen atom, a halogenated alkyl group, or C 3-6 cycloalkyl groups, Or R 1 and R 2 C, along with the carbon atoms attached to them. 3-6 forming a cycloalkyl group, m is 0 or 1; A ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of Aspects 1 to 11, or a pharmaceutically acceptable salt thereof. [Aspect 13] Y is

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Claims

1. General formula (Pc-LYD): 【Chemical 1】 [In the formula, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and each independently selected from a hydrogen atom, a deuterium atom, a halogen atom, or a C 1-6 alkyl group; R 1 is a C 1-6 halogenated alkyl group or a C 3-6 cycloalkyl group; R2 is selected from a hydrogen atom, a C1-6 halogenated alkyl group and a C3-6 cycloalkyl group; or R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group; m is 0 or 1; n is 1 to 10, and n is a decimal or an integer; L is a linker unit, where the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -; where: L1 is 【Chemistry 2】 and s 1 is an integer from 2 to 8; L2 is a chemical bond; L 3 is a tetrapeptide residue; L 4 is —NR 5 (CR 6 R 7 ) t-, where R 5 , R 6 and R 7 are the same or different and each independently represent a hydrogen atom or a C 1-6 alkyl group, and t is 1 or 2; Here, the L 1 end is connected to Pc, and the L 4 end is connected to Y, Pc is an anti-Claudin18.2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; where: iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the sequences of which are set forth in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the sequences of which are set forth in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively; or iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the sequences of which are represented by SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the sequences of which are represented by SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively. A ligand-drug complex represented by the formula: or a pharmaceutically acceptable salt thereof.

2. The ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the anti-Claudin18.2 antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.

3. The anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: (1) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 3 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO: 4 or has at least 90% identity thereto; (2) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 24 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO: 21 or has at least 90% identity thereto; (3) the heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 5 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO: 6 or has at least 90% identity thereto; or (4) The heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 31 or has at least 90% identity thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO: 28 or has at least 90% identity thereto. A ligand-drug complex represented by the general formula (Pc-LYD) according to claim 1 or 2, or a pharmaceutically acceptable salt thereof.

4. The anti-Claudin18.2 antibody is a humanized antibody, and the humanized antibody comprises a framework region derived from a human antibody or a framework region mutant thereof, and the framework region mutant is a back mutation having at most 10 amino acids in the light chain framework region and / or the heavy chain framework region of the human antibody, respectively. A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

5. The ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein the framework region variant comprises a mutation selected from the following (a) or (b): (a) the light chain variable region optionally comprises one or more amino acid backmutations selected from 22S, 85I, and 87H, and / or the heavy chain variable region optionally comprises one or more amino acid backmutations selected from 48I, 82T, and 69M; or (b) the light chain variable region optionally contains one or more amino acid backmutations selected from 4L and 22S, and / or the heavy chain variable region optionally contains one or more amino acid backmutations selected from 38K, 40R, 48I, 66K, 67A, 69L, 71L, and 73K.

6. The ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein the framework region variant comprises a mutation selected from the following: (a-1) the light chain variable region contains the amino acid backmutations 22S, 85I, and 87H, and the heavy chain variable region contains the amino acid backmutations 48I and 82T; or (b-1) The light chain variable region contains 4L amino acid back mutations.

7. The anti-Claudin18.2 antibody or antigen-binding fragment thereof is a ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, which comprises the heavy chain variable region and light chain variable region shown below: (vii) the heavy chain variable region has the sequence represented by SEQ ID NO: 3, and the light chain variable region has the sequence represented by SEQ ID NO: 4; (viii) the heavy chain variable region has the sequence represented by SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27, and the light chain variable region has the sequence represented by SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; (ix) the heavy chain variable region has the sequence represented by SEQ ID NO: 5 and the light chain variable region has the sequence represented by SEQ ID NO: 6; or (x) the heavy chain variable region has a sequence represented by SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, and the light chain variable region has a sequence represented by SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO:

30.

8. The anti-Claudin18.2 antibody or antigen-binding fragment thereof is a ligand-drug conjugate represented by the general formula (Pc-LYD) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, which comprises the heavy chain variable region and light chain variable region shown below: (xi) the heavy chain variable region has the sequence represented by SEQ ID NO: 31, and the light chain variable region has the sequence represented by SEQ ID NO: 29; or (xii) the heavy chain variable region has the sequence represented by SEQ ID NO: 26, and the light chain variable region has the sequence represented by SEQ ID NO: 23;

9. The anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region and a light chain constant region. A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.

10. The heavy chain constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions and common variants thereof, and the light chain constant region is selected from human antibody κ and λ chain constant regions and common variants thereof. A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof.

11. The anti-Claudin18.2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region whose sequence is represented by SEQ ID NO: 7 and a light chain constant region whose sequence is represented by SEQ ID NO:

8. A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof.

12. The anti-Claudin18.2 antibody or antigen-binding fragment thereof a heavy chain having at least 90% sequence identity to the heavy chain represented by SEQ ID NO: 35 or SEQ ID NO: 42, and a light chain having at least 90% sequence identity to the light chain represented by SEQ ID NO: 36 or SEQ ID NO: 39; or a heavy chain having at least 90% sequence identity with the heavy chain represented by SEQ ID NO: 37 or SEQ ID NO: 49, and a light chain having at least 90% sequence identity with the light chain represented by SEQ ID NO: 38 or SEQ ID NO: 46; A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof.

13. The anti-Claudin18.2 antibody or antigen-binding fragment thereof is (c) a heavy chain whose sequence is represented by SEQ ID NO: 35 and a light chain whose sequence is represented by SEQ ID NO: 36; (d) a heavy chain having the sequence represented by SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45 and a light chain having the sequence represented by SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41; (e) a heavy chain having the sequence set forth in SEQ ID NO: 37 and a light chain having the sequence set forth in SEQ ID NO: 38; or (f) a heavy chain having the sequence represented by SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52 and a light chain having the sequence represented by SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48; A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof.

14. The anti-Claudin18.2 antibody h1901-11, which comprises a heavy chain having the amino acid sequence represented by SEQ ID NO: 44 and a light chain having the amino acid sequence represented by SEQ ID NO: 41; or h1902-5, which comprises a heavy chain represented by SEQ ID NO: 49 and a light chain represented by SEQ ID NO: 47; A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

15. A ligand-drug complex or a pharmaceutically acceptable salt thereof, wherein n is a decimal or integer of 2 to 8, and the ligand-drug complex is represented by the general formula (Pc-LYD) according to any one of claims 1 to 14.

16. A ligand-drug complex or a pharmaceutically acceptable salt thereof represented by the general formula (Pc-LYD) described in any one of claims 15, wherein n is a decimal or integer between 3.5 and 4.

5.

17. Y is 【Chemistry 3】 Selected from wherein the O-end of Y is connected to a linker unit L, A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof.

18. The linker unit -L- is -L 1 -L 2 -L 3 -L 4 -where: L 3 is the tetrapeptide residue of GGFG, A ligand-drug complex represented by the general formula (Pc-LYD) according to claims 1 to 17, or a pharmaceutically acceptable salt thereof.

19. -L- is 【Chemistry 4】 That is, A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof.

20. The compound of the general formula (Pc-L) according to any one of claims 1 to 19. b -YD): 【Chemistry 5】 [In the formula, s 1 is an integer between 2 and 8, Pc, R 1 , R 2 , R 5 ~R 7 , m and n are as defined in claim 1. A ligand-drug complex represented by the formula: or a pharmaceutically acceptable salt thereof.

21. The ligand-drug conjugate is 【Chemistry 6】 Selected from where Pc and n are as defined in claim 1. A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof.

22. The ligand-drug conjugate is 【Chemistry 7】 Selected from wherein n is as defined in claim 1 and antibodies h1902-5, h1901-11 are as defined in claim 14. A ligand-drug complex represented by the general formula (Pc-LYD) according to any one of claims 1 to 21, or a pharmaceutically acceptable salt thereof.

23. 23. A drug composition comprising the ligand-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 22, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

24. 24. A pharmaceutical composition according to claim 23 for treating a Claudin18.2-mediated disease or condition.

25. 25. A pharmaceutical composition according to claim 24 for treating and / or preventing tumors and cancer, comprising: The tumors and cancers are head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krueckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light-chain amyloidosis, and Merkel cell carcinoma.

26. A pharmaceutical composition for treating and / or preventing tumors and cancers according to claim 25, wherein the lymphoma is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma and lymphoplasmacytic lymphoma, the lung cancer is selected from non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid cell leukemia.

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