Anti-claudin antibody-drug conjugate and pharmaceutical use thereof

Abstract

This disclosure relates to anti-claudin18.2 antibody-drug conjugates and their pharmaceutical uses. Specifically, this disclosure relates to ligand-drug conjugates represented by the general formula (Pc-LYD), where Pc is an anti-Claudin18.2 antibody or its antigen-binding fragment, and L, Y, and n are as defined in the specification.

Description

Technical Field

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

[0002] This disclosure relates to anti-claudin18.2 antibody-drug conjugates, particularly anti-claudin18.2 antibody-ecetecan analog conjugates, methods of their preparation, pharmaceutical compositions comprising antibody-drug conjugates, and their use in the preparation of medicaments for treating Claudin18.2-mediated diseases or conditions; particularly in the preparation of anticancer medicaments. Prior Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] Claudin-18 (CLDN18) is a protein encoded by the Claudin18 gene in humans. Belonging to the tight junction protein family, it controls molecular flow between lamina cells. The Claudin protein structure includes four transmembrane regions and two extracellular loops, with its N-terminus and C-terminus located in the cytoplasm. Claudin-18 has two splice variants, Claudin 18.1 and Claudin 18.2, differing only in the first extracellular loop by eight amino acids. The expression distribution of Claudin 18.1 and Claudin 18.2 differs; Claudin 18.1 is selectively expressed in normal lung cells, while Claudin 18.2 expression is highly restricted in normal cells but frequently ectopically activated and overexpressed in various tumors (such as gastric, lung, and pancreatic cancer). Claudin 18.2 is considered a potential therapeutic target for gastric cancer and other cancer types, and its discovery provides a new treatment option for gastric cancer.

[0005] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxic agents via stable chemical linker compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of both normal and tumor cells, as well as the high efficiency of cytotoxic agents, while avoiding the lower efficacy of the former and the excessive toxicity of the latter. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can bind to tumor cells more precisely and reduce the impact on normal cells.

[0006] There are already patent reports on antibodies and antibody-drug conjugates (ADCs) targeting Claudin18.2, 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 treatment of Claudin18.2-related tumors. Summary of the Invention

[0007] This disclosure relates to ADCs for anti-Claudin18.2 antibodies and their uses, wherein an ADC drug is provided that is conjugated to an anti-Claudin18.2 antibody or an antigen-binding fragment with a cytotoxic substance eczema analogue.

[0008] Therefore, the purpose of this disclosure is to provide a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof:

[0009] Wherein: Y is selected from -O-(CRaRb)m-CR1R2-C(O)-, -O-CR1R2-(CRaRb)m-, -O-CR1R2-, -NH-(CRaRb)m-CR1R2-C(O)- and -S-(CRaRb)m-CR1R2-C(O)-; Ra and Rb may be the same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxyl, amino, cyano, nitro, hydroxyalkyl, cycloalkyl and heterocyclic groups; or, Ra and Rb together with the carbon atom they are attached to form a cycloalkyl or heterocyclic group. R1 is selected from halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups; R2 is selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups; or, R1 and R2 together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups; or, Ra and R2 together with the carbon atoms they are attached to form cycloalkyl or heterocyclic groups; m is an integer from 0 to 4; n is from 1 to 10, n is a decimal or an integer; L is a linker unit; Pc is an anti-Claudin18.2 antibody or its antigen-binding fragment.

[0010] In some embodiments disclosed herein, the ligand-drug conjugate or its pharmaceutically acceptable salt of the general formula (Pc-LYD) as described in any of the preceding embodiments, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein:

[0011] i) The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO:3, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 with the same sequence as the light chain variable region shown in SEQ ID NO:4; or

[0012] ii) The heavy chain variable region contains HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO:5, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 having the same sequence as the light chain variable region shown in SEQ ID NO:6.

[0013] In some embodiments of this disclosure, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding claims, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein:

[0014] iii) The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively; or

[0015] iv) The heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17 respectively, and the light chain variable region contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 respectively.

[0016] In some embodiments disclosed herein, the ligand-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc-LYD) as described in any of the preceding embodiments is a murine antibody, a chimeric antibody, or a humanized antibody.

[0017] In some embodiments disclosed herein, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding embodiments is used, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein:

[0018] (1) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:3 or has at least 90% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:4 or has at least 90% identity with it;

[0019] (2) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:24 or has at least 90% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:21 or has at least 90% identity with it;

[0020] (3) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:5 or has at least 90% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6 or has at least 90% identity with it; or

[0021] (4) The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:31 or has at least 90% identity with it, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:28 or has at least 90% identity with it.

[0022] In some embodiments disclosed herein, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding embodiments is wherein the anti-Claudin18.2 antibody is a humanized antibody comprising a frame region derived from a human antibody or a frame region variant thereof, the frame region variant having up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid reversion mutations in the light chain frame region and / or heavy chain frame region of the human antibody, respectively;

[0023] Preferably, the frame region variant contains mutations selected from either (a) or (b) below:

[0024] (a) The light chain variable region contains one or more amino acid reversion mutations selected from 22S, 85I, and 87H, and / or the heavy chain variable region contains one or more amino acid reversion mutations selected from 48I, 82T, and 69M; or

[0025] (b) The light chain variable region contains one or more amino acid reversion mutations selected from 4L or 22S, and / or the heavy chain variable region contains one or more amino acid reversion mutations selected from 38K, 40R, 48I, 66K, 67A, 69L, 71L, and 73K;

[0026] Preferably, the frame region variant contains mutations selected from the following:

[0027] (a-1) The light chain variable region contains amino acid reversion mutations of 22S, 85I, and 87H, and the heavy chain variable region contains amino acid reversion mutations of 48I and 82T; or

[0028] (b-1) The variable region of the light chain contains an amino acid reversion mutation selected from 4L.

[0029] In some embodiments disclosed herein, the ligand-drug conjugate or its pharmaceutically acceptable salt of the general formula (Pc-LYD) as described in any of the preceding claims, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as shown below:

[0030] (vii) The heavy chain variable region sequence is shown in SEQ ID NO:3 and the light chain variable region sequence is shown in SEQ ID NO:4;

[0031] (viii) The heavy chain variable region sequence is shown in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27 and the light chain variable region sequence is shown in SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23;

[0032] (ix) The heavy chain variable region sequence is shown in SEQ ID NO:5 and the light chain variable region sequence is shown in SEQ ID NO:6; or

[0033] (x) The heavy chain variable region sequence is shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34 and the light chain variable region sequence is shown in SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30;

[0034] Preferably, the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as shown below:

[0035] (xi) The heavy chain variable region sequence is shown in SEQ ID NO:31 and the light chain variable region sequence is shown in SEQ ID NO:29; or

[0036] (xii) The heavy chain variable region sequence is shown in SEQ ID NO:26 and the light chain variable region sequence is shown in SEQ ID NO:23.

[0037] In some embodiments disclosed herein, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding claims, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3, and IgG4 and their conventional variants, and the light chain constant region is selected from the constant regions of human antibody κ and λ chains and their conventional variants; more preferably, the antibody comprises a heavy chain constant region with a sequence as shown in SEQ ID NO:7 and a light chain constant region with a sequence as shown in SEQ ID NO:8; most preferably, the antibody comprises: a heavy chain having at least 90% identity with a heavy chain having an amino acid sequence as shown in SEQ ID NO:35 or SEQ ID NO:42, and a light chain having at least 90% identity with a light chain having an amino acid sequence as shown in SEQ ID NO:36 or SEQ ID NO:39; or

[0038] A heavy chain having at least 90% identity with a heavy chain having the amino acid sequence shown in SEQ ID NO:37 or SEQ ID NO:49, and a light chain having at least 90% identity with a light chain having the amino acid sequence shown in SEQ ID NO:38 or SEQ ID NO:46.

[0039] In some embodiments disclosed herein, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in any of the preceding claims, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises:

[0040] (c) The heavy chain with sequence as shown in SEQ ID NO:35 and the light chain with sequence as shown in SEQ ID NO:36;

[0041] (d) Heavy chains with sequences as shown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45 and light chains with sequences as shown in SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:41;

[0042] (e) The heavy chain with the sequence shown in SEQ ID NO:37 and the light chain with the sequence shown in SEQ ID NO:38; or

[0043] (f) Heavy chains with sequences such as SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:52 and light chains with sequences such as SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:48.

[0044] In some embodiments disclosed herein, the ligand-drug conjugate of the formula (Pc-LYD) as described in any of the preceding embodiments, wherein the anti-Claudin18.2 antibody is selected from:

[0045] h1901-11: Contains a heavy chain with an amino acid sequence as shown in SEQ ID NO:44, and a light chain with a sequence as shown in SEQ ID NO:41; or

[0046] h1902-5: Contains a heavy chain with an amino acid sequence as shown in SEQ ID NO:49, and a light chain with a sequence as shown in SEQ ID NO:47.

[0047] In some embodiments disclosed herein, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (biantibody), and disulfide bond-stabilized V region (dsFv).

[0048] In some embodiments of this disclosure, the ligand-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc-LYD) as described in any of the preceding claims, wherein n can be an integer or decimal between 1 and 10, and n can be the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. n is a decimal or integer between 2 and 8, preferably a decimal or integer between 3 and 8, more preferably a decimal or integer between 5 and 9, or preferably a decimal or integer between 2 and 7. In some embodiments, n is a decimal or integer between 3.5 and 4.5.

[0049] In some embodiments disclosed herein, the ligand-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc-LYD) as described in the preceding one,

[0050] in:

[0051] Y is -O-(CRaRb)m-CR1R2-C(O)-;

[0052] Ra and Rb may be the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens or alkyl groups;

[0053] R1 is a haloalkyl or C3-6 cycloalkyl;

[0054] R2 is selected from hydrogen atoms, haloalkyl groups, or C3-6 cycloalkyl groups;

[0055] Alternatively, R1 and R2 together with the carbon atoms they are attached to form a C3-6 cycloalkyl group;

[0056] m is 0 or 1.

[0057] In some embodiments disclosed herein, a ligand-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding embodiments, wherein Y is selected from:

[0058]

[0059] The O end of Y is connected to the connector unit L.

[0060] In some embodiments disclosed herein, the provided general formula (Pc-LYD) represents a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit -L- is -L1-L2-L3-L4-.

[0061] In some embodiments, L1 is selected from -(succinimid-3-yl-N)-WC(O)-, -CH2-C(O)-NR3-WC(O)-, or -C(O)-WC(O)-, wherein W is selected from C1-8 alkyl, C1-8 alkyl-C3-6 cycloalkyl, or a straight-chain heteroalkyl of 1 to 8 chain atoms, the heteroalkyl comprising 1 to 3 heteroatoms selected from N, O, or S, wherein each of the C1-8 alkyl, C1-8 alkyl-C3-6 cycloalkyl, or a straight-chain heteroalkyl of 1 to 8 chain atoms is independently and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl.

[0062] In some implementations, L2 is selected from -NR4(CH2CH2O)p1CH2CH2C(O)-, -NR4(CH2CH2O)p1CH2C(O)-, -S(CH2)p1C(O)- or a chemical bond, where p1 is an integer from 1 to 20.

[0063] In some embodiments, L3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are selected from amino acids formed from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl.

[0064] In some implementations, L4 is selected from -NR5(CR6R7)t-, -C(O)NR5-, -C(O)NR5(CH2)t- or a chemical bond, where t is an integer from 1 to 6.

[0065] In some embodiments, R3, R4, and R5 may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl.

[0066] In some embodiments, R6 and R7 may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, and hydroxyalkyl groups.

[0067] In some embodiments disclosed herein, the ligand-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt or solvate thereof, wherein the linker unit -L- is -L1-L2-L3-L4-, L1 is selected from -(succinimid-3-yl-N)-WC(O)-, -CH2-C(O)-NR3-WC(O)-, or -C(O)-WC(O)-, wherein W is selected from C1-8 alkyl, C1-8... Alkyl-cycloalkyl or a straight-chain heteroalkyl group of 1 to 8 atoms, the heteroalkyl group comprising 1 to 3 heteroatoms selected from N, O or S, wherein the C1-8 alkyl, cycloalkyl and straight-chain heteroalkyl groups are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L2 is selected from -NR4(CH2CH2O)p1CH2CH2C(O)-, -NR4(CH2C H2O)p1CH2C(O)-, -S(CH2)p1C(O)-, or a chemical bond, where p1 is an integer from 1 to 20; L3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are selected from amino acids formed from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally further selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. One or more substituents in the alkyl group are substituted; L4 is selected from -NR5(CR6R7)t-, -C(O)NR5, -C(O)NR5(CH2)t- or chemical bonds, where t is an integer from 1 to 6; R3, R4 and R5 may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl; R6 and R7 may be the same or different, and each is independently selected from hydrogen atoms, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl.

[0068] In some embodiments disclosed herein, ligand-drug conjugates or pharmaceutically acceptable salts thereof of the general formula (Pc-LYD) as described in any of the preceding claims, wherein the linker unit -L- is -L1-L2-L3-L4-, and L1 is s1 is an integer from 2 to 8; L2 is a chemical bond; L3 is a tetrapeptide residue; preferably, L3 is a tetrapeptide residue of GGFG (SEQ ID No. 55); L4 is -NR5(CR6R7)t-, where R5, R6, or R7 are the same or different, and each is independently a hydrogen atom or an alkyl group, and t is 1 or 2; wherein the L1 end is connected to Pc, and the L4 end is connected to Y.

[0069] In some embodiments disclosed herein, the ligand-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc-LYD) as described in any of the preceding embodiments, wherein -L- is:

[0070] In some embodiments disclosed herein, a ligand-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding embodiments, wherein -LY- is optionally derived from:

[0071] In some embodiments disclosed herein, the ligand-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc-LYD) as described in the preceding one is a ligand-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc-La-YD):

[0072] Wherein: W, L2, L3, R5, R6, R7 are as defined in the previously described connector unit-L-; Pc, n, R1, R2, m are as defined in the general formula (Pc-LYD).

[0073] In some embodiments disclosed herein, the ligand-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc-LYD) as described in any of the preceding embodiments is a ligand-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc-Lb-YD):

[0074] Where: s1 is an integer from 2 to 8;

[0075] Pc, R1, R2, R5~R7, m and n are as defined in the general formula (Pc-La-YD).

[0076] In some embodiments disclosed herein, the ligand-drug conjugate represented by the general formula (Pc-LYD) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, is selected from:

[0077]

[0078] Where Pc and n are defined as in the general formula (Pc-LYD).

[0079] In some embodiments disclosed herein, the ligand-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof is selected from:

[0080]

[0081] Wherein, n is as defined in the general formula (Pc-LYD); antibodies h1902-5 and h1901-11 are as defined above.

[0082] This disclosure further provides a method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as shown in the general formula (Pc-La-YD), comprising the following steps:

[0083]

[0084] Pc' undergoes a coupling reaction with a compound of general formula (La-YD) to give a compound of general formula (Pc-La-YD);

[0085] in:

[0086] Pc is the anti-Claudin18.2 antibody or its antigen-binding fragment as described above; Pc' is obtained by reducing Pc;

[0087] W, L2, L3, R1, R2, R5~R7, m and n are defined in the general formula (Pc-La-YD).

[0088] This disclosure further provides a method for preparing an antibody-drug conjugate as shown in the general formula (Pc-L'-D), comprising the following steps:

[0089]

[0090] After reduction of Pc, it undergoes a coupling reaction with the general formula (L'-D) to give a compound; wherein:

[0091] Pc is the anti-Claudin18.2 antibody or its antigen-binding fragment as described above;

[0092] n is defined as in the general formula (Pc-LYD).

[0093] On the other hand, this disclosure provides a pharmaceutical composition comprising a ligand-drug conjugate as described in any of the preceding claims or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0094] On the other hand, this disclosure provides the use of ligand-drug conjugates as described in any of the preceding embodiments, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising them, as medicines. In some embodiments, the medicine is used to treat a Claudin18.2-mediated disease or condition; wherein the Claudin18.2-mediated disease or condition is preferably a cancer with high Claudin18.2 expression. In some embodiments, the medicine is used to treat cancer. In some implementation schemes, the preferred cancers include 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, 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, Kuckenberg tumor, and myeloproliferative tumor. Tumors, squamous cell carcinomas, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinomas; 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 / histiocytic rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

[0095] On the other hand, this disclosure provides the use of the ligand-drug conjugate as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising thereus, in the preparation of a medicament for treating a Claudin18.2-mediated disease or condition, wherein the Claudin18.2-mediated disease or condition is a Claudin18.2-overexpressing cancer. In some implementation schemes, the preferred diseases include 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, 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, Kuckenberg tumor, and myeloproliferative tumors. Tumors, squamous cell carcinomas, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinomas; 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 / histiocytic rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

[0096] On the other hand, this disclosure provides the use of the ligand-drug conjugate or its pharmaceutically acceptable salt or pharmaceutical composition comprising the preceding one in the preparation of a medicament for the treatment or prevention of tumors; wherein the tumors and cancers are preferably head and neck squamous cell carcinoma, 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, 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, and myeloma. Multiple myeloma, myelodysplastic syndrome, Kuckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma; 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 / histiocytic rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

[0097] On the other hand, this disclosure further relates to a method for treating and / or preventing tumors, the method comprising administering to a subject in need of the thereof a therapeutically effective dose or a preventatively effective dose of a ligand-drug conjugate as described in any of the preceding claims or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the thereof; preferably wherein the tumor is a cancer associated with high expression of Claudin18.2.

[0098] On the other hand, this disclosure further relates to a method for treating or preventing cancer, the method comprising administering to a subject in need a therapeutically effective dose or a preventatively effective dose of a ligand-drug conjugate as described in any of the preceding claims or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the thereof; wherein the tumor and cancer are preferably 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. Lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Kuckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma; 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 / histiocytic rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

[0099] The active compound (e.g., the ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in this disclosure) may be formulated in a form suitable for administration by any appropriate route, preferably in a unit dose manner or in a manner that allows the subject to self-administer a single dose. The unit dose manner disclosed herein may be in the form of tablets, capsules, sachets, bottled liquids, powders, granules, lozenges, suppositories, regenerated powders, or liquid formulations.

[0100] The dosage of the active compound or composition used in the treatment methods disclosed herein will generally vary depending on the severity of the disease, the subject's weight, and the efficacy of the active compound. However, as a general guideline, a suitable unit dose may be from 0.1 mg to 1000 mg.

[0101] In addition to the active compound, the pharmaceutical composition disclosed herein may contain one or more excipients selected from the following components: fillers, diluents, binders, wetting agents, disintegrants, or excipients. Depending on the route of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0102] The Claudin18.2 antibody and antibody-drug conjugate disclosed herein have good affinity for cell surface antigens, good endocytosis efficiency and strong tumor inhibition efficiency, and have a wider drug application window, making them suitable for clinical drug application. Simple Explanation of the Diagram

[0103] Figure 1 shows the FACS detection results of humanized antibodies binding to human Claudin18.2 at the cellular level.

[0104] Figure 2 shows the endocytosis experiment of humanized antibodies in NUGC4 cells.

[0105] Figures 3A to 3C show the ADCC effect of the antibody in NUGC4 cells with different Claudin18.2 expression levels. Figure 3A shows the ADCC effect of the antibody in wild-type NUGC4 cells (low Claudin18.2 expression); Figure 3B shows the ADCC effect of the antibody in NUGC4 cells with moderate Claudin18.2 expression; and Figure 3C shows the ADCC effect of the antibody in NUGC4 cells with high Claudin18.2 expression.

[0106] Figure 4 shows the tumor suppression experiment results of the ADC-1 disclosed herein.

[0107] Figure 5 shows the tumor suppression experiment results of the ADC-2 disclosed herein. Implementation

[0108] I. Terminology

[0109] Unless otherwise specified, all technical and scientific terms used herein are consistent with the common understanding of one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used to perform or test this disclosure, preferred methods and materials are described herein. In describing and claiming protection for this disclosure, the following terms are used in accordance with the definitions below.

[0110] When a trade name is used in this disclosure, it is intended to include the formulation of the product under that trade name, the generic drug portion of the product under that trade name, and the active pharmaceutical ingredient portion.

[0111] Unless otherwise stated, the terms used in the specification and the scope of the patent application shall have the following meanings.

[0112] The term "drug" refers to a chemical substance that can alter or identify the physiological functions and pathological states of an organism, and can be used to prevent, diagnose, and treat diseases. Drugs include cytotoxic drugs. There is no strict boundary between drugs and poisons; poisons are chemical substances that produce harmful effects on the body even in small doses, damaging human health. Excessive doses of any drug can produce toxic reactions. Cytotoxic drugs are substances that inhibit or prevent cell function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficient concentrations; however, due to their lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals; radioactive isotopes (e.g., radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu); toxic drugs; chemotherapeutic drugs; antibiotics; and ribolysins.

[0113] The terms "connector unit", "connector" or "connecting fragment" refer to a chemical structural fragment or bond that is connected at one end to a ligand (such as an antibody or its antigen-binding fragment) and at the other end to a drug. It can also be connected to other connectors before being linked to a drug.

[0114] The connector may comprise one or more connector components. Exemplary connector components include 6-maleiminohexyl (“MC”), maleiminopropyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimido-4-(2-pyridylthio)valerate (“SPP”), N-succinimido-4-(N-maleiminomethyl)cyclohexane-1-carboxylate (“SMCC”, also referred to herein as “MCC”), and N-succinimido-(4-iodo-acetylated)aminobenzoate (“SIAB”). The linker may include extensions, spacers, and amino acid units, and may be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker may be a “cleavable linker” that facilitates the release of drugs into cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), photostable linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).

[0115] abbreviation

[0116] Connector components include, but are not limited to: MC=6-maleiminohexylated, with the following structure: Val-Cit or “vc” = valine-citrulline (an example dipeptide in a protease-cleavable linker), citrulline = 2-amino-5-ureidovalerate, PAB = p-aminobenzyloxycarbonyl (an example of a “self-sacrificing” linker assembly), Me-Val-Cit = N-methyl-valine-citrulline (where the linker peptide bond is modified to prevent cleavage by cathepsin B), MC(PEG)6-OH = maleiminohexyl-polyethylene glycol (attachable to antibody cysteine), SPP = N-succinimido-4-(2-pyridylthio)valerate, SPDP = N-succinimido-3-(2-pyridyldithio)propionate, SMCC = succinimido-4-(N-maleiminomethyl)cyclohexane-1-carboxylate, IT = iminothionine.

[0117] The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a linker unit. In this disclosure, "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), referring to the linking of a monoclonal antibody or antibody fragment to a biologically active toxic drug via a linker unit. The antibody can be directly or via a linker to the drug. The average number of drug modules per antibody (average drug loading or amount of drug, expressed as n) can range, for example, from about 0 to about 20 drug modules per antibody, in some embodiments from 1 to about 10 drug modules per antibody, and in some embodiments from 1 to about 8 drug modules per antibody.

[0118] The term "average drug loading" or "drug amount" refers to the average number of cytotoxic drugs loaded onto each ligand in a ligand-drug conjugate molecule. It can also be expressed as the ratio of drug amount to antibody amount. The drug loading ranges from 0 to 12 cytotoxic drugs per ligand (Pc), preferably 1 to 10. In embodiments of this disclosure, the drug loading is expressed as n, also known as the DAR (Drug-antibody Ratio) value. n can be a non-zero integer or decimal from 0 to 12, preferably an integer or decimal between 1 and 10; more preferably 2 to 8, and can be an integer or decimal; most preferably 3 to 8, and can be an integer or decimal. Examples include the average values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average number of drugs per ADC molecule after the conjugation reaction can be identified using conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0119] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0120] Claudin 18 (CLD18) molecules (Genbank registry numbers: splice variant 1 (CLD18A1): NP_057453, NM016369, and splice variant 2 (CLD18A2 or Claudin18.2): NM_001002026, NP_001002026) are intrinsic transmembrane proteins located in tight junctions between epithelium and endothelium. In tight junctions, occludin and claudin are the most abundant transmembrane protein components. Due to their strong intercellular adhesion properties, claudins form a primary barrier that prevents and controls paracellular transport of solutes and restricts lateral diffusion of membrane lipids and proteins to maintain cell polarity. Proteins forming tight junctions participate in the tissue structure of the epithelium. These proteins have been reported to be almost inaccessible to antibodies in well-structured epithelium, but become exposed in tumor cells.

[0121] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two heavy chains and two light chains linked by interchain disulfide bonds. Immunoglobulins can be classified into five classes, or isotypes, based on differences in the amino acid composition and sequence of the constant region of the heavy chain: IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains of μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further divide them into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ or λ chain.

[0122] The sequence of approximately 110 amino acids near the N-terminus of both the heavy and light chains of a full-length antibody varies considerably and is known as the variable region (Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and are called constant regions. The variable regions include three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0123] The term "fully human antibody," also known as a "fully human monoclonal antibody," refers to an antibody whose variable and constant regions are both human-derived. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. Related technologies for the preparation of fully human antibodies mainly include: human fusion tumor technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0124] The term "antigen-binding fragment" refers to one or more segments of an antibody that retain its ability to bind to antigens. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. The binding fragments included in the “antigen-binding fragment” are selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (biantibody), disulfide-stabilized V region (dsFv), and antigen-binding fragments of peptides containing CDRs. Examples include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments consisting of two Fab fragments connected by disulfide bridges on hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of an antibody; (v) single-domain or dAb fragments (Ward et al., (1989) Nature 341:544-546) consisting of VH domains; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally connected by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined using recombinant methods via synthetic linkers, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as 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 included in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical fragmentation of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0125] Typically, Fab is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity obtained by treating an IgG antibody molecule with the protease papain (e.g., cleaving the amino acid residue at position 224 of the H chain), wherein the N-terminal portion of the H chain and the L chain are linked together by disulfide bonds.

[0126] Typically, F(ab')2 is obtained by digesting the portion below the disulfide bond in the hinge region of IgG with the enzyme pepsin. It has a molecular weight of approximately 100,000, possesses antigen-binding activity, and is an antibody fragment containing two Fab regions connected at the hinge position.

[0127] Typically, Fab' is an antibody fragment with a molecular weight of approximately 50,000 and possessing antigen-binding activity, obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2.

[0128] In addition, Fab' can be produced by inserting DNA encoding the Fab' fragment into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express Fab'.

[0129] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or VH) of the antibody heavy chain and a variable domain (or VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using 1–4 repeating variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors available for use in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immuno 1.31:94-106, 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.

[0130] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable domain and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable domain. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes. 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 CDRs applies only to CDR1, CDR2, and CDR3 of the light chain variable domain, and CDR2 and CDR3 of the heavy chain variable domain. It also includes the “Chothia” numbering rule, the “ABM” numbering rule, the “contact” numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001) and the ImMunoGenTics (IMGT) numbering rule (Lefranc MP, Dev. Comp. Immunol., 27, 55-77(2003), etc.).

[0131] The term "antibody framework" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0132] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an immunoglobulin or antibody. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0133] The terms “specific binding,” “selective binding,” “selective binding,” and “specific binding” refer to the binding of an antibody to an epitope on a pre-defined antigen. Typically, antibodies bind with an affinity (KD) of approximately less than 10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M, or 10⁻¹⁰ M or less.

[0134] The term "KD" refers to the dissociation equilibrium constant of antibody-antigen interactions. Typically, the antibodies (or antigen-binding fragments) disclosed herein bind to Claudin18.2 (or its epitopes) with a dissociation equilibrium constant (KD) of less than about 10⁻⁷ M, for example less than about 10⁻⁸ M or 10⁻⁹ M. For example, in this disclosure, the affinity of the antibody for cell surface antigens is determined using the FACS method to determine the KD value.

[0135] The term "nucleic acid molecule" refers to either a DNA molecule or an RNA molecule. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to that coding sequence.

[0136] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence during amino acid sequence alignment, where gaps are introduced where necessary to achieve maximum sequence identity, without considering any conserved substitutions as part of sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0137] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0138] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing with the IMGT Human Antibody Variable Region Germplasm Database and MOE software, or from the journal *Immunoglobulin*, Lefranc, G., *The Immunoglobulin Facts Book*, Academic Press, 2001, ISBN 012441351.

[0139] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, 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 NSO cells.

[0140] The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be selected and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the N-terminal site of the Fc region. Positive pure strains are scaled up in a bioreactor medium to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column. Non-specifically bound components are washed away. The bound antibody is then extracted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0141] The term "peptide" refers to a compound segment that lies between an amino acid and a protein. It is composed of two or more amino acid molecules linked together by peptide bonds and is a structural and functional segment of a protein.

[0142] The term "sugar" refers to a biological macromolecule composed of three elements: C, H, and O. It can be classified into monosaccharides, disaccharides, and polysaccharides.

[0143] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tributyl, dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tributyl, dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamine, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and lateral oxy groups.

[0144] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein the alkyl group is as defined above.

[0145] The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples of alkyl groups include, but are not limited to, methylene (-CH2-), 1,1-epenylethyl (-CH(CH3)-), 1,2-epenylethyl (-CH2CH2)-, 1,1-epenylpropyl (-CH(CH2CH3)-), 1,2-epenylpropyl (-CH2CH(CH3)-), 1,3-epenylpropyl (-CH2CH2CH2-), 1,4-epenylbutyl (-CH2CH2CH2CH2-), and 1,5-epenylbutyl (-CH2CH2CH2CH2CH2-). The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamine, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and lateral oxy groups.

[0146] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamine, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0147] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl 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 (containing 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, cyclohepttrienyl, and cyclooctyl; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0148] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer 0, 1, or 2), but excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, wherein 1 to 4 are heteroatoms (1, 2, 3, or 4 heteroatoms); more preferably, the cycloalkyl ring comprises 3 to 10 ring atoms (comprising 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms). Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0149] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it consists of 6 to 14 rings, more preferably 7 to 10 rings. Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or polyspirocyclic groups based on the number of shared spiro atoms between rings, with monospirocyclic and bispirocyclic groups being more preferred. More preferably, it consists of 4 / 4, 4 / 5, 4 / 6, 5 / 5, or 5 / 6 monospirocyclic groups. Non-limiting examples of spirocyclic groups include:

[0150]

[0151] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer 0, 1, or 2), and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (7-, 8-, 9-, or 10-membered rings). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, and more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0152]

[0153] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer 0, 1, or 2), and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (7-, 8-, 9-, or 10-membered rings). If the number of constituent rings can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic, bicyclic, tricyclic, or tetracyclic is preferred, and bicyclic or tricyclic is more preferred. Non-limiting examples of bridged heterocyclic groups include:

[0154]

[0155] The heterocyclic ring can be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0156] and wait.

[0157] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and lateral oxy groups.

[0158] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered (6, 7, 8, 9, or 10-membered), such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, and non-limiting examples include:

[0159]

[0160] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0161] The term "heteroaryl" refers to a heteroaryl system containing 1 to 4 heteroatoms (1, 2, 3 or 4 heteroatoms) and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.

[0162] The heteroaryl group is preferably 5 to 10 members (5, 6, 7, 8, 9, or 10 heteroaryl groups), more preferably 5 or 6 members, such as furanyl, thiophene, pyridyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, and tetrazolyl. The heteroaryl ring can be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0163]

[0164] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0165] The term "amine protecting group" is used to protect the amine group by means of an easily removable group, ensuring that the amine group remains unchanged during reactions at other sites of the molecule. Non-limiting examples include 9-fluorenemethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl, etc. These groups may optionally be substituted with 1 to 3 substituents (one, two, or three substituents) selected from halogens, alkoxy groups, or nitro groups. The amine protecting group is preferably 9-fluorenemethoxycarbonyl.

[0166] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by one or more halogens, wherein the alkyl group is as defined above.

[0167] The term “deuterated alkyl” refers to an alkyl group in which one or more deuterium atoms are replaced by hydrogen atoms, wherein the alkyl group is as defined above.

[0168] The term "hydroxyalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by one or more hydroxyl groups, wherein the alkyl group is as defined above.

[0169] The term "hydroxyl group" refers to the -OH group.

[0170] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0171] The term "amine" refers to -NH2.

[0172] The term "nitro" refers to -NO2.

[0173] The term "cyano" refers to -CN.

[0174] The term "amino group" refers to -C(O)N (alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0175] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the case where the event or environment occurs or does not occur. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0176] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one, two, or three hydrogen atoms that are independently substituted by substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino group or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0177] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of pharmaceutical compositions is to facilitate drug delivery to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0178] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of the ligand-drug conjugate disclosed herein, or a salt of the active compound described herein, which is safe and effective when used in subjects and has the intended biological activity. The ligand-drug conjugate disclosed herein contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of medicinal salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, 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.

[0179] In one embodiment of this disclosure, a cytotoxic drug is coupled to the thiol group of an antibody via a linker unit.

[0180] The loading of ligand-cytotoxic drug conjugates can be controlled using the following non-limiting methods, including:

[0181] (1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

[0182] (2) Control the reaction time and temperature.

[0183] (3) Choose different reaction reagents.

[0184] For the preparation of conventional pharmaceutical components, please refer to the Chinese Pharmacopoeia.

[0185] The term "pharmaceutically acceptable carrier" used in this disclosure refers to a system that can alter the way a drug enters a subject and its distribution in the body, control the rate of drug release, and deliver the drug to the target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, high-molecular-weight surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, with preferred examples including micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers.

[0186] The term "excipient" refers to any additive in a pharmaceutical composition other than the active compound; it can also be called a pharmacological agent. Examples of excipients include binders, fillers, disintegrants, and lubricants in tablets; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants in liquid preparations.

[0187] The term "diluent," also known as a filler, is primarily used to increase the weight and volume of tablets. The addition of diluents not only ensures a specific volume but also reduces dosage deviations of the main components and improves the compressibility of the drug. When the tablet contains oily components, absorbents are added to absorb the oil and maintain a "dry" state, facilitating tablet formation. Examples of absorbents include starch, lactose, inorganic salts of calcium, and microcrystalline cellulose.

[0188] Pharmaceutical formulations may be in the form of sterile injectable aqueous solutions. Water, Ringer's solution, and isotonic sodium chloride solution may be used as acceptable solvents and media. Sterile injectable formulations may be sterile injectable oil-in-water microemulsions in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated with a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion can be injected into the bloodstream of the subject by local large-volume injection. Alternatively, it is preferable to administer the solution and microemulsion in a manner that maintains a constant circulating concentration of the disclosed compound. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous pump.

[0189] Pharmaceutical formulations can be in the form of sterile injectable aqueous or oil suspensions for intramuscular and subcutaneous administration. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions prepared in non-toxic, parenteral-acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Furthermore, sterile fixative oils can be conveniently used as solvents or suspension media. For this purpose, any blended fixative oil, including synthetic mono- or diglycerides of glycerol, can be used. Additionally, fatty acids such as oleic acid can also be used to prepare injectable formulations.

[0190] II. Synthesis Method

[0191] To achieve the synthesis objective, the following synthesis technique was adopted:

[0192] A method for preparing compounds represented by the general formula (Pc-La-YD) includes the following steps:

[0193]

[0194] After reduction, Pc is coupled with a compound of general formula (La-YD) to give a compound of general formula (Pc-La-YD); the reducing agent is preferably TCEP, and in particular, it is preferred to reduce the disulfide bonds on the antibody.

[0195] Where: Pc, W, L2, L3, R1, R2, R5~R7, m and n are defined as in the general formula (Pc-La-YD).

[0196] Details of one or more embodiments of this disclosure are set forth in the foregoing specification. While any methods and materials similar to or the same as those described herein may be used to implement or test this disclosure, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will be apparent from the specification and claims. In the specification and claims, unless the context clearly indicates otherwise, the singular form includes the plural referent. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the specification are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of this disclosure more fully. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

[0197] [Implementation Method]

[0198] [I. Antibody Preparation]

[0199] [Example 1-1: Construction of a cell line with high claudin18.2 expression]

[0200] The pCDH-hClaudin18.2 lentiviral expression vector plasmid and the pVSV-G, pCMV-dR8.91 lentiviral system packaging vector were transfected into virus packaging cells 293T using Lipofectamine 3000 transfection reagent. The culture supernatant containing the virus was collected, filtered, and centrifuged at ultra-high speed. The concentrated virus was used to infect the human gastric signet ring cell carcinoma cell line NUGC4. After two to three weeks of puromycin selection, single-cell sorting was performed using FACS.

[0201] Claudin18.2 expression levels were differentiated based on tumor IHC scores. Cells with Claudin18.2 expression levels comparable to tumors with an IHC score of 3 were considered high-expressing cells; cells with Claudin18.2 expression levels comparable to tumors with an IHC score of 2 were considered moderate-expressing cells. Based on FACS detection of Claudin18.2 expression on the surface of lentivirally infected NUGC4 cells, NUGC4 / hClaudin18.2 monoclonal cell lines with high Claudin18.2 expression were selected. Simultaneously, FACS detection of Claudin18.2 expression on the surface of wild-type NUGC4 cells identified NUGC4 pure-line cell lines with moderate Claudin18.2 expression, while wild-type NUGC4 cells were classified as having low Claudin18.2 expression.

[0202] Selected single cell lines were expanded and cryopreserved for future experiments. Claudin18.2 sequence (Genbank: NP_001002026: (SEQ ID NO:1))

[0203]

[0204] Claudin18.2 DNA sequence: (SEQ ID NO:2)

[0205]

[0206]

[0207]

[0208]

[0209]

[0210] [Example 1-2: Generation of Anti-Human Claudin18.2 Monoclonal Antibody]

[0211] 1 Immunization

[0212] The anti-human Claudin18.2 monoclonal antibody was generated by immunizing mice. The experimental SJL white mice, female, 6 - 8 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd., Animal Production License No.: SCXK(Beijing)2012 - 0001). Feeding environment: SPF level. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle adjustment, temperature 20 - 25 °C; humidity 40 - 60%. The mice that had adapted to the environment were immunized according to the following protocol. The immunization antigen was huClaudin18.2-HEK293 cells (HEK-293 stable transfected cell line transfected with human Claudin18.2 plasmid).

[0213] Immunization protocol: Before the first immunization with cells, inject 0.1 mL / mouse intraperitoneally (IP) with TiterMax® Gold Adjuvant (Sigma Cat No.T2684). Half an hour later, inject each mouse intraperitoneally (IP) with 0.1 mL of cell suspension diluted to a concentration of 1×108 / mL with physiological saline. After the cells were evenly dispersed, they were inoculated at days 0, 14, 28, 42, and 56. Blood was taken at days 21, 35, 49, and 63, and the antibody titer in the mouse serum was determined by ELISA method. After the 4th - 5th immunization, mice with high antibody titer in the serum and a titer tending to plateau were selected for splenocyte fusion. Three days before splenocyte fusion, the mice were immunized intensively by intraperitoneal (IP) injection of 1×107 cells.

[0214] 2 Splenocyte Fusion

[0215] Optimized PEG-mediated fusion procedures were used to fuse spleen lymphocytes with myeloma Sp2 / 0 cells (ATCC® CRL-8287TM) to obtain fusion tumor cells. The resulting fusion tumor cells were resuspended at a density of 0.5–1 × 10⁶ / mL in complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI), and seeded at 100 μL / well in 96-well plates. After incubation at 37°C and 5% CO₂ for 3–4 days, 100 μL / well of HAT complete medium was added, and the cells were cultured for another 3–4 days until pinpoint-sized pure cells were formed. The supernatant was removed, and 200 μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI) was added. The cells were cultured at 37°C and 5% CO₂ for 3 days before ELISA detection.

[0216] 3. Screening of fusion tumor cells

[0217] To assess the growth density of fusion tumor cells, the culture supernatant was analyzed using a combined ELISA method. Cells that strongly bind to huClaudin18.2-HEK293 cells but do not bind to HEK293 cells were promptly expanded and cryopreserved, and subjected to two to three sub-selection processes until a single-cell pure line was obtained.

[0218] Each subselected colony also underwent cell binding assays. Pure fusion tumor strains were obtained through these experiments, and antibodies were further prepared using serum-free cell culture. The antibodies were purified according to the purification example for use in the assays.

[0219] [Examples 1-3: Humanization of mouse antibodies]

[0220] Monoclonal fusion tumor cell lines mAb1901 and mAb1902 with high in vitro activity were selected, and their monoclonal antibody sequences were cloned, then humanized, recombinantly expressed, and their activity evaluated.

[0221] The sequence selection process from fusion tumors is as follows: Log-phase fusion tumor cells were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) (following the kit instructions). Reverse transcription was performed (PrimeScript™ Reverse Transcriptase, Takara, cat # 2680A). The cDNA obtained from reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and then sent to a sequencing company for sequencing. The amino acid sequences corresponding to the obtained fusion tumor cell DNA sequences are shown in SEQ ID NO: 3-6.

[0222] mAb1901 mouse heavy chain variable region (SEQ ID NO:3)

[0223]

[0224] mAb1901 mouse light chain variable region (SEQ ID NO:4)

[0225]

[0226] mAb1902 mouse heavy chain variable region (SEQ ID NO:5)

[0227]

[0228] mAb1902 mouse light chain variable region (SEQ ID NO:6)

[0229]

[0230] The aforementioned murine heavy chain variable region and light chain variable region are respectively linked to the heavy chain constant region and human κ light chain constant region of the human IgG1 antibody to form chimeric antibodies ch1901 and ch1902.

[0231] The constant region is selected from the following sequence:

[0232] Heavy chain constant region of human IgG1 antibody: (SEQ ID NO:7)

[0233]

[0234]

[0235] Human κ light chain constant region: (SEQ ID NO:8)

[0236]

[0237] Humanization of murine monoclonal antibodies is performed using methods disclosed in many publications in the field. In short, a human constant domain is used to replace the parental (murine antibody) constant domain, and a human germline antibody sequence is selected based on the homology between the murine and human antibodies for CDR transplantation. This invention selects candidate molecules with good activity for humanization, and the results are as follows.

[0238] 1. CDR region of murine antibody

[0239] The amino acid residues of VH / VL CDR in Table 1 are determined and annotated using the Kabat numbering system.

[0240] The CDR sequences of the murine antibodies are shown in Table 1:

[0241]

[0242] 2. Select FR region sequences from different ancestral groups

[0243] Based on the obtained typical structure of the murine antibody VH / VLCDR, the variable region sequences of the heavy and light chains were compared with the antibody Germline database to obtain a human germline template with high homology. The human germline light chain framework region was derived from the human κ light chain gene.

[0244] 2.1 Humanization and Reversion Mutation Design of mAb1901

[0245] A suitable human antibody line was selected, and the mouse antibody mAb1901 was humanized. The CDR region of the mouse antibody mAb1901 was transplanted onto a selected humanized template, replacing the humanized variable region. This was then recombined with the IgG constant region to form a complete antibody. Simultaneously, a reverse mutation was performed on the FR region of the V region of the humanized antibody. Exemplary reverse mutation methods and combinations are as follows:

[0246]

[0247] *The positions of all amino acids in the table are numbered according to the Kabat numbering rule. In the N82T of the heavy chain variable region, 82 is the 82A position of the Kabat rule.

[0248]

[0249]

[0250] In the table above, the corresponding heavy chain variable regions are linked to the human IgG1 heavy chain constant region shown in SEQ ID NO:7 to form the heavy chain of the full-length antibody, and the light chain variable regions are linked to the human κ light chain constant region shown in SEQ ID NO:8 to form the light chain of the full-length antibody. In other embodiments, the heavy chain variable regions and light chain variable regions may also be linked to other heavy chain constant regions and light chain constant regions respectively to form the full-length antibody.

[0251] 2.2 Humanization and Reversion Mutation Design of mAb1902

[0252] A suitable human antibody line was selected, and the mouse antibody mAb1902 was humanized. The CDR region of the mouse antibody mAb1902 was transplanted onto a selected humanized template, replacing the humanized variable region. This was then recombined with the IgG constant region to form a complete antibody. Simultaneously, a reverse mutation was performed on the FR region of the V region of the humanized antibody. Exemplary reverse mutation methods and combinations are as follows:

[0253]

[0254] *The positions of all amino acids in the table are numbered according to the Kabat numbering rule.

[0255]

[0256] The corresponding heavy chain variable regions in the table above are linked to the human IgG1 heavy chain constant region shown in SEQ ID NO:7 to form the heavy chain of the full-length antibody, and the light chain variable regions are linked to the human κ light chain constant region shown in SEQ ID NO:8 to form the light chain of the full-length antibody.

[0257] Chimeric antibody ch1901

[0258] ch1901 heavy chain: (SEQ ID NO:35)

[0259]

[0260] ch1901 light chain (SEQ ID NO:36)

[0261]

[0262] Chimeric antibody ch1902

[0263] ch1902 heavy chain (SEQ ID NO:37)

[0264]

[0265]

[0266] ch1902 light chain (SEQ ID NO:38)

[0267]

[0268] Table 6 shows the humanized antibodies against mAb1901:

[0269]

[0270] Note: In the table, the heavy chain of humanized antibody h1901-1 is H1, and the light chain is L1, and so on. The full-length antibody light and heavy chain sequences of the humanized antibody mAb1901 are shown in Table 7 below:

[0271]

[0272]

[0273]

[0274] Table 8 shows the humanized antibodies against mAb1902:

[0275]

[0276] Note: In the table, the heavy chain of humanized antibody h1902-1 is H11, the light chain is L11, and so on.

[0277] The light and heavy chain sequences of the humanized antibody mAb1902 are shown in Table 9 below:

[0278]

[0279]

[0280]

[0281]

[0282] The positive control antibody disclosed in this paper is IMAB-362 (from WO2016166122).

[0283] IMAB-362 heavy chain (SEQ ID NO:53):

[0284]

[0285]

[0286] IMAB-362 light chain (SEQ ID NO:54):

[0287]

[0288] The antibodies were cloned, expressed, and purified using conventional gene cloning and recombinant expression methods.

[0289] [II. Preparation of Compounds]

[0290] Experimental methods not specifying specific conditions in the embodiments disclosed herein are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, standard reagents.

[0291] 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 NMR spectrometer with deuterated dimethyl monoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD) as solvents, and tetramethylsilane (TMS) as the internal standard. Chemical shifts are given in units of 10⁻⁶ ppm.

[0292] MS measurements were performed using a Finnigan LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

[0293] The UPLC determination was performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometry system.

[0294] HPLC determinations were performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C18 150×4.6mm column).

[0295] The UV-HPLC determination was performed using a Thermo nanodrop2000 UV spectrophotometer.

[0296] The proliferation inhibition rate and IC50 value were determined using a PHERA starFS microplate reader (BMG GmbH, Germany).

[0297] Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The silica gel plates used in TLC have a diameter of 0.15 mm to 0.2 mm, while those used for TLC separation and purification of products have a diameter of 0.4 mm to 0.5 mm.

[0298] Column chromatography typically uses Yantai Huanghai 200-300 mesh silicone as a carrier.

[0299] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0300] Unless otherwise specified in the examples, the reactions were carried out under an argon or nitrogen atmosphere.

[0301] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0302] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

[0303] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0304] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0305] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0306] Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

[0307] Unless otherwise specified in the examples, the reaction temperature is room temperature.

[0308] Room temperature is the optimal reaction temperature, with a range of 20°C to 30°C.

[0309] Preparation of PBS buffer solution with pH=6.5 in the example: Take 8.5g of KH2PO4, 8.56g of K2HPO4·3H2O, 5.85g of NaCl, and 1.5g of EDTA and place them in a bottle. Make up to 2L, sonicate to dissolve completely, and shake well to obtain the solution.

[0310] The eluent systems for column chromatography and the developing solvent systems for thin-layer chromatography used to purify the compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and can also be adjusted by adding a small amount of triethylamine and acidic or basic reagents.

[0311] The compounds disclosed herein were characterized by Q-TOF LC / MS. The Q-TOF LC / MS was performed using an Agilent 6530 Precision Mass Quadrupole-Time-of-Flight Mass Spectrometer and an Agilent 1290-Infinity Ultra-High Performance Liquid Chromatography System (Agilent Poroshell 300SB-C8 5μm, 2.1×75mm column).

[0312] The YD drug portion of this disclosure of antibody-drug conjugates is referenced in PCT / CN2019 / 107873, and the related compound synthesis and testing are cited in this patent. The non-limiting synthetic examples are cited below:

[0313] [Example 1]

[0314] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclopropane-1-methylamine [1]

[0315]

[0316] Add 1 mL of N,N-dimethylformamide to eczema sulfonate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application "EP0737686A1"), cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxycyclopropylformic acid 1a (1.4 mg, 3.7 μmol, prepared by the known method "Tetrahedron Letters, 25(12), 1269-72; 1984") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (3.8 mg, 13.7 μmol) to the reaction solution in sequence. After the addition is complete, stir the reaction solution at 0-5 °C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution 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 with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give title product 1 (1.6 mg, yield: 82.1%).

[0317] MS m / z(ESI): 520.2 [M+1]

[0318] 1H NMR (400MHz, 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),3.40-3.1 5(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).

[0319] [Example 2]

[0320] (S)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide [2-A]

[0321] (R)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide [2-B]

[0322]

[0323] Towards [1b] 2 mL of ethanol and 0.4 mL of N,N-dimethylmethamide were added to (4 mg, 7.53 μmol) and the mixture was purged with argon three times. The mixture was then cooled to 0-5 °C in an ice-water bath. 0.3 mL of N-methylmorpholine was added dropwise, and the mixture was stirred until the reaction solution became clear. 2-Cyclopropyl-2-hydroxyacetic acid was added to the reaction solution sequentially. [2a] (2.3 mg, 19.8 μmol, prepared by 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, and 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 solution was concentrated under reduced pressure to obtain the crude compound. [2] The product was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A-water (10mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18mL / min), the corresponding components were collected, and the product was concentrated under reduced pressure to obtain the title product (2-A: 1.5mg, 2-B: 1.5mg).

[0324] MS m / z(ESI): 534.0 [M+1].

[0325] Single-configuration compound 2-B (shorter retention time)

[0326] UPLC analysis: retention time 1.06 min, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7μm 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0327] 1H NMR (400MHz, DMSO-d 6): 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).

[0328] Compound 2-A with a single configuration (longer retention time)

[0329] UPLC analysis: retention time 1.10 min, purity: 86% (chromatographic column: ACQUITY UPLC BEHC18 1.7μm 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0330] 1H NMR (400MHz, DMSO-d 6): 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).

[0331] [Example 3]

[0332] (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropylamine [3-A]

[0333] (R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropylamine [3-B]

[0334]

[0335] Towards [1b] (5.0 mg, 9.41 μmol) was reacted with 2 mL of ethanol and 0.4 mL of N,N-dimethylmethamide, cooled to 0-5 °C in an ice-water bath, and 0.3 mL of N-methylmorpholine was added dropwise. The mixture was stirred until the reaction solution became clear. 3,3,3-trifluoro-2-hydroxypropionic acid was then added to the reaction solution sequentially. [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, and 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 solution was concentrated under reduced pressure to obtain the crude compound. [3] The product was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A-water (10mmol NH4OAc):B-acetonitrile, gradient elution, flow rate: 18mL / min), the corresponding components were collected, and the product was concentrated under reduced pressure to obtain the title product (3-A: 1.5mg, 3-B: 1.5mg).

[0336] MS m / z(ESI): 561.9 [M+1].

[0337] Single-configuration compounds (shorter retention time)

[0338] UPLC analysis: retention time 1.11 min, purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7μm 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0339] 1H NMR (400MHz, DMSO-d 6): 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).

[0340] Single-configuration compounds (longer retention time)

[0341] UPLC analysis: retention time 1.19 min, purity: 90% (chromatographic column: ACQUITY UPLC BEHC18 1.7μm 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0342] 1H NMR (400MHz, DMSO-d 6): 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).

[0343] [Example 4]

[0344] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclopentane-1-methylamine [4]

[0345]

[0346] Towards [1b] Add 1 mL of N,N-dimethylformamide (3.0 mg, 5.64 μmol), cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxy-cyclopentanecarboxylic acid sequentially to the reaction solution. [4a] (2.2 mg, 16.9 μmol, prepared by the method disclosed in patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (4.7 mg, 16.9 μmol) were added, and the mixture was stirred at 0-5 °C for 1 hour. The reaction was quenched with 5 mL of water, and the reaction mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to obtain the title product. [4](2.5mg, yield: 80.9%).

[0347] MS m / z(ESI): 548.0 [M+1].

[0348] 1H NMR (400MHz, 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),2.4 3(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).

[0349] [Example 5]

[0350] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-methylamine [5]

[0351]

[0352] Towards [1b] Add 1 mL of N,N-dimethylformamide (2.0 mg, 3.76 μmol), cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(hydroxymethyl)-cyclopentanecarboxylic acid sequentially to the reaction solution. [5a] (0.87 mg, 7.5 μmol, prepared by the method disclosed in patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (2 mg, 7.24 μmol), after which the reaction was stirred at 0-5 °C for 2 hours. The reaction was quenched with 5 mL of water, and the reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to obtain the title product. [5] (1.0 mg, yield: 50%).

[0353] MS m / z(ESI): 533.9 [M+1].

[0354] 1H NMR (400MHz, 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).

[0355] [Example 6]

[0356] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclobutane-1-methylamine [6]

[0357]

[0358] Towards [1b] Add 1 mL of N,N-dimethylformamide (3.0 mg, 5.64 μmol), cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, and stir until the reaction solution becomes clear. Then, add 1-(hydroxymethyl)cyclobutane-1-carboxylic acid sequentially to the reaction solution. [6a] (2.2 mg, 16.9 μmol; prepared by the method disclosed in the literature "Journal of the American Chemical Society, 2014, vol. 136, #22, p. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (4.7 mg, 16.9 μmol), after addition, the reaction was stirred at 0-5 °C for 1 hour. The reaction was quenched with 5 mL of water, and the reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to obtain the title product. [6](2.1 mg, yield: 67.9%).

[0359] MS m / z(ESI): 548.0 [M+1].

[0360] 1H NMR (400MHz, DMSO-d 6): 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).

[0361] [Example 7]

[0362] N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclobutane-1-methylamine [7]

[0363]

[0364] Towards [1b] (3.0 mg, 5.64 μmol) was reacted with 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice-water bath. 0.3 mL of N-methylmorpholine was added dropwise, and the mixture was stirred until the reaction solution became clear. 1-Hydroxycyclobutanecarboxylic acid was then added sequentially to the reaction solution. [7a] (2.0 mg, 17.22 μmol, supplier's reagent), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) were added, and 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 solution was concentrated under reduced pressure, and the residue was purified by thin-layer chromatography using solvent system B to obtain the title product. [7](2.5mg, yield: 83.1%).

[0365] MS m / z(ESI): 534.0 [M+1].

[0366] 1H NMR (400MHz, DMSO-d 6): 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).

[0367] [Example 8]

[0368] 1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentazaeicosyl)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':6,71indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-methylamine [8]

[0369]

[0370]

[0371] first step

[0372] 1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamino)methoxy)cyclopropane-1-carboxylic acid benzyl ester [8c]

[0373] 1-hydroxycyclopropane-1-carboxylic acid benzyl ester [8a](104 mg, 0.54 mmol; prepared by the method disclosed in patent application “US2005 / 20645”) and 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamino)methyl acetate) [8b] (100 mg, 0.27 mmol; prepared by the method disclosed in patent application “CN105829346A”) was added to a reaction flask, followed by 5 mL of tetrahydrofuran. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and then potassium terbutoxide (61 mg, 0.54 mmol) was added. The ice bath was removed, and the mixture was brought to room temperature and stirred for 10 minutes. 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 3 mL of 1,4-dioxane, and 0.6 mL of water was added. Sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorene methyl chloroformate (70 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Add 20 mL of water, extract with ethyl acetate (8 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry to anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography with solvent system B to obtain the title product. [8c](100mg, yield: 73.6%).

[0374] MS m / z(ESI):501.0[M+1].

[0375] Step 2

[0376] 1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamino)methoxy)cyclopropane-1-carboxylic acid [8d]

[0377] Will [8c] (50 mg, 0.10 mmol) was dissolved in 3 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (25 mg, 10%) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with tetrahydrofuran, and the filtrate was concentrated to give the title product. [8d] (41mg, yield: 100%).

[0378] MS m / z(ESI):411.0[M+1].

[0379] Step 3

[0380] (9H-fluorene-9-yl)methyl(2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bis(oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropoxy)methyl)amino)-2-bis(oxyethyl)aminocarbamate [8e]

[0381] Will [1b] (7 mg, 0.013 mmol) was added to the reaction flask, followed by 1 mL of N,N-dimethylformamide. The mixture was purged three times with argon gas, cooled to 0-5°C in an ice-water bath, and then one drop of triethylamine was added. [8d] (7 mg, 0.017 mmol) of 0.5 mL N,N-dimethylmethamide solution was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (7 mg, 0.026 mmol) was added. The mixture was stirred in an ice bath for 35 minutes. 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 residue was purified by thin-layer chromatography using solvent system B to give the title product. [8e](8.5mg, yield 78.0%).

[0382] MS m / z(ESI):828.0[M+1].

[0383] Step 4

[0384] 1-((2-aminoacetamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-methamide [8f]

[0385] Will [8e] (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, and 0.1 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred. The upper n-hexane layer was decanted. This process was repeated three times, and the crude product was concentrated under reduced pressure to obtain the title product. [8f] (2.9 mg), the product was used directly in the next reaction without purification.

[0386] MS m / z(ESI): 606.0 [M+1].

[0387] Step 5

[0388] 1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentazaeicosyl)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]indolazino[1,2-b]quinolino-1-yl)cyclopropane-1-methylamine [8]

[0389] crude products [8f] (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and then (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrolo-1-yl)hexamyl)acetamido)acetamido)-3-phenylpropionic acid was added. [8g] (2.7mg, 5.80μmol, prepared using the method disclosed in patent application "EP2907824") in 0.3mL of N,N-dimethylmethamide solution was added, along with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2.7mg, 9.67μmol). The mixture was stirred in an ice bath for 30 minutes, then the ice bath was removed, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product. [8] (2 mg, yield: 39.0%).

[0390] MS m / z(ESI):1060.0[M+1].

[0391] 1H NMR (400MHz, DMSO-d 6): 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).

[0392] [Implementation Example 9]

[0393] 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxo-5,8,11,14-tetraazahexadecyl-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexylamine [9-A]

[0394] N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxy-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-bioxy-2,5-dihydro-1H-pyrrole-1-yl)hexylamine [9-B]

[0395]

[0396] first step

[0397] 2-Cyclopropyl-2-hydroxyacetic acid benzyl ester [9a]

[0398] Will [2a] (1.3 g, 11.2 mmol; prepared by 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 solution was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with solvent system C to obtain the title product. [9a](2g, yield: 86.9%).

[0399] Step 2

[0400] 10-Cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabut-11-acid benzyl ester [9b]

[0401] Will [9a](120.9 mg, 0.586 mmol) and [8b] (180 mg, 0.489 mmol) was added to the reaction flask, followed by 4 mL of tetrahydrofuran. The mixture was purged three times with argon gas, cooled to 0-5 °C in an ice-water bath, and then potassium terbutoxide (109 mg, 0.98 mmol) was added. The ice bath was removed, and the mixture was brought to room temperature and stirred for 40 minutes. 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). The organic phases were combined and concentrated. The residue was dissolved in 4 mL of dioxane, and 2 mL of water was added. Sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorene chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 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 residue was purified by silica gel column chromatography with solvent system C to obtain the title product. [9b](48mg, yield: 19%).

[0402] MS m / z(ESI): 515.0 [M+1].

[0403] Step 3

[0404] 10-Cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabutane-11-acid [9c]

[0405] Will [9b] (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (12 mg, 10% purity, dry type) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with ethyl acetate, and the filtrate was concentrated to obtain the crude product. [9c](13mg), the product was directly proceeded to the next reaction without purification.

[0406] MS m / z(ESI):424.9[M+1].

[0407] Step 4

[0408] (9H-fluorene-9-yl)methyl(2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-epoxyethoxy)methyl)amino)-2-epoxyethyl)aminocarbamate [9d]

[0409] Will [1b] (10 mg, 18.8 μmol) was added to the reaction flask, followed by 1 mL of N,N-dimethylformamide. The mixture was purged with argon three times, cooled to 0-5 °C in an ice-water bath, and then one drop of triethylamine was added. The crude product was then added. [9c] (13 mg, 30.6 μmol) was added to 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (16.9 mg, 61.2 μmol), and the mixture was stirred in an ice bath for 40 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phases were washed with saturated sodium chloride solution (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography using solvent system B to give the title product. [9d] (19mg, yield: 73.6%).

[0410] MS m / z(ESI):842.1[M+1].

[0411] Step 5

[0412] 2-((2-aminoacetamino)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)acetaminophen [9e]

[0413] Will [9d] (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue and stirred. After standing, the supernatant was decanted, and the solid was retained. The solid residue was concentrated under reduced pressure and then dried using an oil pump to obtain the crude product. [9e](17mg), the product is used directly in the next reaction without purification.

[0414] MS m / z(ESI): 638.0 [M+18].

[0415] Step 6

[0416] N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxy-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-bioxy-2,5-dihydro-1H-pyrrole-1-yl)hexylamine [9-A]

[0417] N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-bioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxy-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-bioxy-2,5-dihydro-1H-pyrrole-1-yl)hexylamine [9-B]

[0418] crude products [9e] (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and then added... [8 g] (21.2 mg, 44.8 μmol) of 0.3 mL N,N-dimethylmethamide solution was added, along with 18.5 mg (67.3 μmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride. The mixture was stirred in an ice bath for 10 minutes, then the ice bath was removed, and the mixture was stirred at room temperature for 1 hour to produce the compound. [9]. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A-water (10mmol NH4OAc):B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4mg, 9-B: 1.7mg).

[0419] MS m / z(ESI):1074.4[M+1].

[0420] Single-configuration compound 9-A (shorter retention time):

[0421] UPLC analysis: retention time 1.14 min, purity: 85% (chromatographic column: ACQUITY UPLC BEHC18 1.7μm 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0422] 1H NMR (400MHz, DMSO-d 6): 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),7.2 6-7.15(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).

[0423] Single-configuration compound 9-B (longer retention time):

[0424] UPLC analysis: retention time 1.16 min, purity: 89% (chromatographic column: ACQUITY UPLC BEHC18 1.7μm 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0425] 1H NMR (400MHz, DMSO-d 6): 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).

[0426] [III. Preparation of anti-Claudin18.2 antibody ADC conjugate]

[0427] Drug loading analysis of ADC stock solution

[0428] I. UV-HPLC Method

[0429] The DAR value n of the ADC embodiments disclosed herein was calculated using UV-HPLC, as detailed below:

[0430] 1. Measurement method:

[0431] After placing cuvettes containing sodium succinate buffer into the reference absorption cell and the sample measurement absorption cell respectively, and subtracting the solvent blank, the cuvette containing the test solution was placed into the sample measurement absorption cell, and the absorbance at 280 nm and 370 nm was measured.

[0432] 2. Result Calculation: The ADC stock solution loading was determined using ultraviolet spectrophotometry (instrument used: Thermo Nanodrop 2000 ultraviolet spectrophotometer). The principle is that the total absorbance of the ADC stock solution at a certain wavelength is equal to the sum of the absorbance values ​​of the drug and the monoclonal antibody at that wavelength, i.e.:

[0433] (1)A280nm=εmab-280bCmab+εDrug-280bCDrug

[0434] εDrug-280: The drug has an average molar extinction coefficient of 5100 at 280 nm;

[0435] CDrug: Drug concentration;

[0436] εmab-280: The average molar extinction coefficient of the single antigen solution at 280 nm is 214,600;

[0437] Cmab: Concentration of the single antigen solution;

[0438] b: The optical path length is 1cm.

[0439] Similarly, the equation for the total absorbance of the sample at 370 nm can be obtained:

[0440] (2)A370nm=εmab-370bCmab+εDrug-370bCDrug

[0441] εDrug-370: The drug has an average molar extinction coefficient of 19000 at 370 nm;

[0442] CDrug: Drug concentration;

[0443] εmab-370: The extinction coefficient of the single antigen solution is 0 at 370 nm;

[0444] Cmab: Concentration of the single antigen solution;

[0445] b: The optical path length is 1cm.

[0446] The drug loading can be calculated by combining equations (1) and (2) with the extinction coefficients and concentration data of the monoclonal antibody and drug at two detection wavelengths.

[0447] Drug load = CDrug / Cmab.

[0448] II. RP-HPLC Method

[0449] The method for calculating the DAR value in some of the ADC embodiments disclosed herein uses RP-HPLC (reversed-phase high-performance liquid chromatography), as detailed below.

[0450] 1. Measurement method:

[0451] Naked antibody (unconjugated antibody) and the ADC sample (concentration 1 mg / mL) were reduced with 4 μL of DDT (sigma) and incubated at 37℃ for 1 hour. After the reaction, the sample was transferred to an inner tube. Detection was performed using an Agilent 1200 high-performance liquid chromatograph (HPLC). The chromatographic column used was an Agilent PLRP-S 1000A 8μm 4.6*250mm. The column temperature was 80℃; the DAD detector wavelength was 280nm; the flow rate was 1 mL / min; and the injection volume was 40 μL. The positions of the light and heavy chains were then distinguished by comparing the spectra of the sample and the naked antibody. The DAR value n was calculated by integrating the spectra of the detected sample.

[0452] 2. Solution preparation

[0453] 1) 0.25M DTT solution:

[0454] Preparation example: Take 5.78 mg of DTT, add 150 μL of purified water to dissolve it completely, and prepare a 0.25 M DTT solution. Store at -20℃.

[0455] 2) Mobile phase A (0.1% TFA aqueous solution):

[0456] Preparation example: Measure 1000mL of purified water with a graduated cylinder, add 1mL of TFA (sigma), mix thoroughly before use, and store at 2-8℃ for 14 days.

[0457] 3) Mobile phase B (0.1% TFA acetonitrile solution):

[0458] Preparation example: Measure 1000 mL of acetonitrile with a graduated cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8℃ for 14 days.

[0459] 3. Data Analysis

[0460] By comparing the spectra of the sample with those of the bare antibody, the positions of the light and heavy chains are distinguished. Then, the spectrum of the sample is integrated to calculate the DAR value (n).

[0461] The calculation formula is as follows:

[0462]

[0463] HC+2 4

[0464] HC+3 6

[0465] Total LC peak area = LC peak area + LC+1 peak area

[0466] Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area

[0467] LC DAR = Σ(number of connected drugs * percentage of peak area) / total LC peak area

[0468] HC DAR = Σ(Number of drugs linked * Percentage of peak area) / Total peak area of ​​HC

[0469] DAR = LC DAR + HC DAR.

[0470] Claudin 18.2 Antibody-Drug Conjugate Preparation Examples

[0471] [Examples 3-1, 3-2: ADC-1, ADC-2]

[0472]

[0473] At 37°C, tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 11.03 mL, 110.3 μmol) was added to PBS buffer containing antibody h1902-5 (pH=6.5, 0.05 M PBS buffer solution; 10.0 mg / mL, 320.0 mL, 21.62 μmol). The mixture was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0474] Compound 9-A (350 mg, 303 mol) was dissolved in 13.2 mL of acetonitrile and 6.6 mL of DMSO, and added to the above reaction solution which had been cooled to 25 °C. The mixture was placed in a water bath shaker and shaken at 25 °C for 3 hours before the reaction was stopped.

[0475] The obtained reaction solution 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 a concentration of 60 mg / mL, and Tween-20 was added to a concentration of 0.2 mg / mL. Finally, the exemplary product ADC-1 (10 mM, pH=5.3 succinate; 10 mg / mL, 2.626 g) of the general formula antibody-drug conjugate h1902-5-9-A was prepared. Yield: 81.81%.

[0476] UV-HPLC calculated average: n=6.8.

[0477] Using the above method, an exemplary product ADC-2 of the general formula antibody-drug conjugate h1901-11-9-A can be prepared by replacing h1902-5 with antibody h1901-11 and compound 9-A, with a DAR value n=7.1.

[0478] [Example 3-3 ADC-3]

[0479] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 10.1 μL, 101 nmol) was added to the PBS buffered aqueous solution of antibody h1901-11 (pH=6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1 mL, 67.5 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0480] compound [9-A] (0.58 mg, 540 nmol) was dissolved in 34 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then 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 of the antibody-drug conjugate h1901-11-9-A. [ADC-3] in PBS buffer (0.72 mg / mL, 11.2 mL) was stored at 4 °C. RP-HPLC calculated average: n = 2.51.

[0481] [Example 3-4 ADC-4]

[0482] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 16.9 μL, 169 nmol) was added to a PBS buffer solution of antibody h1901-11 (pH=6.5, 0.05 M PBS buffer solution; 10.0 mg / mL, 1 mL, 67.5 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0483] compound [9-A] (0.73 mg, 680 nmol) was dissolved in 43 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product of the antibody-drug conjugate h1901-11-9-A. [ADC-4] in PBS buffer (0.62 mg / mL, 12.5 mL) was stored at 4 °C. RP-HPLC calculated mean: n = 4.06.

[0484] [Example 3-5 ADC-5]

[0485] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 35.8 μL, 358 nmol) was added to a PBS buffer solution of antibody h1901-11 (pH=6.5, 0.05 M PBS buffer solution; 10.0 mg / mL, 1 mL, 67.5 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0486] compound [9-A] (1.09 mg, 10¹⁵ nmol) was dissolved in 64 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product of the antibody-drug conjugate h1901-11-9-A. [ADC-5] in PBS buffer (0.54 mg / mL, 12.5 mL) was stored at 4 °C. RP-HPLC calculated mean: n = 6.8.

[0487] [Example 3-6 ADC-6]

[0488] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 10.9 μL, 109 nmol) was added to a PBS buffer solution of antibody h1902-5 (pH=6.5, 0.05 M PBS buffer solution; 10.0 mg / mL, 1.08 mL, 72.9 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0489] compound [9-A] (0.63 mg, 587 nmol) was dissolved in 40 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then 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 h1902-5-9-A. [ADC-6] in PBS buffer (0.7 mg / mL, 13.0 mL) was stored at 4 °C. RP-HPLC calculated mean: n = 2.69.

[0490] [Example 3-7 ADC-7]

[0491] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 18.3 μL, 183 nmol) was added to a PBS buffer solution of antibody h1902-5 (pH=6.5, 0.05 M PBS buffer solution; 10.0 mg / mL, 1.08 mL, 72.9 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0492] compound [9-A] (0.79 mg, 736 nmol) was dissolved in 50 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting 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 h1902-5-9-A. [ADC-7] in PBS buffer (0.6 mg / mL, 14.0 mL), stored at 4 °C. RP-HPLC calculated mean: n = 4.25.

[0493] [Example 3-8 ADC-8]

[0494] At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 38.7 μL, 387 nmol) was added to a PBS buffer solution of antibody h1902-5 (pH=6.5, 0.05 M PBS buffer solution; 10.0 mg / mL, 1.08 mL, 72.9 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0495] compound [9-A] (1.18 mg, 1099 nmol) was dissolved in 70 μL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then 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 h1902-5-9-A. [ADC-8] in PBS buffer (0.56 mg / mL, 14.2 mL) was stored at 4 °C. RP-HPLC calculated mean: n = 7.01.

[0496] [Example 3-9 ADC-9]

[0497] At 12°C, a prepared TCEP histidine buffer (10mM histidine-acetic acid-Tris / EDTA buffer at pH 7.2, containing 10mM histidine-acetic acid-Tris and 2.5mM EDTA; 20.6g / L, 6.49L, 0.91mmol) was added to a histidine-acetic acid-Tris / EDTA buffer containing antibody h1902-5. The mixture was placed in a constant temperature water bath and stirred at 12°C for 2 hours. The reaction was then stopped to obtain intermediate I solution.

[0498] Compound 9-A (4.72 g, 4.39 mmol) was dissolved in 0.38 L of DMSO to generate a DMSO solution of compound 9-A. 0.38 L of DMSO was pre-added to the above intermediate I solution, followed by the DMSO solution of compound 9-A. The mixture was placed in a constant temperature water bath and stirred at 12 °C for 1 hour, after which the reaction was stopped.

[0499] The above reaction solution was purified using a Capto S Impact cation exchange column. Washing was performed with 9 column volumes of 0.05 M acetate buffer (pH=5.0) containing 10% (v / v) DMSO and 6 column volumes of 0.05 M acetate buffer (pH=5.0), followed by elution with 0.05 M acetic acid and 0.30 M sodium chloride buffer (pH=5.5) to remove free toxins and residual solvents. The cation eluent was then subjected to 7-fold equal-volume ultrafiltration (using a 30 KD polycellulose membrane) at 22 °C to obtain the exemplary product ADC-9 of h1902-5-9-A. RP-HPLC calculated average: n=4.1.

[0500] The drug loading obtained in this embodiment is a non-limiting embodiment. Those skilled in the art can obtain conjugates with different DAR values ​​(1-10, preferably 1-8, more preferably 2-8, 2-7) by adjusting the reaction conditions and reagents.

[0501] [Biological Evaluation]

[0502] [Test Example 1: Cell-level ELISA Binding Assay]

[0503] Cell-based ELISA was used to detect the binding properties of Claudin18.2 antibody. NUGC4 cells stably expressing Claudin18.2 were cultured in 96-well plates. When the cells reached 90% confluence, 4% paraformaldehyde was added to fix the cells for 1 hour. The plates were washed three times with PBST buffer (pH 7.4, PBS containing 0.05% Tween-20), and then 200 μL / well of blocking buffer (5% skim milk, Bright Dairy skim milk powder) diluted in PBS was added. The plates were incubated at 37°C for 2.5 hours or overnight (16-18 hours) at 4°C. After blocking, the blocking buffer was discarded, and the plates were washed three times with PBST buffer. Then, 50 μL / well of the antibody of different concentrations diluted with sample dilution buffer (pH 7.4, PBS containing 1% γ-milk) was added, and the plates were incubated at 37°C for 2 hours. After incubation, wash the plate 5 times with PBST, add 100 μL / well of HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, 109-035-003) diluted with sample dilution buffer, and incubate at 37°C for 1 hour. After washing the plate 6 times with PBST, add 50 μL / well of TMB chromogenic substrate (KPL, 52-00-03), incubate at room temperature for 10-15 minutes, and stop the reaction by adding 50 μL / well of 1M H2SO4. Read the absorbance at 450 nm using an MD Versa Max™ microplate reader and calculate the EC50 value of Claudin18.2 antibody binding to Claudin18.2.

[0504]

[0505]

[0506]

[0507] [Test Example 2: Antibody Cellular Binding Experiment]

[0508] NUGC4 cells stably expressing Claudin18.2 were prepared into a 1×10⁶ / mL cell suspension using FACS buffer (2% fetal bovine serum (Gibco, 10099141), pH 7.4, PBS (Sigma, P4417-100TAB)). 100 μL / well was added to each well of a 96-well round-bottom plate (Corning, 3795). After centrifugation to remove the supernatant, 50 μL / well of different concentrations of Claudin18.2 antibody diluted with FACS buffer was added, and the plates were incubated at 4°C in the dark for 1 hour. After washing three times with FACS buffer at 300g, the working concentration of Alexa Fluor 488 goat anti-human IgG (H+L) (Invitrogen, A-11013) was added, and the plates were incubated at 4°C in the dark for 40 minutes. After washing three times with FACS buffer at 300g, the geometric mean fluorescence intensity was detected on a BD FACS CantoII flow cytometer. The EC50 value of Claudin18.2 antibody binding to NUGC4 cells stably expressing Claudin18.2 was calculated. The results are shown in Figure 1.

[0509] [Test Example 3: Antibody Internalization Assay]

[0510] The Claudin18.2 antibody pre-labeled with DyLight 488 NHS Ester (thermofisher, 46403) was added to NUGC4 cells stably expressing Claudin18.2 at a final concentration of 5 μg / mL. The cells were incubated on ice in the dark for 1 hour. After washing three times with pre-chilled FACS buffer (pH 7.4 PBS, 2% fetal bovine serum), the supernatant was discarded, and the cells were added to pre-warmed complete culture medium. The cells were then incubated at 37°C in a 5% CO2 cell culture incubator. Cells were harvested at 0, 0.5, 1, 2, and 4 hours and stored on ice in the dark. After all samples were collected, the supernatant was removed by centrifugation at 300g at low temperature. Evaporation buffer (pH 1.7, 0.05M glycine, 0.1M sodium chloride) was added, and the mixture was incubated at room temperature for 7 minutes. The cells were washed once by centrifugation at 300g with FACS buffer. The geometric mean fluorescence intensity was detected using a BD FACS CantoII flow cytometer, and the endocytosis efficiency of the Claudin18.2 antibody on NUGC4 cells stably transfected with Claudin18.2 was calculated. The results (see Figure 2) showed that the humanized antibody had good endocytosis efficiency.

[0511] [Test Example 4: Antibody Affinity Assay Based on Flow Cytometry]

[0512] On the day of the experiment, HEK293 / hClaudin18.2 cells were collected in 96-well U-bottom plates, with 1-2 × 10⁵ cells per well. Human Claudin18.2 antibody at an initial concentration of 5 μg / mL, serially diluted 2× (12 concentration points), was added and incubated at 4°C for 1 hour. IMAB362 was used as a positive control, and a negative control without antibody was also included. After centrifugation to remove the antibody, 100 μL / well of FITC anti-human IgG Fc antibody (200×) was added, and the plates were incubated at 4°C in the dark for 30 minutes. The plates were washed twice with PBS + 2% FBS before flow cytometry analysis. BD FACS CantoII was started, and after warm-up, BD FACSDiva software was opened to create a new experiment to analyze the HEK293 / hClaudin18.2 negative control sample. The FSC and SSC voltages were adjusted to appropriate values ​​and saved. According to the Quantum™ FITC-5 MESF Kit instructions, blank sample B and standard curve 1 were analyzed, and the FITC voltage was adjusted to appropriate values ​​and saved. Samples in 96-well U-bottom plates were analyzed at the preserved voltage, and data were recorded. The Geo-mean was obtained by analyzing the experimental data using Flowjo software. A MESF-Geo Mean standard curve was fitted according to the Quantum™ FITC-5 MESF Kit instructions. The molar concentration of human Claudin18.2 antibody binding to HEK293 / hClaudin18.2 cells and the concentration of free antibody were calculated based on the concentration fluorescence value 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.

[0513]

[0514] [Test Example 5: Evaluation of Antibody ADCC Effect]

[0515] Various NUGC4 cells (high, medium, and low expression of Claudin 18.2) were digested, centrifuged at 1000 rpm, and resuspended for counting. Cells were resuspended at a density of 3 × 10⁵ 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) was added to each well of a 96-well plate (Corning, 3903). Antibody was diluted in the above phenol red-free medium to prepare a 3× antibody dilution, and 25 μL of antibody was added to each well of the cell culture plate. The cells were incubated at 37°C in a 5% CO₂ incubator for 0.5 hours.

[0516] Effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected, centrifuged at 1000 rpm, and resuspended for counting. Cells were resuspended at a density of 3 × 10⁶ cells / mL in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum), and 25 μL of cells (7.5 × 10⁴ cells / well) was added to each well of the experimental plate. The plates were incubated at 37°C in a 5% CO₂ incubator for 6 hours.

[0517] Add 75 μL of Bright-Glo (Promega, E2610) to each well of the experimental plate and detect chemiluminescence using a microplate reader (PerkinElmer, VITOR3).

[0518] The results showed (see Table 14 and Figures 3A-3C) that antibodies h1901-11 and h1902-5 exhibited strong ADCC activity in NUGC4 cells with different levels of Claudin18.2 expression, ranging from low (Figure 3A) to medium (Figure 3B) to high (Figure 3C).

[0519]

[0520] [Test Example 6: In vitro proliferation inhibition test of compounds on tumor cells]

[0521] I. Purpose of the Test

[0522] The purpose of this experiment was to detect the inhibitory activity of the disclosed drug compound 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, catalog number HTB-30). Cells were treated with different concentrations of the compound in vitro, and after 6 days of culture, cell proliferation was detected using the CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, catalog number: G7573) reagent. The in vitro activity of the compound was evaluated based on the IC50 value.

[0523] II. Experimental Methods

[0524] The following example, using the in vitro proliferation inhibition assay of U87MG cells, illustrates the method for testing the in vitro proliferation inhibition activity of the disclosed compounds against tumor cells. This method is also applicable to, but not limited to, testing the in vitro proliferation inhibition activity of other tumor cells.

[0525] 1. Cell culture: U87MG and SK-BR-3 cells were cultured in EMEM medium (GE, catalog number SH30024.01) with 10% FBS and McCoy's 5A medium (Gibco, catalog number 16600-108) with 10% FBS, respectively.

[0526] 2. Cell preparation. Take U87MG and SK-BR-3 cells in the logarithmic growth phase, wash them once with PBS (phosphate-buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3 mL of trypsin (0.25% Trypsin-EDTA (1x), Gibico, Life Technologies) to digest for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to wash off the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 mL of cell culture medium to resuspend the cells to prepare a single-cell suspension.

[0527] 3. Cell Plating. Mix U87MG and SK-BR-3 single-cell suspensions thoroughly. Adjust the viable cell density to 2.75 × 10³ cells / mL and 8.25 × 10³ cells / mL respectively using cell culture medium. Mix the adjusted cell suspensions thoroughly and add 180 μL / well to a 96-well cell culture plate. Add only 200 μL of culture medium to the outer wells of the 96-well plate. Incubate the plates in an incubator for 24 hours (37℃, 5% CO₂).

[0528] 4. Compound preparation. The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a storage solution with an initial concentration of 10 mM.

[0529] The initial concentration of the small molecule compound is 500 nM, and the preparation method is as follows.

[0530] Add 30 μL of each different test sample (100 μM) to the first well of a 96-well U-bottom preparation plate. Add 20 μL of DMSO to each well in columns 2 through 11. Take 10 μL of the sample from the first column and add it to the 20 μL of DMSO in the second column, mix well, and then add 10 μL to the third column, and so on up to the tenth column. Transfer 5 μL of the drug from each well of the preparation plate to 95 μL of EMEM medium, mix well, and set aside for later use.

[0531] The initial concentration of the ADC is 10 nM or 500 nM, and the preparation method is as follows.

[0532] Add 100 μL of different test samples to the first column of a 96-well plate, with a sample concentration of 100 nM or 5 μM; add 100 μL of PBS to each well in columns 2 through 11. Take 50 μL of the sample from the first column and add it to 100 μL of PBS in the second column, mix well, take 50 μL and add it to the third column, and so on, diluting 3-fold up to the 10th column.

[0533] 5. Sample addition procedure. Add 20 μL of the prepared test sample at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37℃, 5% CO2).

[0534] 6. Colorimetric procedure. Take out the 96-well cell culture plate, add 90 μL of CTG solution to each well, and incubate at room temperature for 10 minutes.

[0535] 7. Plate reading procedure. Remove the 96-well cell culture plate and place it in a microplate reader (BMG Labtech, PHERAstar FS) to measure the chemiluminescence.

[0536] III. Data Analysis

[0537] The data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. The results of the example are shown in the table below.

[0538]

[0539] Conclusion: The small molecule fragment disclosed in this study exhibits significant inhibitory activity against the proliferation of SK-BR-3 and U87 cells, and the chiral center has a certain influence on the inhibitory activity of the compound.

[0540] [Test Example 7: ADC Molecular Cellular Activity Assay]

[0541] This experiment used the CellTiter-Glo Luminescence Cell Viability Assay to detect the in vitro killing effect of ADC molecules on human gastric cancer cell lines. On day one, cells expressing low, medium, and high levels of NUGC4-claudin18.2 were collected and their density adjusted to 2.5 × 10⁴ / mL. 90 μL / well (approximately 2500 cells per well) was added to each 96-well white transparent plate. The plates were incubated overnight at 37°C with 5% CO₂. On day two, the sample was diluted in a U-bottom 96-well plate with an initial concentration of 5 μM, using a 4× serial dilution method (9 concentration points). 10 μL / well of the diluted sample was added to each well. The plates were incubated at 37°C with 5% CO₂ for 6 days. On day eight, the cell culture plates were removed, and 50 μL / well of Cell Titer-Glo Reagent was added. After incubation at room temperature for 2–3 minutes, fluorescence values ​​were read using a Pherastar FS plate reader. Data analysis was performed using GraphPad Prism software. See Table 16.

[0542]

[0543] In vivo bioactivity evaluation

[0544] [Test Example 8: In vivo efficacy evaluation of ADC molecules]

[0545] Balb / c cells were subcutaneously inoculated with human gastric cancer cells NUGC4 (Claudin 18.2 moderately expressed) (5 × 10⁶ cells / cell containing 50% Matrigel matrix gel) in the right rib area. Cells were grouped on day 0, with 8 cells per group, for a total of 5 groups. The average tumor volume was approximately 84.41 mm³.

[0546] The ADC was administered via intraperitoneal injection three times, with each animal receiving 10g / 0.1mL of the drug based on its body weight, on days 0, 4, and 11.

[0547] On the day of grouping, ADC was injected intraperitoneally, and a total of 4 doses were administered, with an interval of 5 days between doses. Each animal was injected with 10g / 0.1mL based on its body weight.

[0548] Tumor volume and body weight were measured twice a week, and the data were recorded.

[0549] Using Excel 2003 statistical software: the mean was calculated as avg; the SD value was calculated as STDEV; the SEM value was calculated as STDEV / SQRT; and the p-value for differences between groups was calculated as TTEST.

[0550] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short²

[0551] Relative volume (RTV) = VT / V0

[0552] Tumor inhibition rate (%) = (CRTV - TRTV) / CRTV (%)

[0553] V0 and VT represent the tumor volume at the start of the experiment (day 0, the day of the first dose) and at the time of measurement and counting, respectively. CRTV and TRTV represent 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.

[0554]

[0555] vs blank:** p<0.01.

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

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[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

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[0599]

Claims

1. A ligand-drug conjugate of general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, wherein: Y is -O-(CRaRb)m-CR1R2-C(O)-; Ra and Rb may be the same or different, and each is independently selected from hydrogen, deuterium, halogen, or C1-6 alkyl; R1 is a C3-6 cycloalkyl; R2 is selected from hydrogen, C1-6 haloalkyl, or C3-6 cycloalkyl; or, R1 and R2 together with the carbon atom they are attached to form a C3-6 cycloalkyl; m is 0 or 1; n is 1 to 10, n It can be a decimal or an integer; -L- is -L1-L2-L3-L4-, where L1 is -(succinimino-3-yl-N)-WC(O)-, and W is a C1-8 alkyl group; L2 is a chemical bond; L3 is a peptide residue composed of 2 to 7 amino acids, wherein the amino acid is selected from amino acids formed from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid. L4 is -NR5(CR6R7)t-, where t is an integer from 1 to 6; R5 is a hydrogen atom or a C1-6 alkyl group; R6 and R7 may be the same or different, and each is independently selected from hydrogen atom, halogen, C1-6 alkyl group, C1-6 haloalkyl group, C1-6 deuterated alkyl group, and C1-6 hydroxyalkyl group; Pc is an anti-Claudin18.2 antibody or its antigen-binding fragment, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region, wherein: iii) the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; or iv) the heavy chain variable region contains sequences as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. The light chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in NO:17, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively.

2. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in claim 1, wherein the anti-Claudin18.2 antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

3. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in claim 1, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein: (1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; (2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21; (3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; or (4) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

28.

4. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in claim 1, wherein the anti-Claudin18.2 antibody is a humanized antibody comprising a frame region derived from a human antibody or a variant thereof, the frame region variant having a reversion mutation of up to 10 amino acids in the light chain frame region and / or the heavy chain frame region of the human antibody, respectively.

5. A ligand-drug conjugate of the general formula (Pc-LYD) as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the frame region variant contains a mutation selected from (a) or (b) of the following: (a) the light chain variable region contains one or more amino acid reversion mutations selected from 22S, 85I, and 87H, and / or the heavy chain variable region contains one or more amino acid reversion mutations selected from 48I, 82T, and 69M; or (b) the light chain variable region contains one or more amino acid reversion mutations selected from 4L and 22S, and / or the heavy chain variable region contains one or more amino acid reversion mutations selected from 38K, 40R, 48I, 66K, 67A, 69L, 71L, and 73K.

6. A ligand-drug conjugate of the general formula (Pc-LYD) as claimed in claim 1, wherein the frame region variant contains a mutation selected from the following: (a-1) an amino acid reversion mutation containing 22S, 85I, and 87H in the light chain variable region, and an amino acid reversion mutation containing 48I and 82T in the heavy chain variable region; or (b-1) an amino acid reversion mutation containing 4L in the light chain variable region.

7. A ligand-drug conjugate of the general formula (Pc-LYD) as claimed in claim 1, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as shown below: (vii) the heavy chain variable region sequence is shown in SEQ ID NO: 3 and the light chain variable region sequence is shown in SEQ ID NO: 4; (viii) the heavy chain variable region sequence is shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27 and the light chain variable region sequence is shown in SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23; (ix) the heavy chain variable region sequence is shown in SEQ ID NO: 5 and the light chain variable region sequence is shown in SEQ ID NO: 6; or (x) the heavy chain variable region sequence is shown in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 and the light chain variable region sequence is shown in SEQ ID NO:

6. As shown in NO: 28, SEQ ID NO: 29 or SEQ ID NO:

30.

8. A ligand-drug conjugate of the formula (Pc-LYD) as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as shown below: (xi) the heavy chain variable region sequence is shown in SEQ ID NO: 31 and the light chain variable region sequence is shown in SEQ ID NO: 29; or (xii) the heavy chain variable region sequence is shown in SEQ ID NO: 26 and the light chain variable region sequence is shown in SEQ ID NO:

23.

9. A ligand-drug conjugate of the formula (Pc-LYD) as claimed in claim 1, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, and the light chain constant region is selected from the constant regions of human antibody κ and λ chains and their conventional variants.

10. A ligand-drug conjugate of the general formula (Pc-LYD) as claimed in claim 1, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO: 7 and a light chain constant region as shown in SEQ ID NO:

8.

11. The ligand-drug conjugate of the general formula (Pc-LYD) as claimed in claim 1, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises: a heavy chain as shown in SEQ ID NO: 35 or SEQ ID NO: 42, and a light chain as shown in SEQ ID NO: 36 or SEQ ID NO: 39; or a heavy chain as shown in SEQ ID NO: 37 or SEQ ID NO: 49, and a light chain as shown in SEQ ID NO: 38 or SEQ ID NO:

46.

12. A ligand-drug conjugate of the general formula (Pc-LYD) as claimed in claim 1, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment comprises: (c) a heavy chain as shown in SEQ ID NO: 35 and a light chain as shown in SEQ ID NO: 36; (d) a heavy chain as shown in SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO: 45 and a light chain as shown in SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 41; (e) a heavy chain as shown in SEQ ID NO: 37 and a light chain as shown in SEQ ID NO: 38; or (f) a heavy chain as shown in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 52 and a light chain as shown in SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO:

48.

13. The ligand-drug conjugate of the general formula (Pc-LYD) as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the anti-Claudin18.2 antibody is selected from: h1901-11: comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 44, and a light chain as shown in SEQ ID NO: 41; or h1902-5: comprising a heavy chain with an amino acid sequence as shown in SEQ ID NO: 49, and a light chain as shown in SEQ ID NO:

47.

14. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in claim 1, wherein n is a decimal or integer from 2 to 8.

15. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in claim 1, wherein n is a decimal or integer from 3.5 to 4.

5.

16. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in claim 1, wherein Y is selected from: wherein the O end of Y is connected to the connector unit L.

17. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as claimed in claim 1, wherein the linker unit -L- is -L1-L2-L3-L4-, L1 is , s1 is an integer from 2 to 8; L2 is a chemical bond; L3 is a tetrapeptide residue; L4 is -NR5(CR6R7)t-, R5, R6 or R7 are the same or different and are each independently a hydrogen atom or an alkyl group, t is 1 or 2; wherein the L1 end is connected to Pc and the L4 end is connected to Y.

18. A ligand-drug conjugate of the general formula (Pc-LYD) as described in claim 17, or a pharmaceutically acceptable salt thereof, wherein L3 is a tetrapeptide residue of GGFG.

19. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-LYD) as described in claim 1, wherein -L- is:

20. A ligand-drug conjugate of the general formula (Pc-LYD) as described in claim 1, or a pharmaceutically acceptable salt thereof, wherein -LY- is optionally selected from:

21. A ligand-drug conjugate of the general formula (Pc-LYD) as described in claim 1, or a pharmaceutically acceptable salt thereof, wherein: W, L2, L3, R5, R6, R7, Pc, n, R1, R2, m are as defined in request item 1.

22. A ligand-drug conjugate of the general formula (Pc-LYD) as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein: s1 is an integer from 2 to 8; Pc, R1, R2, R5~R7, m and n are as defined in request item 21.

23. A ligand-drug conjugate of the general formula (Pc-LYD) as described in claim 1, or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate is selected from: wherein Pc and n are as defined in claim 1.

24. A ligand-drug conjugate of the general formula (Pc-LYD) as described in claim 1, or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate is selected from: where, n is as defined in request item 1; antibodies h1902-5 and h1901-11 are as defined in request item 13.

25. A method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as shown in the general formula (Pc-La-YD), comprising the following steps: Pc' is coupled with a compound of general formula (La-YD) to obtain a compound of general formula (Pc-La-YD); wherein: Pc is an anti-Claudin18.2 antibody or its antigen-binding fragment, wherein the anti-Claudin18.2 antibody or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region, wherein: iii) the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; or iv) the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and the light chain variable region contains sequences as shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 10, respectively. NO:20 shows LCDR1, LCDR2 and LCDR3; Pc' is obtained by reducing Pc; W, L2, L3, R1, R2, R5~R7, m and n are as defined in request item 21.

26. A pharmaceutical composition comprising a ligand-drug conjugate as described in any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers.

27. Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 24 in the preparation of a medicament for treating a disease or condition mediated by Claudin 18.

2.

28. The use as described in claim 27, wherein the Claudin18.2-mediated disease or condition is a cancer with high Claudin18.2 expression.

29. Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 24 in the preparation of a medicament for treating tumors and cancers, wherein the tumors and cancers are selected from: head and neck cancer, brain cancer, 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, gastrointestinal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, myelodysplastic syndrome, Kuckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma.

30. The use as described in claim 29, wherein the tumor and cancer are selected from: squamous cell carcinoma of the head and neck, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, gastric cancer, intestinal cancer, colon cancer, colorectal cancer, clear cell renal cell carcinoma, multiple myeloma, 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, large B-cell lymphoma rich in T-cells / histocytes, lymphoplasmacytic lymphoma, non-small cell lung cancer and small cell lung cancer, chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia.