Antibody-drug conjugate as well as preparation method and application thereof

Abstract

The invention provides an antibody-drug conjugate as well as a preparation method and application thereof. The antibody-drug conjugate comprises an antibody, an effective load with cytotoxicity and a connexon shown in a formula I, and the connexon shown in the formula I is in thioether bond connection with a sulfydryl part reduced by a disulfide chain between antibody chains through a succinimide group; the carbonyl group in the ester group of the linker is connected with the amino group in the effective load. The antibody-drug conjugate can be used for preparing a therapeutic agent for diagnosing, preventing and treating tumor diseases.

Description

Technical Field

[0001] The present disclosure relates to an antibody-drug conjugate which connects an antibody and an anti-tumor active drug or toxin through a linker and exerts an anti-tumor effect targeting tumor cells. Background Art

[0002] The statements in this section only provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] An antibody-drug conjugate (ADC) is a carrier-based chemotherapy that can selectively deliver an effective cytotoxic drug to tumors / cancer cells (Antibody-Drug Conjugates: The Last Decade, Nicolas Joubert et al., Pharmaceuticals (Basel). 2020 Sep 14;13(9):245.). With the development of drug delivery technology, the ADC targeted delivery technology can effectively overcome the side effects caused by the poor water solubility and insufficient tissue distribution of camptothecin drugs. The marketed ADC drugs Enhertu and Sacituzumab govitecan have superior effects in treating tumors, especially malignant tumors. Both Enhertu and Sacituzumab govitecan use a camptothecin derivative, a DNA topoisomerase inhibitor with stronger hydrophobicity than tubulin inhibitors (such as MMAE, MMAF), as the cytotoxic drug. Sacituzumab govitecan uses MCC-triazole spacer-PEG7-lysine-PABC as the linker and releases camptothecin SN38 in the cell lysosome (US13 / 948,732). Enhertu developed by AstraZeneca / Daiichi Sankyo uses a cathepsin B-activated GGFG (an amino acid sequence composed of glycine-glycine-phenylalanine-glycine connected by peptide bonds) tetrapeptide as the linker and introduces a self-cleaving structure to release the Exatecan derivative Dxd (Yusuke Ogitani et al., Clin Cancer Res (2016) 22(20):5097–5108.). However, the above cytotoxic drugs MMAE, SN38, and Dxd are all substrates of P-glycoprotein (P-gp) (Front Pharmacol 2019;10:749), and there may be a problem of drug resistance for some tumors with high expression of P-gp.

[0004] Therefore, there is still a need for a new antibody-drug conjugate for delivering cytotoxic drugs to tumors / cancer cells. Summary of the Invention

[0005] The present disclosure provides an antibody-drug conjugate, or an isomer, isotope variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, comprising an anti-MUC18 or anti-CD44v7 / 8 antibody or an antigen-binding fragment thereof, a payload, and a linker represented by Formula I,

[0006]

[0007] The linker represented by Formula I forms a thioether bond connection with the thiol group portion after reduction of the interchain disulfide bond of the antibody or its antigen-binding fragment through a succinimidyl group;

[0008] The carbonyl group in the ester group of the linker represented by Formula I is connected to the amino group in the payload;

[0009] R 1 and R 2 are each independently selected from hydrogen, methyl, or isopropyl;

[0010] R 3 represents -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 or a single bond, R 5 is selected from hydrogen or benzyl, n 1 represents an integer from 0 to 2, and n 2 represents an integer from 0 to 2;

[0011] R 4 represents methylamino or -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , and n 3 represents an integer from 1 to 20.

[0012] In some embodiments, R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , and n 3 represents an integer from 8 to 15.

[0013] In some embodiments, R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , and n 3Represents an integer from 10 to 12.

[0014] In some embodiments, R 3 represents -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 - or a single bond, R 5 is selected from benzyl, n 1 represents 1 or 2, n 2 represents 1 or 2, R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 8 to 15.

[0015] In some embodiments, R 3 represents a single bond; R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 8 to 15.

[0016] In some embodiments, R 3 represents a single bond; R 4 represents methylamino.

[0017] In some embodiments, the linker is selected from one of the following groups:

[0018]

[0019] or a combination thereof.

[0020] In some embodiments, the payload is selected from at least one of the group consisting of cytotoxic agents, markers, nucleic acids, radionuclides, hormones, immunomodulators, prodrug converting enzymes, ribonucleases, agonistic antibodies, antagonistic antibodies and fragments thereof, fusion proteins or derivatives.

[0021] In some embodiments, the cytotoxic agent includes a tubulin inhibitor and / or a topoisomerase inhibitor, the tubulin inhibitor includes auristatin or a derivative thereof, maytansine or a derivative thereof, and the topoisomerase inhibitor includes camptothecin and its derivatives.

[0022] In some embodiments, the payload is irinotecan as shown in Formula II, and the nitrogen atom of the amino group on the cyclohexane ring is connected to the linker.

[0023]

[0024] In some embodiments, the anti-MUC18 antibody or its antigen-binding fragment includes any one of the following structures:

[0025] Heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO:4, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO:16, heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO:28, and light chain CDR1 with the amino acid sequence shown in SEQ ID NO:38, light chain CDR2 with the amino acid sequence STS, light chain CDR3 with the amino acid sequence shown in SEQ ID NO:52;

[0026] Heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO:1, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO:11, heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO:23, and light chain CDR1 with the amino acid sequence shown in SEQ ID NO:34, light chain CDR2 with the amino acid sequence LAS, light chain CDR3 with the amino acid sequence shown in SEQ ID NO:46;

[0027] Heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO:1, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO:12, heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO:23, and light chain CDR1 with the amino acid sequence shown in SEQ ID NO:35, light chain CDR2 with the amino acid sequence LAS, light chain CDR3 with the amino acid sequence shown in SEQ ID NO:47;

[0028] Heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO:2, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO:13, heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO:24, and light chain CDR1 with the amino acid sequence shown in SEQ ID NO:36, light chain CDR2 with the amino acid sequence NAK, light chain CDR3 with the amino acid sequence shown in SEQ ID NO:48;

[0029] The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:3, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:14, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:25, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:37, the light chain CDR2 with the amino acid sequence FAS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:49;

[0030] The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:4, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:15, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:26, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:38, the light chain CDR2 with the amino acid sequence STS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:50;

[0031] The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:4, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:15, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:27, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:39, the light chain CDR2 with the amino acid sequence STS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:51;

[0032] The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:5, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:17, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:29, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:40, the light chain CDR2 with the amino acid sequence WAS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:53;

[0033] The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:6, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:18, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:29, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:41, the light chain CDR2 with the amino acid sequence WAS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:53;

[0034] The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:7, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:19, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:30, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:42, the light chain CDR2 with the amino acid sequence RTS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:54;

[0035] The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:8, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:20, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:31, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:43, the light chain CDR2 with the amino acid sequence WAS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:55;

[0036] The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:9, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:21, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:32, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:44, the light chain CDR2 with the amino acid sequence WAS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:56; and

[0037] The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:10, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:22, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:33, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:45, the light chain CDR2 with the amino acid sequence LMS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:57.

[0038] In some embodiments, the anti-MUC18 antibody or its antigen-binding fragment comprises:

[0039] The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:84, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:85;

[0040] The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:86, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:87; or

[0041] The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:89, or a conservative variant thereof.

[0042] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof specifically binds to a human CD44v7 / 8 binding peptide, and the binding peptide comprises the amino acid sequence shown in SEQ ID NO:90.

[0043] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof does not bind to a human CD44 v7 / 8 binding peptide having the amino acid sequence shown in SEQ ID NO:91 and / or SEQ ID NO:92.

[0044] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises: HCDR1 of the amino acid sequence shown in SEQ ID NO:93, HCDR2 of the amino acid sequence shown in SEQ ID NO:94, HCDR3 of the amino acid sequence shown in SEQ ID NO:95; and LCDR1 of the amino acid sequence shown in SEQ ID NO:96, LCDR2 having the amino acid sequence RAN, LCDR3 of the amino acid sequence shown in SEQ ID NO:97.

[0045] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises:

[0046] The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:100, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:101; or

[0047] The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:100, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:104, or a conservative variant thereof.

[0048] In some embodiments, the DAR is 1 to 10. In some embodiments, the DAR is 4 to 10.

[0049] The present application provides a method for preparing the above-mentioned antibody-drug conjugate, or an isomer, isotope variant, pharmaceutically acceptable salt, prodrug, solvate thereof or a combination thereof, comprising the following steps: the anti-MUC18 antibody or CD44v7 / 8 antibody or antigen-binding fragment thereof is reductively treated so that at least part of the disulfide bonds are reduced, and reacted with the 3-position carbon atom of the maleimide-N-group of the linker shown in formula III.

[0050]

[0051] In the linker-payload, the carbonyl group in the ester group of the linker shown in Formula III is linked to the amino group of the payload.

[0052] R 1 , R 2 Each independently selected from hydrogen, methyl or isopropyl;

[0053] R 3 represents -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 - or a single bond, R 5 is selected from hydrogen or benzyl, n 1 represents an integer from 0 to 2, n 2 represents an integer from 0 to 2;

[0054] R 4 represents methylamino or -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 1 to 20.

[0055] In some embodiments, the method further comprises: reacting the antibody or its antigen-binding fragment with a reducing agent in a buffer solution containing a chelating agent, adding a solution of the linker-payload of the linker shown in Formula III, and adjusting the pH of the reaction solution.

[0056] In some embodiments, the payload is irinotecan shown in Formula II, and the nitrogen atom of the amino group on the cyclohexane ring is linked to the carbonyl group of the ester group in Formula III.

[0057]

[0058] In some embodiments, the DAR is from 1 to 10, and optionally, from 4 to 10.

[0059] The present disclosure provides a pharmaceutical composition comprising the above-mentioned antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate thereof or a combination thereof, and a pharmaceutically acceptable excipient.

[0060] The present disclosure provides a kit comprising the above-mentioned antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate thereof or a combination thereof.

[0061] The present disclosure provides the above-mentioned antibody-drug conjugate, the antibody-drug conjugate prepared by the above method, and the use of the pharmaceutical composition or kit containing the above antibody-drug conjugate in the manufacture of a therapeutic agent for diagnosing, preventing and treating tumor diseases. In some embodiments, the tumor diseases include benign and malignant tumors expressing MUC18 and / or CD44v7 / 8.

[0062] In some embodiments, the tumor diseases include melanoma, pharyngeal cancer, triple-negative breast cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, urothelial cancer, bladder neuroendocrine tumor, small cell lung cancer, non-small cell lung cancer, cutaneous squamous cell carcinoma, cholangiocarcinoma, metastatic pancreatic cancer, squamous cell carcinoma of the lung, squamous cell carcinoma of the head and neck, and / or esophageal squamous cell carcinoma.

[0063] The present disclosure provides the above-mentioned antibody-drug conjugate, the antibody-drug conjugate prepared by the above method, and the use of the pharmaceutical composition or kit containing the above antibody-drug conjugate in the preparation of a therapeutic agent targeting MUC18 and / or CD44v7 / 8.

[0064] The present disclosure provides a method for diagnosing, preventing and treating neoplastic diseases, which comprises administering to a subject a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent comprises the above-mentioned antibody-drug conjugate, the antibody-drug conjugate prepared by the above method, or the above pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1A Showing the size exclusion chromatogram of 02-1 naked antibody; Figure 1B Showing the size exclusion chromatogram of Hu1H2-2 naked antibody.

[0066] Figure 2A Showing the hydrophobic chromatography chromatogram of 02-1 naked antibody; Figure 2B Showing the hydrophobic chromatography chromatogram of Hu1H2-2 naked antibody.

[0067] Figure 3A Showing the size exclusion chromatogram in the antibody-drug conjugate 02-1-LP1 prepared in Example 5; Figure 3B Showing the hydrophobic chromatography detection map of the antibody-drug conjugate 02-1-LP1.

[0068] Figure 4A Showing the size exclusion chromatogram of the antibody-drug conjugate Hu1H2-2-LP1 prepared in Example 6; Figure 4B Showing the hydrophobic chromatography detection map of the antibody-drug conjugate Hu1H2-2-LP1.

[0069] Figure 5AShows the size exclusion chromatogram of the antibody-drug conjugate Hu1H2-2-LP1 prepared in Example 7; Figure 5B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Hu1H2-2-LP1.

[0070] Figure 6A Shows the size exclusion chromatogram of the antibody-drug conjugate Hu1H2-2-LP2 prepared in Example 8; Figure 6B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Hu1H2-2-LP2.

[0071] Figure 7A Shows the size exclusion chromatogram of the antibody-drug conjugate Hu1H2-2-LP3 prepared in Example 9; Figure 7B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Hu1H2-2-LP3.

[0072] Figure 8A Shows the size exclusion chromatogram of the antibody-drug conjugate 02-1-vc-MMAE prepared in Comparative Example 1; Figure 8B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate 02-1-vc-MMAE.

[0073] Figure 9A Shows the size exclusion chromatogram of the antibody-drug conjugate Rituximab-vc-MMAE prepared in Comparative Example 2; Figure 9B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Rituximab-vc-MMAE.

[0074] Figure 10A Shows the size exclusion chromatogram of the antibody-drug conjugate Rituximab-LP1 prepared in Comparative Example 3; Figure 10B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Rituximab-LP1.

[0075] Figure 11A Shows the size exclusion chromatogram of the antibody-drug conjugate Human IgG-DXD prepared in Comparative Example 4; Figure 11B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Human IgG-DXD.

[0076] Figure 12A Shows the size exclusion chromatogram of the antibody-drug conjugate Human IgG-LP1 prepared in Comparative Example 5; Figure 12B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Human IgG-LP1.

[0077] Figure 13AShows the size exclusion chromatogram of the antibody-drug conjugate Human IgG-LP1 prepared in Comparative Example 6; Figure 13B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Human IgG-LP1.

[0078] Figure 14A Shows the size exclusion chromatogram of the antibody-drug conjugate Human IgG-LP2 prepared in Comparative Example 7; Figure 14B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Human IgG-LP2.

[0079] Figure 15A Shows the size exclusion chromatogram of the antibody-drug conjugate Human IgG-LP3 prepared in Comparative Example 8; Figure 15B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Human IgG-LP3.

[0080] Figure 16A Shows the size exclusion chromatogram of the antibody-drug conjugate Hu1H2-2-DXD prepared in Comparative Example 9; Figure 16B Shows the hydrophobic chromatography detection map of the antibody-drug conjugate Hu1H2-2-DXD.

[0081] Figure 17 Shows the time-fluorescence intensity statistical chart of the endocytosis of the antibody-drug conjugate 02-1-LP1 by A375 cells, HMVII cells, SK-MEL-2 cells and GAK cells.

[0082] Figure 18A1 Shows the tumor volume-time change curve of melanoma model mice treated with the antibody-conjugated drugs Rituximab-vc-MMAE, 02-1-vc-MMAE, Rituximab-LP1 and 02-1-LP1; Figure 18A2 Is a partial view of Figure 16A1, showing the tumor volume-time change curve of 02-1-LP1, 02-1-vc-MMAE and the solvent group.

[0083] Figure 18B1 Shows the body weight-time change curve of melanoma model mice treated with the antibody-conjugated drugs Rituximab-vc-MMAE, 02-1-vc-MMAE, Rituximab-LP1 and 02-1-LP1; Figure 18B2 Is Figure 18B1 A partial view of, showing the body weight-time change curve of 02-1-LP1, 02-1-vc-MMAE and the solvent group.

[0084] Figure 19Shows the in vitro killing curve of the antibody-drug conjugate against the head and neck squamous cell carcinoma cell line Detroit562; Figure 19A1 Is Figure 19 A partial view showing the in vitro killing curves of Hu1H2-2-LP2, Hu1H2-2-LP1, and Hu1H2-2-DXD; Figure 19A2 Is Figure 19 A partial view showing the in vitro killing curves of Human IgG-LP2, Human IgG-LP1, and Human IgG-DXD.

[0085] Figure 20 Shows the tumor volume-time change curve graph of the head and neck squamous cell carcinoma model mice after receiving the antibody-drug conjugates Hu1H2-2-LP1, Hu1H2-2-LP2, and Human IgG-LP1.

[0086] Figure 21 Shows the body weight-time change curve graph of the head and neck squamous cell carcinoma model mice after receiving the antibody-drug conjugates Hu1H2-2-LP1, Hu1H2-2-LP2, and Human IgG-LP1;

[0087] Figure 22 Shows the tumor volume-time change curve graph of the lung cancer model mice after receiving the antibody-drug conjugates Hu1H2-2-LP1, Hu1H2-2-LP1 (DAR4), Hu1H2-2-LP3, Human IgG-LP1, Human IgG-LP1 (DAR4), and Human IgG-LP3.

[0088] Figure 23 Shows the body weight-time change curve graph of the lung cancer model mice after receiving the antibody-drug conjugates Hu1H2-2-LP1, Hu1H2-2-LP1 (DAR4), Hu1H2-2-LP3, Human IgG-LP1, Human IgG-LP1 (DAR4), and Human IgG-LP3.

[0089] Figure 24A Shows the tumor volume-time change curve graph of the SCC-9 head and neck squamous cell carcinoma CDX model after receiving the antibody-drug conjugates Rituximab-LP1 and 02-1-LP1. Figure 24B Shows the body weight-time change curve graph of the SCC-9 head and neck squamous cell carcinoma CDX model after receiving the antibody-drug conjugates Rituximab-LP1 and 02-1-LP1.

[0090] Figure 25AShows the tumor volume-time change curve of the Huh-7 human liver cancer CDX model after treatment with the antibody-drug conjugate Human IgG-LP1 and 02-1-LP1. Figure 25B Shows the body weight-time change curve of the Huh-7 human liver cancer CDX model after treatment with the antibody-drug conjugate Human IgG-LP1 and 02-1-LP1.

[0091] Figure 26A Shows the tumor volume-time change curve of the LD1-0015-200617 human esophageal squamous cell carcinoma PDX model after treatment with the antibody-drug conjugate IgG-LP1 and 02-1-LP1. Figure 26B Shows the body weight-time change curve of the LD1-0015-200617 human esophageal squamous cell carcinoma PDX model after treatment with the antibody-drug conjugate Human IgG-LP1 and 02-1-LP1.

[0092] Figure 27A Shows the tumor volume-time change curve of the LD1-0016-390730 human esophageal adenocarcinoma PDX model after treatment with the antibody-drug conjugate 02-1-LP1. Figure 27B Shows the body weight-time change curve of the LD1-0016-390730 human esophageal adenocarcinoma PDX model after treatment with the antibody-drug conjugate 02-1-LP1.

[0093] Figure 28A Shows the tumor volume-time change curve of the LD1-2025-362797 human small cell lung cancer PDX model after treatment with the antibody-drug conjugate 02-1-LP1. Figure 28B Shows the body weight-time change curve of the LD1-2025-362797 human small cell lung cancer PDX model after treatment with the antibody-drug conjugate 02-1-LP1.

[0094] Figure 29A Shows the tumor volume-time change curve of the LD1-2009-362263 human triple-negative breast cancer PDX model after treatment with the antibody-drug conjugate 02-1-LP1. Figure 29B Shows the body weight-time change curve of the LD1-2009-362263 human triple-negative breast cancer PDX model after treatment with the antibody-drug conjugate 02-1-LP1.

[0095] Figure 30A Shows the tumor volume-time change curve of the LD1-0060-200770 human cholangiocarcinoma PDX model after treatment with the antibody-drug conjugate 02-1-LP1. Figure 30BShows the weight-time change curve of the LD1-0060-200770 human cholangiocarcinoma PDX model after treatment with the antibody-drug conjugate 02-1-LP1.

[0096] Figure 31A Shows the tumor volume-time change curve of the OV-10-0073 human ovarian cancer PDX model after treatment with the antibody-drug conjugates Human IgG-LP1 and 02-1-LP1. Figure 31B Shows the weight-time change curve of the OV-10-0073 human ovarian cancer PDX model after treatment with the antibody-drug conjugates Human IgG-LP1 and 02-1-LP1. Detailed implementation mode

[0097] Unless otherwise specified, all numbers representing content, concentration, ratio, mass, volume, time, temperature, thickness, technical effect, etc. used in this specification and claims shall be understood to be modified by the term "about" or "substantially" in any case. Therefore, unless there is a contrary indication, the numerical parameters listed in the following specification and appended claims are approximate values. For those skilled in the art, they can vary according to the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant digits and the conventional rounding method or the way understood by those skilled in the art.

[0098] Although the numerical ranges and parameters of the broad scope of this disclosure are approximate values, the values set forth in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain certain errors, which are necessarily caused by the standard deviations found in their corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range that falls within that broader numerical range, as if these narrower numerical ranges were all expressly written herein.

[0099] In an ADC molecule, the linker, as the connecting structure between the antibody and the payload, is a key factor in the successful construction of an ADC molecule. In terms of pharmacokinetics (PK) / pharmacodynamics (PD) and the therapeutic window, the molecular design and properties of the linker are key determinants of the efficacy of an ADC. To exert optimal potency, an ideal linker should possess the following characteristics: (1) The linker is sufficiently stable in plasma so that the ADC molecule can circulate in the bloodstream and localize to the tumor site without premature cleavage. An unstable linker will cause premature release of the toxic payload and damage non-target healthy cells, resulting in systemic toxicity and adverse reactions. (2) The linker needs to have the ability to be rapidly cleaved so that once the ADC is internalized into the target tumor cells, the free toxic payload can be rapidly released. (3) The linker design also needs to consider hydrophobicity. The combination of a hydrophobic linker and a hydrophobic payload usually promotes the aggregation of the ADC molecule, which is not only unfavorable for the efficacy of the ADC but may also lead to liver toxicity or trigger unwanted immune responses (Kyoji Tsuchikama et al., Antibody-drug conjugates: recent advances in conjugation and linker chemistrie, Protein Cell. 2018 Jan; 9(1):33-46).

[0100] Antibody-drug conjugate

[0101] The present disclosure provides an antibody-drug conjugate (ADC), or an isomer, isotope variant, pharmaceutically acceptable salt, prodrug, solvate thereof, or a combination thereof, comprising an antibody or an antigen-binding fragment thereof, a payload, and a linker represented by Formula I,

[0102]

[0103] The linker represented by Formula I forms a thioether bond connection with the thiol moiety after reduction of the interchain disulfide bond of the antibody or its antigen-binding fragment through a succinimidyl group;

[0104] The carbonyl group in the ester group of the linker represented by Formula I is connected to the amino group in the payload;

[0105] In Formula I, R 1 , R 2 are each independently selected from hydrogen, methyl, or isopropyl;

[0106] R 3 represents -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 -, or a single bond, R 5Selected from hydrogen or benzyl, n 1 represents an integer from 0 to 2, n 2 represents an integer from 0 to 2;

[0107] R 4 represents methylamino or -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 1 to 20.

[0108] The term "antibody-drug conjugate" or "ADC" refers to a conjugate of an anti-MUC18 / CD44v7 / 8 antibody or an antigen-binding fragment thereof covalently linked to a payload as described herein. Generally, an antibody-drug conjugate can include an antibody or an antigen-binding fragment thereof, a payload, and optionally a linker between the antibody or an antibody-binding fragment and the payload. An ADC can provide a therapeutic effect by delivering the payload to MUC18 and / or CD44v7 / 8 cells targeted by the antibody or an antigen-binding fragment thereof, particularly MUC18 or CD44v4 / 8 tumor cells. Antibody-drug conjugates can be prepared by various methods known in the art for preparing antibody-drug conjugates.

[0109] As used herein, "antibody" refers to a polypeptide of the immunoglobulin (Ig) family that binds to an antigen. For example, a naturally occurring IgG-type "antibody" is a tetramer that comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region consists of one domain. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen.

[0110] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including diabody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain Fv (scFv), scFv dimer (bivalent diabody), multispecific antibody formed by a part of an antibody, the antibody including one or more CDRs, camelized single-domain antibody, nanobody, domain antibody, bivalent domain antibody or any other antibody fragment that binds to an antigen but does not include the complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen as the parental antibody or parental antibody fragment (e.g., parental scFv).

[0111] The term "isomer" refers to compounds with the same molecular formula but different structures, also known as structural isomers, and generally includes structural isomers and stereoisomers. Structural isomers refer to isomers caused by different connection orders or bonding properties of atoms in a molecule, preferably including tautomers. Tautomers refer to functional group isomers produced by the rapid movement of atoms between two positions in a molecule. Stereoisomers refer to isomers caused by the atoms or atomic groups in a molecule being connected to each other in the same order and bonds but having different spatial arrangements, preferably including optical isomers. Optical isomers refer to stereoisomers with different optical properties due to the absence of an anti-axis of symmetry in the molecule, such as enantiomers, diastereomers, racemates, and mesomers.

[0112] The term "prodrug" refers to a compound obtained by changing the chemical structure of a drug, which is inactive or has low activity in vitro and releases the active drug through enzymatic or non-enzymatic conversion in vivo to exert its pharmacological effect. In this article, the prodrug can be an ADC molecule or a payload.

[0113] The term "payload" includes compounds that are cytotoxic or capable of killing cells after being released from an antibody-drug conjugate, compounds with radioactive labels, radionuclides or polypeptides, fluorophores, chromophores, imaging agents and / or metal ions as detection labels or for cell killing, compounds, nucleic acids, polypeptides or proteins, enzymes, hormones or nucleic acids that regulate the immune activity of the body (including activation or inhibition).

[0114] In some ideal cases, the conjugated payload in an antibody-drug conjugate has little or negligible cytotoxicity such that administration of the ADC at an effective therapeutic dose does not cause systemic toxic reactions in the subject due to the conjugated payload. The payload can be a drug that has been clinically validated for treating a specific disease, or a compound, radionuclide, nucleic acid, protein, or polypeptide having acceptable pharmacological activity under clinical use conditions.

[0115] In the present disclosure, the terminal succinimidyl group shown in Formula I forms a thioether bond connection with the thiol moiety after reduction of the interchain disulfide bond of the antibody or its antigen-binding fragment. The succinimidyl group is which forms a thioether bond connection with the thiol moiety after reduction of the interchain disulfide bond of the antibody or its antigen-binding fragment at the 3-position. In the present disclosure, the bond with in the structural formula represents a chemical bond connecting to other groups.

[0116] In the present disclosure, the disulfide bonds of the antibody or its antigen-binding fragment include interchain disulfide bonds and intrachain disulfide bonds. Preferably, the interchain disulfide bonds are treated, for example, activated to become thiol groups and then bonded to the linker. The amino acids that form a chemical bond with the succinimidyl group in the linker in the antibody or its antigen-binding fragment include one or a combination of lysine, histidine, tyrosine, and cysteine. Optionally, the chemically bound amino acid in the antibody or its antigen-binding fragment is cysteine. In some embodiments, the linker shown in Formula I can be connected to the hinge region, variable region, and / or constant region of the antibody.

[0117] In some embodiments, in the linker shown in Formula I, R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 1 to 20. The n 3 can be selected as any integer from, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0118] In some embodiments, R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 8 to 15. In some embodiments, R 4 represents -(NCH 3 COCH2 )n 3 -NCH 3 COCH 3 ,n 3 represents an integer from 10 to 12. In some embodiments, R 4 represents methylamino.

[0119] In the present disclosure, the linker shown in Formula I contains a hydrophilic amino group R 4 , which contains a poly(sarcosine) group or methylamino, increasing the hydrophilicity of the antibody-drug conjugate. The introduction of the hydrophilic amino group is beneficial to improving the hydrophilicity of the ADC conjugated with the hydrophobic payload. The increase in the hydrophilicity of the ADC molecule helps to reduce the aggregation of the ADC molecule during the preparation process, thereby improving the stability, homogeneity and purity of the antibody-drug conjugate.

[0120] In some embodiments, in the linker shown in Formula I, R 3 represents a single bond.

[0121] In some embodiments, R 3 represents -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 -, R 5 is benzyl, n 1 represents an integer from 1 to 2, n 2 represents an integer from 1 to 2.

[0122] In some embodiments, R 3 represents -CR 5 HCONH-, -CH 2 CONH-, -CR 5 HCONH-CH 2 CONH-, -(CR 5 HCONH) 2 -CH 2 CONH-, -CR 5 HCONH-(CH 2 CONH) 2 -, or, -(CR 5 HCONH) 2 -(CH 2 CONH) 2 -; R 5 is benzyl.

[0123] In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is isopropyl.

[0124] In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is methyl.

[0125] In some embodiments, the linker in the antibody-drug conjugate is selected from the group

[0126]

[0127] and

[0128]

[0129] In some embodiments, the payload in the antibody-drug conjugate is a label containing a radioactive label, fluorophore, chromophore, imaging agent, and / or metal ion as a detection label, and the label includes, but is not limited to, a chemically synthesized organic compound, radionuclide, metal complex, or polypeptide. Among them, a radioactive label refers to a labeled compound in which one or several atoms of a compound molecule are replaced by a radionuclide so that it can be recognized and used as a tracer, including amino acids, polypeptides, proteins, saccharides, nucleotides, nucleosides, purines, pyrimidines, steroids, lipid compounds, and tumor antigens, hormones, receptors, vitamins, and drugs for medical research. Radionuclides are usually nuclides that can spontaneously emit rays, including, but not limited to, tritium, iodine-125, iodine-131, sulfur-35, phosphorus-32, and carbon-14. A fluorophore is usually a group containing conjugated double bonds, and when the molecule returns from the excited state to the ground state, the fluorophore emits fluorescence. A chromophore refers to an unsaturated group and its related chemical bonds contained in a molecule that can absorb light radiation and have a transition. An imaging agent usually refers to a radiopharmaceutical that can perform organ, tissue, or molecular imaging after being introduced into the body in nuclear medicine.

[0130] In some embodiments, the payload in the antibody-drug conjugate is a nucleic acid, and the nucleic acid can be ribonucleic acid and / or deoxyribonucleic acid.

[0131] In some embodiments, the payload in the antibody-drug conjugate is a hormone, growth factor, clotting factor, fibrinolytic protease (such as a prodrug converting enzyme that can convert a prodrug into an active drug, ribonuclease).

[0132] In some embodiments, the payload in the antibody-drug conjugate is an immunomodulator (including cytokines and chemokines that have an impact on immunity), an agonistic antibody or antagonistic antibody with biological activity.

[0133] In some embodiments, the payload in the antibody-drug conjugate is a cytotoxic compound. In some embodiments, the payload in the antibody-drug conjugate has anti-tumor activity or is an anti-tumor drug. The payload is selected from DNA topoisomerase inhibitors or from tubulin inhibitors. The DNA topoisomerase inhibitor can be a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0134] In the present disclosure, the term "topoisomerase inhibitor" generally refers to a compound that inhibits the activity of topoisomerase. A compound that is a topoisomerase I inhibitor is active against topoisomerase I, and a topoisomerase II inhibitor is active against topoisomerase II. Some compounds are active against both topoisomerase I and topoisomerase II and are referred to as topoisomerase I / II inhibitors.

[0135] The term "tubulin inhibitor" generally refers to a compound that inhibits the eukaryotic cell microtubule system, interferes with cell division, and inhibits cell proliferation.

[0136] In some embodiments, the payload is camptothecin or its derivatives having topoisomerase inhibitory activity. The term "derivative" refers to a compound formed by replacing an atom or group of atoms in the molecule of a parent compound with other atoms or groups of atoms, and is called a derivative of the parent compound. The term "camptothecin and its derivatives" generally includes camptothecin and camptothecin derivatives. Camptothecin exerts its pharmacological effect by irreversibly inhibiting topoisomerase I. The camptothecin derivatives include exatecan, irinotecan, topotecan, lurtotecan, silatecan, etirinotecan pegol, TAS 103, 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10,11-methylenedioxycamptothecin, 9-amino-10,11-methylenedioxycamptothecin, 9-chloro-10,11-methylenedioxycamptothecin, 7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin, 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-camptothecin, 7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin, etc., and their stereoisomers, salts and esters. Methods for synthesizing camptothecin and camptothecin analogs or derivatives are known and are summarized and described in U.S. Patent No. 5,244,903, which is incorporated herein by reference in its entirety.

[0137] In some embodiments, the payload is auristatin or its derivative having tubulin inhibitory effect, maytansine or its derivative. The term "auristatin and its derivatives" generally includes auristatin F and auristatin F derivatives, and the auristatin F derivatives include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). The term "maytansine and its derivatives" generally includes maytansine and maytansine derivatives, and the maytansine derivatives include maytansine DM1, maytansine DM2 and maytansine DM4.

[0138] In some embodiments, the payload is exatecan, a camptothecin derivative, which can act on the entire cell cycle as a topoisomerase inhibitor and has strong penetrability and good efficacy against slow-growing solid tumors. Moreover, the number of intracellular targets is much lower than that of tubulin inhibitors, and better killing effects can be achieved when the ADC molecule carries the same amount of payload into the cell. The exatecan molecule is not a substrate of P-gp, which is beneficial to reducing or alleviating the problem of drug resistance.

[0139] In some embodiments, the payload is camptothecin represented by formula II, which is connected to the linker through the nitrogen atom of the amino group on the cyclohexane ring.

[0140]

[0141] The structure of the exatecan molecule is rigid and has poor hydrophilicity. When it is connected to the commonly used GGFG tetrapeptide linker in the prior art to prepare ADC, it is easy to cause aggregation between ADC molecules, which does not meet the development requirements of ADC drugs (Bioorg. Med. Chem. Lett. 26 (2016) 1542-1545). Therefore, the selection and matching of the linker and the payload have an impact on the safety and stability of ADC drugs.

[0142] Without being bound by any theory, in the antibody-drug conjugate provided by the present disclosure, since there are multiple hydrophilic groups in the linker, the hydrophilicity in the linker-payload structure is improved, and the aggregation, precipitation and other phenomena of ADC molecules caused by the hydrophobic payload can be reduced to a certain extent.

[0143] After the ADC molecule is endocytosed into cells, compounds of the linker-payload structure are released according to whether the linker is degraded or the linker (or part of the linker)-payload structure. In some embodiments, after the amino group on the cyclohexane ring of irinotecan shown in Formula II is bonded to the carbonyl group in the ester group of the linker shown in Formula I, a linker-payload structure including a carbamate is formed. Without being bound by any theory, in the linker-payload structure provided by the present disclosure, after the ADC molecule is endocytosed into cells, the linker is enzymatically cleaved by cathepsin (e.g., Cathepsin B) to form an intermediate or active metabolite shown in Formula IV below,

[0144]

[0145] R 4 represents methylamino or -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 1 to 20.

[0146] Subsequently, the PABC (para-aminobenzyloxycarbonyl) group in the intermediate or active metabolite shown in Formula IV undergoes 1,6-elimination and releases irinotecan (see, Angew. Chem. Int. Ed. 2015, 54, 7492-7509.). Therefore, the linker-payload structure in the ADC molecule provided by the present disclosure has good in vivo stability and biological activity.

[0147] The cleavage site in the linker-payload structure, without being bound by any theory, can be an amide bond in the linker, for example, the amide bond between the carbon atom where the substituent represented by R 2 is located and the group represented by R 3 , or the amide bond in the group represented by R 3 .

[0148] In some embodiments, in the antibody-drug conjugate, the ratio of the number of conjugated payload molecules to each molecule of antibody or antigen binding (drug-antibody ratio or DAR) is 1 to 10. In some embodiments, the DAR is 1 to 2, 2 to 4, 4 to 6, 2 to 8, 4 to 8, 4 to 10, 6 to 10, 7 to 10 or 8 to 10, and exemplary DAR values are 4, 6, 7.8, 9.2 or 9.92.

[0149] DAR represents the average number of conjugated payloads or drug molecules per antibody molecule, i.e., the average number of conjugated drug molecules. In an antibody-drug conjugate, DAR is a key factor affecting its efficacy and safety. The production of an antibody-drug conjugate is carried out by specifying reaction conditions such as the amounts of starting materials and reagents used in the reaction to achieve a constant number of conjugated payload molecules. Usually, a mixture containing different numbers of conjugated payload molecules is obtained when preparing an antibody-drug conjugate. Unless otherwise specified, DAR in the present disclosure is defined as the average value, i.e., the average number of conjugated payloads or drug molecules.

[0150] In some embodiments, the antibody-drug conjugate comprises any one of the following structures:

[0151]

[0152]

[0153] Ab represents an antibody or an antigen-binding fragment; n is the same as DAR.

[0154] The antibody or its antigen-binding fragment is linked to the linker through a reduced reactive thiol group. Optionally, the disulfide bond in the hinge region of the antibody or its antibody-binding fragment is reduced to a reactive thiol group and then linked to the linker.

[0155] In some embodiments, the antibody or its antigen-binding fragment in the antibody-drug conjugate targets MUC18. MUC18, also known as CD146 or melanoma cell adhesion molecule (MCAM), is a transmembrane glycoprotein that mainly functions in cell adhesion. It is expressed at detectable levels in endothelial cells within vascular tissues (including vascular smooth muscle). Notably, MUC18 is overexpressed in human malignant melanoma, especially in metastatic lesions and advanced primary tumors.

[0156] In the present disclosure, the anti-MUC18 antibody or its antigen-binding fragment comprises a heavy chain (H) and a light chain (L), and the CDRs of the heavy chain and the light chain are shown in Table 1.

[0157] Table 1: CDR sequences (numbered according to the IMGT scheme)

[0158]

[0159]

[0160] As used herein, the term "CDR" or "complementary determining region" refers to the non - contiguous antigen - binding sites within the variable regions of the heavy and / or light chains. CDR residue numbering follows the IMGT nomenclature, see Lefranc M.P. et al., Dev. Comp. Immunol., 27:55 - 77(2003), and when compared to each other, the definitions include overlapping or subsets of amino acid residues. However, the use of any of the definitions to refer to the CDRs of an antibody or its antigen - binding fragment or its grafted antibody or variant is intended to be within the scope of the terms defined and used herein.

[0161] In some embodiments, the anti - MUC18 antibody or its antigen - binding fragment comprises the heavy - chain CDRs and light - chain CDRs of the antibodies numbered CL070336, CL070335, CL070333, CL070319, CL070321, CL070320, CL070324, CL070341, CL070350, CL070349, CL070348, CL070370 or J253.

[0162] In some embodiments, the anti - MUC18 antibody or its antigen - binding fragment comprises an HCDR1 (heavy - chain CDR1) having the amino - acid sequence shown in SEQ ID NO:4, an HCDR2 (heavy - chain CDR2) having the amino - acid sequence shown in SEQ ID NO:16, an HCDR3 (heavy - chain CDR3) having the amino - acid sequence shown in SEQ ID NO:28; and an LCDR1 (light - chain CDR1) having the amino - acid sequence shown in SEQ ID NO:38, a light - chain CDR2 with the amino - acid sequence STS, and an LCDR3 (light - chain CDR3) having the amino - acid sequence shown in SEQ ID NO:52.

[0163] In some embodiments, the anti - MUC18 antibody or its antigen - binding fragment comprises a heavy - chain variable region (VH) having at least 85% or 90% identity to any of the VH sequences of the reference antibody sequences shown in Table 2, and / or a light - chain variable region (VL) having at least 85% or 90% identity to any of the VL sequences of the reference antibody sequences shown in Table 2.

[0164] The identity can be determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. A BLAST protein search can be performed with the BLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to the protein molecule of interest. In the case where there are gaps between two sequences, Gapped BLAST can be employed as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0165] In some embodiments, there are no mutations in the framework region or there are mutations that do not affect the binding of the antibody variable region to the antigen, which mutations can increase the binding affinity of the antibody to the antigen or remain substantially unchanged. In some embodiments, the anti-MUC18 or CD44v7 / 8 antibody or its antigen-binding fragment further comprises conservatively modified variants, which conservatively modified variants include single substitutions, deletions or additions to the polypeptide sequence, which result in the substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. Such conservatively modified variants are complementary to polymorphic variants, interspecies homologs and alleles and do not exclude these variants. The following eight groups contain amino acids that are conservatively substituted for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence.

[0166] Table 2 Reference Antibody Sequences

[0167]

[0168] In some embodiments, the anti-MUC18 antibody or its antigen-binding fragment comprises a VH sequence having at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identity to the VH sequences shown in Table 3; and a VL sequence having at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identity to the VL sequences shown in Table 3. The antibodies shown in Table 3 are the humanized sequences of CL070324 and J253, respectively.

[0169] Table 3 Humanized Antibody Sequences

[0170] Antibody number VH VL Humanized CL070324(02-1) SEQ ID NO:84 SEQ ID NO:85 Humanized hJ253-03-1 SEQ ID NO:86 SEQ ID NO:87 Humanized hJ253-03-7 SEQ ID NO:88 SEQ ID NO:89

[0171] In some embodiments, the anti-MUC18 antibody or its antigen-binding fragment comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO:84, and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:85.

[0172] In some embodiments, the anti-MUC18 antibody or its antigen-binding fragment is a humanized CL070324, humanized hJ253-03-1, or humanized hJ253-03-7, or a conservative variant thereof.

[0173] Among them, the CDR sequences and antibody variable region sequences of the anti-MUC18 antibodies shown in SEQ ID NOs: 1-89 are described in US Patent US2022041749 (SEQ ID NOs: 8-90, SEQ ID NOs: 1-6), which is incorporated herein by reference in its entirety.

[0174] In some embodiments, the antibody or antigen-binding fragment targets CD44v7 / 8. CD44, also known as HCAM (homing cell adhesion molecule), Pgp-1 (phagocytic glycoprotein-1), Hermes antigen, lymphocyte homing receptor, ECM-III, or HUTCH-1, is a cell surface glycoprotein that functions mainly in cell adhesion and cell-cell interaction. Several splice variants of CD44 are known, including CD44 v7 / 8, which is a variant containing exon 7 and exon 8. CD44 is a protein receptor for hyaluronic acid and has further been shown to bind to or otherwise interact with osteopontin, collagen, matrix metalloproteinases, and other similar ligands.

[0175] In some embodiments, the anti-CD44 v7 / 8 antibody or its antigen-binding fragment specifically binds to a binding peptide comprising the amino acid sequence QAGRRMDMDSSHSIT (SEQ ID NO:90) in human CD44v7 / 8.

[0176] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof does not bind to a binding peptide of the amino acid sequences PIS HPMGRGHQAGRR (SEQ ID NO:91) and / or SHSITLQPTANPNTG (SEQ ID NO:92) in human CD44 v7 / 8.

[0177] In some embodiments, the heavy chain of the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises: heavy chain CDR1 of the amino acid sequence as shown in SEQ ID NO:93, heavy chain CDR2 of the amino acid sequence as shown in SEQ ID NO:94, and heavy chain CDR3 of the amino acid sequence as shown in SEQ ID NO:95; the light chain comprises: light chain CDR1 of the amino acid sequence as shown in SEQ ID NO:96, CDR2 with the amino acid sequence RAN, and light chain CDR3 of the amino acid sequence as shown in SEQ ID NO:97. The CDR sequences of the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof are shown in Table 4:

[0178] Table 4 CDR sequences (numbered according to the IMGT scheme)

[0179]

[0180] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises a VH having at least 85% or 90% identity with the VH of the reference antibody sequence shown in Table 5, and / or a VL having at least 85% or 90% identity with the VL of the reference antibody sequence shown in Table 5.

[0181] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises a VH having at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identity with the VH of HIS1H2-2a or HIS1H2-2 of the reference antibody humanized sequence shown in Table 5; and a VL having at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identity with the VL sequence of HIS1H2-2a or HIS1H2-2.

[0182] Table 5 Light / heavy chain variable region sequences of anti-CD44 v7 / 8 antibody

[0183] Antibody number VH VL Reference antibody HIS1H2-2 SEQ ID NO:98 SEQ ID NO:99 Humanized antibody HIS1H2-2a SEQ ID NO:100 SEQ ID NO:101 Humanized antibody HIS1H2-2 SEQ ID NO:100 SEQ ID NO:104

[0184] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 100, and a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 101 or SEQ ID NO: 104, or a conservative variant thereof.

[0185] The binding peptide sequence, non-binding peptide sequence, CDR sequence, and antibody variable region sequences of the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof shown in SEQ ID NOs: 90-101 have been described in PCT patent application WO2020159754 (SEQ ID NOs: 2-14), which is incorporated herein by reference in its entirety.

[0186] The antibody-drug conjugate provided by the present disclosure exhibits excellent killing activity against tumor cells, and extremely high killing activity against tumor cells with high expression of MUC18 or CD44v7 / 8.

[0187] The antibody-drug conjugate provided by the present disclosure exhibits excellent anti-tumor activity. In some embodiments, the antibody-drug conjugate exhibits excellent anti-tumor activity against subjects suffering from neoplastic diseases with different MUC18 expression levels (low, medium, high). The expression level of MUC18 can be measured by the H-Score method known to those skilled in the art. The score range is 0-300, and the higher the score, the higher the expression. For example, low (H-Score = 10-99), medium (H-Score = 100-99), and high (H-Score = 200-300). In addition, the antibody-drug conjugate does not show obvious adverse reactions in vivo.

[0188] It should be noted that the antibody-drug conjugate of the present disclosure may absorb moisture, retain adsorbed water, or form a hydrate due to being placed in the atmosphere or recrystallized. Such water-containing compounds and salts are also included in the present disclosure. In addition, the present disclosure also includes isotopically variant compounds labeled with various radioactive or non-radioactive isotopes. One or more of the atoms constituting the antibody-drug conjugate of the present disclosure may contain non-natural proportions of atomic isotopes. Examples of atomic isotopes include deuterium (2H), tritium (3H), iodine-125 (125I), or carbon-14 (14C), etc. In addition, the compounds of the present disclosure can be radioactively labeled with radioactive isotopes such as tritium (3H), iodine-125 (125I), or carbon-14 (14C). The radioactively labeled compounds are used as therapeutic or prophylactic agents, research reagents such as assay reagents, and diagnostic agents such as in vivo imaging diagnostic agents. All isotopically variant forms of the antibody-drug conjugate of the present disclosure, whether radioactive or not, are included within the scope of the present disclosure.

[0189] Preparation method

[0190] The present disclosure provides a preparation method of an antibody-drug conjugate, or an isomer, isotope variant, pharmaceutically acceptable salt, prodrug, solvate thereof or a combination thereof, and a pharmaceutically acceptable excipient, comprising the following steps: after the antibody or its antigen-binding fragment is reductively treated, at least part of the interchain disulfide bonds are reduced, and it reacts with the 3-position carbon atom of the maleimide-N-group of the linker in the linker-payload shown in Formula III,

[0191]

[0192] the carbonyl group in the ester group of the linker shown in Formula III is connected to the amino group of the payload in the antibody-drug conjugate;

[0193] R 1 and R 2 are each independently selected from hydrogen, methyl or isopropyl;

[0194] R 3 represents -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 - or a single bond, R 5 is selected from hydrogen or benzyl, n 1 represents an integer from 0 to 2,

[0195] and n 2 represents an integer from 0 to 2;

[0196] R 4 represents methylamino or -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 and n 3 represents an integer from 1 to 20.

[0197] In an antibody-drug conjugate, the interchain disulfide bonds are reduced to thiol groups, which then react with the reactive groups of the linker of the linker-payload represented by Formula III. In many practical cases, the linker of the structure shown in Formula III with the payload is attached to the same antibody or its antigen-binding fragment molecule having reactive thiol groups. In some embodiments, the antibody or its antigen-binding fragment reacts with a reducing agent such as dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to form reactive thiol groups from the disulfide bonds on the antibody or its antigen-binding fragment. The amount of the reducing agent used can be 0.3 to 10 times the equivalent of the molar amount of the antibody or its antigen-binding fragment. For example, it can be 1 to 10, 3 to 10, 5 to 10, 7 to 10 times the molar equivalent of the antibody or its antigen-binding fragment.

[0198] In some embodiments, the method further includes: reacting the antibody or its antigen-binding fragment with a reducing agent in a buffer solution containing a chelating agent, and then adding a solution of the linker-payload for reaction. The linker-payload is specifically a compound formed by bonding the linker shown in Formula III and the payload, wherein the amino group (primary amino group) in the payload is connected to the carbonyl group in the ester group of the linker shown in III. In the linker-payload, the payload is selected from the payloads described in the [antibody-drug conjugate] section. The term "chelating agent" refers to a substance that can bind to a metal atom or ion through a coordination bond to form a complex with a cyclic structure.

[0199] In some embodiments, the reducing agent reacts with the antibody or its antigen-binding fragment in a buffer solution containing a chelating agent to obtain an antibody or its antigen-binding fragment in which the interchain disulfide bonds are partially or completely reduced. The chelating agent includes, but is not limited to, ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). The use concentration of the chelating agent is 1 mM to 20 mM. For example, it can be 2 mM to 20 mM, 5 mM to 20 mM, 8 mM to 20 mM, 1 mM to 15 mM, or 1 mM to 10 mM. The components of the buffer solution can be buffer salts commonly used in the art, such as sodium phosphate, sodium borate, sodium acetate, or similar buffer salts.

[0200] The reaction of the antibody or its antigen-binding fragment with the reducing agent is carried out under adjusted pH conditions. In some embodiments, the pH of the solution when the antibody or its antigen-binding fragment reacts with the reducing agent is 5 to 9, and can be optionally pH 6 to 8, pH 6 to 7, pH 6.5 to 7.5, or pH 7 to 8. For example, the reaction is carried out when the solution is about pH 7. Chemical substances with acidity or alkalinity can be used to adjust the pH value of the solution. Exemplary chemical substances with acidity or alkalinity include acetic acid, hydrochloric acid, phosphoric acid, sulfuric acid, sodium bicarbonate, sodium carbonate, sodium hydroxide, and triethylamine.

[0201] The reaction of the antibody or its antigen-binding fragment with the reducing agent is carried out under regulated temperature conditions. Exemplary reaction temperatures are -10 to 40 °C, -10 to 10 °C, 5 to 40 °C, 10 to 40 °C, 25 to 40 °C, 30 to 40 °C, 35 to 38 °C, such as about 37 °C.

[0202] The linker-payload can be dissolved in an organic solvent selected from any one or a combination of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP).

[0203] In some embodiments, the solution of the linker-payload is added in an amount of 1% to 20% by volume to the buffer solution that has been reductively treated or contains an antibody or its antigen-binding fragment with a reactive thiol group, based on the volume of the buffer solution of the antibody or its antigen-binding fragment. In some embodiments, the volume ratio of the added solution of the linker-payload is 1 to 20%, 2 to 20%, 5 to 20%, 10 to 20%, 15 to 20%, 1 to 18%, 1 to 15%, 1 to 13%, 1 to 10%, or 5 to 15%, based on the volume of the buffer solution of the antibody or its antigen-binding fragment.

[0204] In some embodiments, the molar dosage ratio of the linker-payload to the antibody or its antigen-binding fragment is 4 to 20, optionally 8 to 20. In some embodiments, the molar dosage ratio of the linker-payload to the antibody or its antigen-binding fragment is 10 to 20, 14 to 20, 16 to 20, or 18 to 20.

[0205] In some embodiments, the temperature at which the antibody or its antigen-binding fragment reacts with the linker-payload is -10 to 40 °C or 0 to 37 °C. In some embodiments, the reaction temperature is -10 to 10 °C, 5 to 40 °C, 5 to 37 °C, 10 to 37 °C, 10 to 25 °C, or 15 to 30 °C.

[0206] In some embodiments, the reaction time of the antibody or its antigen-binding fragment with the linker-payload is 0.5 to 2 hours. In some embodiments, the reaction time of the antibody or its antigen-binding fragment with the linker-payload is 0.5 to 1.75 hours, 0.5 to 1.5 hours, 0.5 to 1.25 hours, 0.75 to 2 hours, or 1 to 2 hours.

[0207] The reaction can be terminated by inactivating the reactivity of the unreacted linker-payload using a thiol-containing reagent. The thiol-containing reagents include, but are not limited to, cysteine or N-acetyl-(L)-cysteine (NAC). More specifically, a thiol-containing reagent in a molar equivalent of 1 to 2 times the molar equivalent of the linker-payload is added to the reaction solution and incubated at room temperature (10 - 30 °C) for 10 - 30 minutes to terminate the reaction.

[0208] In the case where the antibody or antigen-binding fragment has a thiol group, the antibody-drug conjugate can also be obtained by reacting compounds using known methods (for example, it can be obtained by the methods described in the patent publication US2016 / 297890 (for example, it can be obtained by the methods described in paragraphs

[0336] to

[0374] )). The antibody or its antigen-binding fragment having a thiol group can be obtained by methods well-known to those skilled in the art (Hermanson, G.T, Bioconjugate Techniques, pp. 56 - 136, pp. 456 - 493, Academic Press (1996)).

[0209] The antibody-drug conjugate provided by the present disclosure can be obtained by the above preparation method. In some embodiments, the prepared antibody-drug conjugate is subjected to purification treatment, and the purification treatment includes, but is not limited to, gel filtration, for example, purification using a gel column.

[0210] Kit

[0211] The present disclosure provides a kit, which includes the above antibody-drug conjugate, or its isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates, or combinations thereof. The kit may further include instructions for the use of the antibody-drug conjugate provided by the present disclosure in the methods of the present disclosure, for example, instructions for use in the method of treating neoplastic diseases.

[0212] The kit may further include a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., single-chamber or dual-chamber syringes), and test tubes. The container can be formed of various materials such as glass or plastic. The container contains a formulation. The container containing the formulation can be a disposable vial or a multi-use vial, allowing repeated administration of the reconstituted formulation.

[0213] The kit may further include a label or a package insert, which is on or related to the container and can indicate the instructions for reconstitution and / or use of the formulation. The label or the package insert may further indicate that the formulation is for or intended for subcutaneous, intravenous (e.g., intravenous infusion), or other administration modes for treating a neoplastic disease (e.g., cancer) of a subject. The kit may also include other materials required from commercial, therapeutic, and user perspectives, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0214] Drug composition

[0215] The present disclosure provides a drug composition comprising the antibody-drug conjugate described above, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate thereof, or a combination thereof, and a pharmaceutically acceptable excipient.

[0216] The antibody-drug conjugate or the drug composition of the present disclosure can select a suitable administration mode according to the specific applicable form, physicochemical characteristics, etc. of the pharmaceutically acceptable excipient. In some embodiments, the drug composition can be formulated in the form of a lyophilized preparation or a liquid preparation, and the preparation may contain appropriate formulation additives in the art. For example, in the above drug composition, typically, it contains more than one pharmaceutical carrier, such as a sterilized liquid, such as water and oil (including oils of petroleum, animal, plant, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)). In the case of intravenous administration of the above drug composition, water is a more representative carrier. In addition, saline solution, glucose aqueous solution, and glycerol solution can also be used as liquid carriers, especially for injection solutions. Appropriate pharmaceutical excipients are known in the art. Optionally, the above composition may also contain trace amounts of wetting agents or emulsifiers, or pH buffering agents. The administration mode of the drug composition is generally parenteral administration, which can be intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous injection, but is not limited to these. For example, the drug can be administered by infusion or bolus injection. See, for example, the Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), the entire content of which is incorporated herein by reference. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), the entire content of which is incorporated herein by reference.

[0217] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the antibody-drug conjugate of the present application, or a pharmaceutical composition containing the antibody-drug conjugate and at least one other therapeutic agent (such as a cancer therapeutic agent). In some embodiments, the antibody-drug conjugate of the present disclosure may also be administered together with other cancer therapeutic agents to enhance the anti-cancer effect. Other anti-cancer agents used for this purpose may be administered to an individual simultaneously with, separately from, or sequentially with the antibody-drug conjugate, and the administration intervals may also be varied. Exemplary other cancer therapeutic agents include paclitaxel, cisplatin, vinblastine, etc., and any agent with anti-tumor activity is not limited.

[0218] Regarding the present disclosure, the active agent or the pharmaceutical composition containing the active agent may be administered to a subject by any suitable administration route. For example, the active agent may be administered to a subject by parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following discussion of administration routes is provided only to illustrate various embodiments and should not be construed as limiting the scope in any way.

[0219] In some embodiments, the antibody-drug conjugate of the present disclosure is administered to a subject. As used herein, the term "subject" refers to humans and non-human animals. Non-human animals include all vertebrates, such as mice, rabbits, cats, dogs, pigs, monkeys, chimpanzees, gorillas, etc. In some examples, the subject is a human.

[0220] Method of Use

[0221] The present disclosure provides the use of an antibody-drug conjugate, an antibody-drug conjugate prepared by the above method, a pharmaceutical composition comprising the antibody-drug conjugate, or a kit provided herein in the preparation of a therapeutic agent for diagnosing, preventing, and treating neoplastic diseases.

[0222] Neoplastic diseases include benign tumors and malignant tumors (such as cancer). In some embodiments, the benign tumors and malignant tumors express MUC18 and / or CD44v7 / 8.

[0223] The type of neoplastic disease to which the antibody-drug conjugate is applied is not limited to the above cancer cells, as long as it is a cancer cell expressing a protein recognizable by the antibody or its antigen-binding fragment of the antibody-drug conjugate. In some embodiments, the neoplastic disease is a solid tumor expressing MUC18 and / or CD44v7 / 8.

[0224] In some embodiments, the neoplastic disease includes melanoma, pharyngeal cancer, triple-negative breast cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, urothelial cancer, bladder neuroendocrine tumor, small cell lung cancer, non-small cell lung cancer, cutaneous squamous cell carcinoma, cholangiocarcinoma, metastatic pancreatic cancer, lung squamous cell carcinoma, head and neck squamous cell carcinoma, and / or esophageal squamous cell carcinoma.

[0225] The present disclosure provides the use of an antibody-drug conjugate, an antibody-drug conjugate prepared by the above method, a pharmaceutical composition comprising the antibody-drug conjugate, or a kit in the preparation of a therapeutic agent targeting MUC18 and / or CD44v7 / 8.

[0226] The present disclosure provides methods for diagnosing, preventing, and treating neoplastic diseases, including administering to a subject a therapeutically effective amount of the above therapeutic agent.

[0227] As used herein, the term "therapeutically effective amount" refers to the amount of the active ingredient of the ADC described herein sufficient to induce the desired effect, including but not limited to the treatment of the diseases defined herein. The therapeutic dose of the antibody-drug conjugate varies depending on factors such as the specific condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, build, gender, and weight), the duration of treatment, the nature of any combination therapy (if any), the specific route of administration, and the knowledge of the health care practitioner. In some embodiments, the dose of the above antibody-drug conjugate can be determined empirically in an individual who has received one or more administrations of the antibody or its antigen-binding fragment. In some embodiments, an acceptable therapeutic dose of the antibody-drug conjugate is 0.1 to 30 mg / kg, 0.5 to 30 mg / kg, 1 to 30 mg / kg, 1 to 25 mg / kg, 0.1 to 25 mg / kg, 0.1 to 20 mg / kg, 1 to 20 mg / kg, or 0.5 to 20 mg / kg. In some embodiments, the dosing frequency is once every 12 hours, once a day, once a week, once every 2 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, or once every 10 weeks; or once a month, once every 2 months, or once every 3 months, or longer. The therapeutic dose and dosing frequency can be changed with the change of the treatment regimen.

[0228] The various embodiments and preferences of the present disclosure can be combined with each other as long as they are not inherently contradictory to each other, and all the various embodiments formed by such combination are regarded as part of the disclosure of this application.

[0229] The technical solutions of the present disclosure will be more clearly and specifically illustrated below in the form of examples by way of illustration. It should be understood that these examples are only for the purpose of illustration and are by no means intended to limit the protection scope of the present disclosure. The protection scope of the present disclosure is only defined by the claims.

[0230] Example

[0231] The present disclosure is specifically illustrated by the following examples, but the present disclosure is not limited by them. In addition, no restrictive interpretation is made in any way for these examples. In addition, in this specification, reagents, solvents and starting materials not specifically described can be easily obtained from commercially available sources.

[0232] Example 1: Preparation of Compound LP-1

[0233]

[0234] Step 1: Synthesis of Intermediate 11-1

[0235] In a mixed solution of Compound 11-1A (Mc-Val-Ala-OH, purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., 2.4 g, 6.29 mmol) and Compound 11-1B (3.18 g, 6.29 mmol), DCM (dichloromethane):MeOH (methanol) (v:v = 2:1, 90 mL) and EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline) (1.86 g, 7.55 mmol) were added at room temperature. The reaction solution was stirred at room temperature for 24 hours, the solvent was removed under vacuum, and the crude residue was further purified by flash chromatography to obtain Compound 11-1 (3.9 g, 71%). LC-MS (ESI, m / z): 868.49 (M+H).

[0236] Step 2: Synthesis of Intermediate 11-2

[0237] Compound 11-1 (2 g, 2.3 mmol) was dissolved in anhydrous tetrahydrofuran THF (50 mL), hydrogen fluoride-pyridine (4.6 g, 46 mmol) was added at 0 °C under an argon atmosphere, and then the reaction mixture was stirred at 0 °C for 2 hours. The reaction was quenched by adding water, extracted with DCM, the organic phase was dried and concentrated. The residue was purified by silica gel chromatography to obtain Compound 11-2 (1.1 g, 76%). LC-MS (ESI, m / z): 630.31 (M+H).

[0238] Step 3: Synthesis of Intermediate 11-3

[0239] Compound 11-2 (700 mg, 1.11 mmol) was dissolved in anhydrous N,N-dimethylformamide (DMF, 4 mL). Under argon atmosphere, N,N-diisopropylethylamine (DIPEA, 0.39 mL, 2.23 mmol) and 4,4'-dinitrophenyl carbonate (406 mg, 1.33 mmol) were added at room temperature. Then the reaction mixture was stirred overnight at ambient temperature. The solvent was removed by concentration to obtain a methyl tert-butyl ether (MTBE) precipitated product. The yellow solid was collected by filtration, washed with diethyl ether, and dried to obtain Compound 11-3. LC-MS (ESI, m / z): 795.41 (M+H).

[0240] Step 4: Synthesis of Intermediate 11-4

[0241] Compound 11-3 (300 mg, 0.44 mmol) was dissolved in anhydrous DMF (4 mL), dry pyridine (1 mL) was added, and then irinotecan mesylate (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., 234 mg, 0.44 mmol) and 1-hydroxybenzotriazole (HOBt, 60 mg, 0.44 mmol) were added. The reaction mixture was stirred overnight at room temperature under argon. The product was purified by pre-HPLC to obtain Intermediate 11-4 (230 mg, 48%). LC-MS (ESI, m / z): 1091.53 (M+H).

[0242] Step 5: Synthesis of Intermediate 11-5

[0243] Compound 11-4 (200 mg, 0.183 mmol) was dissolved in 1 mL of anhydrous DCM. 300 μL of TFA (trifluoroacetic acid) was added at 0 °C. The reaction mixture was stirred at room temperature for 30 min. The solvent was removed by concentration to obtain the TFA salt of Intermediate 11-5, which was used in the next step without further purification. LC-MS (ESI, m / z): 991.47 (M+H).

[0244] Step 6: Synthesis of Compound LP-1

[0245] Compound 11-5 (120 mg, 0.109 mmol) was dissolved in 1 mL of anhydrous DMF, Ac-Sar10-COOH (N-Acetyl Decasarcosine, 84 mg, 0.109 mmol) was added, then 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU (50 mg, 0.130 mmol) and DIPEA (38 μL, 0.22 mmol) were added. The reaction mixture was stirred overnight at room temperature, the solvent was removed by concentration, and the crude product was purified by preparative high performance liquid chromatography (pre-HPLC) to obtain Compound LP-1 (74 mg, 38%). LC-MS (ESI, m / z): 1743.85 (M+H).

[0246] Example 2: Preparation of Compound LP-2

[0247]

[0248] The synthesis of Compound LP-2 was carried out according to the procedure of Compound LP-1, replacing the starting material 11-1A with Mc-GGFG-OH (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., GGFG represents an amino acid sequence consisting of glycine-glycine-phenylalanine-glycine connected by peptide bonds) to obtain Compound LP-2, which is a beige amorphous solid. LC-MS (ESI, m / z): 1891.90 (M+H).

[0249] Example 3: Preparation of Compound LP-3

[0250]

[0251] Compound LP-3 is an intermediate of LP-1. Remove step 6, and intermediate 11-5 is LP-3.

[0252] Example 4: Detection method for antibody-drug conjugate

[0253] The antibody-drug conjugate was concentrated, buffer exchanged, purified, and the antibody concentration was measured and the average number of drug molecules carried by each antibody was calculated to identify the antibody-drug conjugate according to the following method.

[0254] Operation A: Concentration of antibody or antibody-drug conjugate

[0255] Take an ultrafiltration tube (Amicon Ultra, 50000 MWCO, Millipore), add the solution of the antibody or antibody-drug conjugate to be concentrated, and centrifuge until the volume of the antibody or antibody-drug conjugate solution reaches the required volume and then take it out.

[0256] Operation B: Measurement of antibody concentration

[0257] Using a microplate reader (Multiskan GO, Thermo Fisher Scientific), measure the absorbance value of the antibody according to the method defined by the manufacturer. The ratio of this absorbance value to the absorption coefficient of the antibody at this wavelength is the concentration of the antibody.

[0258] Operation C: Antibody buffer exchange

[0259] According to the instructions provided by the manufacturer (Thermo Fisher Scientific), pre-equilibrate the Zeba desalting column (5 mL, 40K MWCO) with phosphate buffer containing sodium chloride (50 mM) and EDTA (2 mM) (referred to as "PBS7.0 / EDTA", 50 mM, pH 7.0). Load 2 mL onto each Zeba desalting column, centrifuge (1000 g, 4 minutes), collect the flow-through fraction and concentrate it by Operation A, measure the antibody concentration by Operation B, and adjust the antibody concentration with PBS7.0 / EDTA.

[0260] Operation D: Purification of antibody-drug conjugate

[0261] According to the instructions provided by the manufacturer (Thermo Fisher Scientific), pre-equilibrate the Zeba desalting column (5 mL, 40K MWCO) with the storage buffer. The storage buffer is either histidine-acetate buffer containing 150 mM sodium chloride (20 mM histidine, pH 5.5) or phosphate buffer containing 50 mM sodium chloride (50 mM, pH 7.0). Add the reaction solution containing the antibody-drug conjugate (about 2 mL) to the Zeba desalting column, centrifuge (1000 g, 4 minutes), and collect the flow-through fraction (about 2 mL). Repeat the elution process twice to remove unbound linker-payload and low molecular weight compounds including reducing agents.

[0262] Operation E: Measurement of the antibody concentration and the number of drug molecules conjugated per antibody (DAR value) in the antibody-drug conjugate - (1)

[0263] The drug concentration conjugated to the antibody-drug conjugate can be obtained by measuring the ultraviolet absorbance values of the antibody-drug conjugate aqueous solution at 280 nm and 370 nm and calculating using the following formula.

[0264] At any given wavelength, the total absorbance of a system is equal to the sum of the absorbances of all the absorbing chemical substances present in the system (additivity of absorbance). Therefore, assuming that the molar absorption coefficients of the antibody and the drug remain unchanged before and after conjugation, the antibody concentration and drug concentration in the antibody-drug conjugate can be expressed by the following formula.

[0265] A 280 = A D,280+ A A,280 = ε D,280 C D + ε A,280 C A Equation (1)

[0266] A 370 = A D,370 + A A,370 = ε D,370 C D + ε A,370 C A Equation (2)

[0267] A 280 represents the total absorbance value of the aqueous solution of the antibody-drug conjugate at 280 nm, A 370 represents the total absorbance value of the aqueous solution of the antibody-drug conjugate at 370 nm. A A,280 represents the absorbance value of the antibody at 280 nm, A A,370 represents the absorbance value of the antibody at 370 nm, A D,280 represents the absorbance value of the conjugate precursor (drug) at 280 nm, A D,370 represents the absorbance value of the conjugate precursor at 370 nm, ε A,280 represents the molar extinction coefficient of the antibody at 280 nm, ε A,370 represents the molar extinction coefficient of the antibody at 370 nm, ε D,280 represents the molar extinction coefficient of the conjugate precursor at 280 nm, ε D,370 represents the molar extinction coefficient of the conjugate precursor at 370 nm, C A represents the concentration of the antibody in the antibody-drug conjugate, C D represents the concentration of the drug molecules in the antibody-drug conjugate.

[0268] In this case, ε A,280 , ε A,370 , ε D,280 and ε D,370 are all known values (calculated from the sequence of the antibody or measured by UV absorption of the compound). For example, ε A,280 can be calculated by a known method using the amino acid sequence of the antibody (Protein Science, 1995, Vol. 4, pp. 2411-2423). The antibody usually has no absorption at 370 nm, so ε A,370 is usually 0. ε D,280 and ε D,370The value can be calculated by measuring the absorbance changes of the binding precursor at 280 nm and 370 nm with different concentrations and using the Lambert-Beer law (Absorbance = Molar concentration × Molar extinction coefficient × Optical path length). C A and C D The absorbance values A 280 and A 370 of the antibody-drug conjugate at 280 nm and 370 nm can be measured, and then the binary linear equations (1) and (2) can be solved to obtain the value. Additionally, by dividing C D by C A , the number of drug molecules conjugated to each antibody (DAR value) can be obtained.

[0269] Operation F: Number of drug molecules conjugated to each antibody (DAR value) - (2)

[0270] The number of drug molecules conjugated to each antibody molecule in the antibody-drug conjugate, in addition to the aforementioned "Operation E", can also be determined by liquid chromatography-mass spectrometry (LC-MS) analysis method as follows:

[0271] Preparation of high performance liquid chromatography-mass spectrometry (LC-MS) analysis sample (reduction of antibody-drug conjugate)

[0272] Add 3 μL of 100 mM reducing agent dithiothreitol (DTT) and 21 μL of deionized water to the antibody-drug conjugate (about 5 mg / mL, 6 μL). The reaction solution is incubated in a water bath at 37 °C for 30 minutes, and the disulfide bonds between the heavy and light chains and between the heavy chains in the antibody-drug conjugate are completely opened. The obtained sample can be used for LC-MS detection and analysis.

[0273] High performance liquid chromatography parameters

[0274] Column model: Agilent PLRP-S, 50×2.1 mm, 8 μm

[0275] Detection wavelength: 280 nm

[0276] Band width: 4 nm

[0277] Column temperature: 80 °C

[0278] Sample tray temperature: 5 °C

[0279] Flow rate: 0.5 mL / min

[0280] Injection volume: 5 μL

[0281] Mobile phase A: 0.05% TFA, H 2 O

[0282] Mobile phase B: 0.05% TFA, ACN

[0283] Elution program (B%: 25% - 34% (0 - 0.7 min), 34% - 45% (0.7 - 5 min), 45% - 90% (5 - 6 min), 90% (6 - 7 min), 90% - 25% (7 - 7.10 min), 25% (7.10 - 10 min)

[0284] MS parameters

[0285] Nebulizer gas temperature: 350 °C

[0286] Nebulizer gas flow: 13 L / min

[0287] Sprayer: 45 psig

[0288] Capillary voltage: 5000 V

[0289] Fragmentation voltage: 350 V

[0290] m / z range: 500 - 8000 m / z

[0291] Acquisition rate: 1 spectra / s

[0292] Data analysis

[0293] The light chain coupled with i drug molecules is denoted as Li, and the heavy chain coupled with i small molecules is denoted as Hi, which can be determined according to the ESI scan of the mass spectrum.

[0294] Substituting the peak area percentage (%) of each chain into the following formula can obtain the corrected peak area ratio.

[0295] Peak area ratio of the light chain coupled with i drug molecules = 100% × A Li / (A L0 + A L1 )

[0296] Peak area ratio of the heavy chain coupled with i drug molecules = 100% × A Hi / (A H0 + A H1 + A H2 + A H3 )

[0297] The average number of drug molecules conjugated to each antibody can be calculated by the following formula.

[0298] Average number of conjugated drug molecules = (peak area ratio of L0 × 0 + peak area ratio of L1 × 1 + peak area ratio of H0 × 0 + peak area ratio of H1 × 1 + peak area ratio of H2 × 2 + peak area ratio of H3 × 3) × 2

[0299] Operation G: Number of drug molecules conjugated to each antibody (DAR value) - (3)

[0300] For the number of drug molecules conjugated to each antibody molecule in an antibody-drug conjugate, in addition to being measurable by the aforementioned "Operation E" and "Operation F", it can also be determined by the following hydrophobic interaction chromatography (HIC) analysis method.

[0301] The elution of the antibody-drug conjugate on the hydrophobic chromatography column is separated based on the difference in the salt ion concentration in the eluent - as the salt ion concentration decreases, the number of bound small molecule drugs in the eluted antibody-drug conjugate increases, that is, the antibody-drug conjugate with a low DAR value is preferentially eluted. The elution order of each component is D0 (antibody not conjugated with any linker-payload), D2 (antibody conjugated with 2 linkers-payload), D4 (antibody conjugated with 4 linkers-payload), D6 (antibody conjugated with 6 linkers-payload), D8 (antibody conjugated with 8 linkers-payload). The percentage content of each component can be obtained by measuring the proportion of the peak area of each peak. Then the calculation method of the HIC-DAR of the corresponding sample is as follows:

[0302] Average number of conjugated drug molecules = Peak area ratio of D0 × 0 + Peak area ratio of D2 × 2 + Peak area ratio of D4 × 4 + Peak area ratio of D6 × 6 + Peak area ratio of D8 × 8

[0303] Operation H: Measurement of aggregates in antibody-drug conjugates

[0304] The aggregates in the antibody-drug conjugate are detected using size exclusion chromatography in high performance liquid chromatography, and the method is as follows:

[0305] High performance liquid chromatography system: Agilent 1260 Infinity II HPLC system

[0306] Detector: Ultraviolet absorption spectrometer (Detection wavelength: 280 nm)

[0307] Column model: TOSOH TSKgel G3000SWXL (7.8 × 300 mm, 5 μm)

[0308] Mobile phase: 200 mmol / L KHPO 4 , 150 mmol / L NaCl, 15% (v / v) isopropanol, pH 7.0

[0309] Flow rate: 0.75 mL / min

[0310] Analysis time: 18 min

[0311] Column temperature: room temperature

[0312] Sample injection volume: 50 μg

[0313] Data analysis:

[0314] The size exclusion chromatogram of the quality control product (QC, 02-1 naked antibody, i.e., the antibody numbered 02-1 without conjugated linker-payload) is as Figure 1A shown; the size exclusion chromatogram of the quality control product (QC, Hu1H2-2 naked antibody, i.e., the antibody numbered hu1H2-2 without conjugated linker-payload) is as Figure 1B shown, and the aggregate content of the Hu1H2-2 naked antibody is 1.17%. The retention time of the main peak (single peak) of the 150 kDa quality control product is between 9.5 - 10.5 minutes. The retention time of the aggregate should be earlier than that of the above monomer.

[0315] Operation I: Comparison of the hydrophobicity of antibody-drug conjugates

[0316] The hydrophobicity of the antibody-drug conjugate was analyzed using high performance liquid chromatography hydrophobic interaction chromatography (HIC) as follows:

[0317] High performance liquid chromatography system: Agilent 1260 Infinity II HPLC system

[0318] Detector: Ultraviolet absorption spectrometer (detection wavelength: 280 nm)

[0319] Column model: TOSOH TSKgel Butyl-NPR (4.6 mm I.D.×3.5 cm, 2.5 μm)

[0320] Mobile phase A: mol / L (NH 4 ) 2 SO 4 , 50 mmol / L KHPO 4 , pH 7.0

[0321] Mobile phase B: 50 mmol / L KHPO 4 , 25% (v / v) isopropanol, pH 7.0

[0322] Analysis time: 25 min

[0323] Column temperature: room temperature

[0324] Elution program (B%): 0% - 25% (0 - 1 minute), 25% (1 - 3 minutes), 25% - 80% (3 - 13 minutes), 80% (13 - 17 minutes), 80% - 0% (17 - 17.10 minutes), 0% (17.10 - 25 minutes)

[0325] Sample injection volume: 10 μL

[0326] Data analysis: The hydrophobic chromatography of the quality control product (QC, 02 - 1 naked antibody) is as Figure 2A shown. The hydrophobic chromatography of the quality control product (QC, Hu1H2 - 2 naked antibody) is as Figure 2B shown, and its retention time is 3.387 min. The shorter the retention time of a sample, the weaker its hydrophobicity. Compared with the naked antibody without conjugated drug molecules, the antibody - drug conjugate has higher hydrophobicity, so its retention time is longer.

[0327] Example 5: Preparation of antibody - drug conjugate 02 - 1 - LP1 (DAR 8)

[0328] Reduction of antibody: By operation B (the extinction coefficient of the antibody at 280 nm is 1.523 mL mg - 1 cm -1 ) and operation C in Example 4, the antibody was exchanged with PBS7.0 / EDTA, and the antibody concentration after exchange was 23.2 mg / mL. To 2.155 mL of the aqueous solution of 02 - 1 antibody, 0.482 mL of 5 mM TCEP solution (equivalent to 7 - fold equivalent of the antibody content) was added, and at the same time, 4.31 mL of 50 mM phosphate buffer (pH 7.0, PBS7.0) and 69.47 μL of 200 mM EDTA solution were added. After confirming that the solution pH was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours.

[0329] The VH sequence of the 02 - 1 antibody is the amino acid sequence shown in SEQ ID NO:84, and the VL sequence is the amino acid sequence shown in SEQ ID NO:85. For the purpose of example preparation and / or detection, the constant region of the 02 - 1 antibody uses the human IgG1 constant region sequence, and the heavy - chain constant region sequence selects the amino acid sequence shown in SEQ ID NO:102; the light - chain constant region sequence selects the amino acid sequence shown in SEQ ID NO:103.

[0330] Antibody-conjugate payload coupling: The above mixture was incubated at 4 °C for 10 minutes. The conjugate payload LP-1 prepared in Example 1 was dissolved in N,N-dimethylacetamide (DMA) and then added to the mixture. The added volume was 0.489 mL (equivalent to 14.2-fold equivalent of the antibody content). The mixture was further reacted at 22 °C for 30 minutes.

[0331] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method of Operation D in Example 4 to obtain the antibody-drug conjugate 02-1-LP1.

[0332] Characterization of the antibody-drug conjugate: The obtained antibody-drug conjugate was characterized by Operation E (ε D,280 = 6384 and ε D,370 = 16180), Operation F, Operation H, and Operation I.

[0333] The concentration of the antibody-drug conjugate measured and calculated by Operation E was 12.13 mg / mL, and the average number of conjugate payloads per antibody measured and calculated by Operation E was 7.78. The average number of conjugate payloads per antibody measured by Operation F was 7.80. Figure 3A The detection spectrum showing aggregates was presented. The aggregate content of the antibody-drug conjugate 02-1-LP1 measured by Operation H was 1.18%. Figure 3B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate 02-1-LP1 was presented. The retention time of the antibody-drug conjugate 02-1-LP1 measured by Operation I was 6.499 min.

[0334] Example 6: Preparation of the antibody-drug conjugate Hu1H2-LP1 (DAR 8)

[0335] Reduction of the antibody: By Operation B (the extinction coefficient of the antibody at 280 nm was 1.54 mL mg -1 cm -1 ) and Operation C in Example 4, the antibody was exchanged with PBS7.0 / EDTA. The concentration of the antibody after the exchange was 9.37 mg / mL. To 640.34 μL of an aqueous solution of Hu1H2-2 (HIS1H2-2) antibody, 80 μL of a 5 mM TCEP solution (equivalent to 10-fold equivalent of the antibody content) was added, along with 0.2 mL of 50 mM PBS7.0 and 79.66 μL of deionized water. After confirming that the solution pH was 7.0 ± 0.1, the mixture was reacted at 37 °C for 2 hours.

[0336] The VH sequence of the Hu1H2-2 antibody is the amino acid sequence shown in SEQ ID NO: 100, and the VL sequence is the amino acid sequence shown in SEQ ID NO: 104. For the purpose of preparation and / or detection in the examples, the heavy chain constant region sequence of the Hu1H2-2 antibody is selected as the amino acid sequence shown in SEQ ID NO: 102; the light chain constant region sequence is selected as the amino acid sequence shown in SEQ ID NO: 103.

[0337] Coupling between the antibody and the linker-payload: The above-mentioned mixture was incubated at 4 °C for 10 minutes. The linker-payload LP-1 prepared in Example 1 was dissolved in DMA and then added to the mixture. The added volume was 72 μL (equivalent to 18-fold equivalents of the antibody content), and the mixture was allowed to react at 22 °C for 30 minutes.

[0338] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method of Operation D in Example 4 to obtain the antibody-drug conjugate Hu1H2-2-LP1.

[0339] Characterization of the antibody-drug conjugate: Using Operation E (ε D,280 = 6384 and ε D,370 = 16180), Operation H and Operation I to characterize the obtained antibody-drug conjugate.

[0340] The concentration of the antibody-drug conjugate was calculated to be 6.74 mg / mL by measurement with Operation E, and the average number of payloads conjugated to each antibody was 9.20 by measurement with Operation E. Figure 4A The detection spectrum showing aggregates was presented, and the aggregate content of the antibody-drug conjugate Hu1H2-2-LP1 was measured to be 1.24% by Operation H. Figure 4B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate Hu1H2-2-LP1 was presented, and the retention time of the antibody-drug conjugate Hu1H2-2-LP1 measured by Operation I was 6.239 min.

[0341] Example 7: Preparation of the antibody-drug conjugate Hu1H2-2-LP1 (DAR 4)

[0342] Reduction of the antibody: By Operation B in Example 4 (the extinction coefficient of the antibody at 280 nm was 1.54 mL mg -1 cm -1) and operation C replaced the liquid of the antibody with PBS 7.0 / EDTA, and the concentration of the antibody after liquid replacement was 23 mg / mL. 23.52 μL of 5 mM TCEP solution was added to 456.52 μL of the aqueous solution of Hu1H2-2 antibody, and at the same time, 0.28 mL of 50 mM PBS 7.0 and 639.96 μL of deionized water were added. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours.

[0343] Coupling between the antibody and the linker-payload: The above mixture was incubated in an environment at 4 °C for 10 minutes. The linker-payload LP-1 prepared in Example 1 was dissolved in DMA and then added to the mixture. The added volume was 112 μL (equivalent to 8-fold equivalents of the antibody content), and the mixture continued to react in an environment at 22 °C for 30 minutes.

[0344] Characterization of the antibody-drug conjugate: Using operation E of Example 4 (ε D,280 = 6384 and ε D,370 = 16180) and operation H to characterize the obtained antibody-drug conjugate.

[0345] The prepared antibody-drug conjugate Hu1H2-2-LP1 (DAR4) had a concentration of 6.14 mg / mL calculated by operation E for the antibody-drug conjugate, and the average number of payloads conjugated to each antibody was 3.59 calculated by operation E. Figure 5A The detection spectrum showing aggregates was presented, and the aggregate content of the antibody-drug conjugate Hu1H2-2-LP1 (DAR4) was 0.44% measured by operation H. Figure 5B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate Hu1H2-2-LP1 was presented.

[0346] Example 8: Preparation of antibody-drug conjugate Hu1H2-2-LP2 (DAR 8)

[0347] Reduction of the antibody: The reduction method in Example 8 was the same as that of the antibody in Example 6. The amino acid sequence of the Hu1H2-2 antibody in Example 8 was the same as that of the Hu1H2-2 antibody in Example 6.

[0348] Coupling between the antibody and the linker-payload: The mixture in the antibody reduction step was incubated in an environment at 4 °C for 10 minutes. The linker-payload LP-2 prepared in Example 2 was dissolved in DMA and then added to the mixture. The added volume was 72 μL (equivalent to 18-fold equivalents of the antibody content), and the mixture continued to react in an environment at 22 °C for 30 minutes.

[0349] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method of Operation D in Example 4 to obtain the antibody-drug conjugate Hu1H2-2-LP2.

[0350] Characterization of the antibody-drug conjugate: The obtained antibody-drug conjugate was characterized by the above Operation E (ε D,280 = 5814 and ε D,370 = 14742), Operation F, Operation H and Operation I.

[0351] The concentration of the antibody-drug conjugate was calculated to be 6.64 mg / mL by measurement with Operation E, and the average number of payloads conjugated to each antibody was 9.92 by measurement with Operation E. Figure 6A The detection spectrum showing aggregates was presented, and the aggregate content of the antibody-drug conjugate Hu1H2-2-LP2 was measured to be 1.37% by Operation H. Figure 6B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate Hu1H2-2-LP2 was presented, and the retention time of the antibody-drug conjugate Hu1H2-2-LP2 measured by Operation I was 6.561 min.

[0352] Example 9: Preparation of the antibody-drug conjugate Hu029-LP3 (DAR 8)

[0353] Reduction of the antibody: By Operation B of Example 4 (the extinction coefficient of the antibody at 280 nm was 1.54 mL mg -1 cm -1 ) and Operation C, the antibody was exchanged with PBS7.0 / EDTA, and the concentration of the antibody after the exchange was 23.07 mg / mL. 70 μL of a 5 mM TCEP solution (equivalent to 7-fold equivalent of the antibody content) was added to 325.10 μL of an aqueous solution of the Hu1H2-2 antibody, and at the same time, 0.3 mL of 50 mM PBS7.0 and 804.90 μL of deionized water were added. After confirming that the solution pH was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours.

[0354] The amino acid sequence of the Hu1H2-2 antibody was the same as that of the Hu1H2-2 antibody in Example 6.

[0355] Conjugation of the antibody with the linker-payload: The above mixture was incubated at 4 °C for 10 minutes, and the linker-payload LP-3 prepared in Example 3 was dissolved in DMA and then added to the mixture. The added volume was 90 μL (equivalent to 18-fold equivalent of the antibody content), and the mixture was continued to react at 22 °C for 30 minutes.

[0356] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method described in Operation D, and finally the antibody-drug conjugate Hu1H2-2-LP3 was obtained.

[0357] Characterization of the antibody-drug conjugate: The obtained antibody-drug conjugate was characterized by the above Operation E (ε D,280 = 5186 and ε D,370 = 13688), Operation F, Operation H and Operation I.

[0358] The concentration of the antibody-drug conjugate calculated by measurement through Operation E was 4.10 mg / mL, and the average number of payloads conjugated to each antibody calculated by measurement through Operation E was 8.50. Figure 7A The detection spectrum showing aggregates was presented, and the aggregate content of the antibody-drug conjugate Hu1H2-2-LP3 measured by Operation H was 3.37%. Figure 7B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate Hu1H2-2-LP3 was presented, and the retention time of the antibody-drug conjugate Hu1H2-2-LP3 measured by Operation I was 5.878 min.

[0359] Comparative Example 1: Preparation of the antibody-drug conjugate 02-1-vc-MMAE (DAR 4)

[0360] Reduction of the antibody: By Operation B in Example 4 (the extinction coefficient of the antibody at 280 nm was 1.523 mL mg - 1 cm -1 ) and Operation C, the antibody was exchanged with PBS7.0 / EDTA, and the concentration of the antibody after the exchange was 31.6 mg / mL. 0.076 mL of 5 mM TCEP solution (equivalent to 2.3-fold equivalents of the antibody content) was added to 0.759 mL of the aqueous solution of 02-1 antibody, and at the same time, 0.48 mL of 50 mM PBS7.0 and 1.084 mL of deionized water were added. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours. The 02-1 antibody sequence was the same as the 02-1 antibody sequence in Example 5.

[0361] Conjugation of the antibody with the linker-payload: The above mixture was incubated at 4 °C for 10 minutes, and the linker-payload vc-MMAE (DC Chemicals, DC50025) was dissolved in DMA and then added to the mixture. The added volume was 0.322 mL (equivalent to 7-fold equivalents of the antibody content), and the mixture continued to react at 22 °C for 30 minutes.

[0362] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method of Operation D in Example 4 to obtain the antibody-drug conjugate 02-1-vc-MMAE.

[0363] Characterization of the antibody-drug conjugate: The obtained antibody-drug conjugate was characterized by Operation B, Operation G, Operation H and Operation I in Example 4.

[0364] The concentration of the antibody-drug conjugate was calculated to be 9.71 mg / mL by measurement in Operation B. Figure 8A The detection chromatogram showing aggregates was presented. The aggregate content of the antibody-drug conjugate 02-1-vc-MMAE was measured to be 2.74% by Operation H. Figure 8B The hydrophobic chromatography detection chromatogram of the antibody-drug conjugate 02-1-vc-MMAE was presented. The average number of payloads conjugated to each antibody in the antibody-drug conjugate 02-1-vc-MMAE was calculated to be 3.99 (HIC-DAR) by Operation G and Operation I.

[0365] Comparative Example 2: Preparation of the antibody-drug conjugate Rituximab-vc-MMAE (DAR 4)

[0366] Reduction of the antibody: The antibody was buffer-exchanged with PBS7.0 / EDTA by Operation B (the extinction coefficient of the antibody at 280 nm was 1.5 mL mg - 1 cm -1 ) and Operation C in Example 4, and the concentration of the buffer-exchanged antibody was 12 mg / mL. 0.015 mL of 5 mM TCEP solution (equivalent to 2.2-fold equivalents of the antibody content) was added to 0.438 mL of an aqueous solution of Rituximab antibody (purchased from Shanghai Minkai Biotech Co., Ltd.), and at the same time, 0.14 mL of 50 mM PBS7.0 and 0.107 mL of deionized water were added. After confirming that the solution pH was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours.

[0367] Conjugation of the antibody with the linker-payload: The above mixture was incubated at 4 °C for 10 minutes, and the linker-payload vc-MMAE dissolved in DMA was added to the mixture. The added volume was 0.049 mL (equivalent to 7-fold equivalents of the antibody content), and the mixture was continuously reacted at 22 °C for 30 minutes.

[0368] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method of Operation D in Example 4 to obtain the antibody-drug conjugate Rituximab-vc-MMAE.

[0369] Characterization of antibody-drug conjugate: The obtained antibody-drug conjugate was characterized by Operation B, Operation G, Operation H, and Operation I in Example 4.

[0370] The concentration of the antibody-drug conjugate calculated by measurement in Operation B was 3.78 mg / mL. Figure 9A The detection chromatogram showing aggregates was presented. The aggregate content of the antibody-drug conjugate Rituximab-vc-MMAE measured by Operation H was 3.78%. Figure 9B The hydrophobic chromatography detection chromatogram of the antibody-drug conjugate Rituximab-vc-MMAE was presented. The average number of payloads conjugated to each antibody in the antibody-drug conjugate Rituximab-vc-MMAE was calculated to be 4.39 (HIC-DAR) by Operation G and Operation I.

[0371] Comparative Example 3: Preparation of antibody-drug conjugate Rituximab-LP1 (DAR 8)

[0372] Reduction of antibody: The antibody was exchanged with PBS7.0 / EDTA by Operation B (the extinction coefficient of the antibody at 280 nm was 1.5 mL mg - 1 cm -1 ) and Operation C in Example 4. The concentration of the antibody after the exchange was 11.8 mg / mL. To 6.05 mL of an aqueous solution of Rituximab antibody, 0.667 mL of 5 mM TCEP solution (equivalent to 7-fold equivalent of the antibody content) was added, and at the same time, 1.02 mL of 100 mM PBS7.0 and 2.46 mL of deionized water were added. After confirming that the solution pH was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours.

[0373] Conjugation of antibody with linker-payload: The above mixture was incubated at 4 °C for 10 minutes. The linker-payload LP-1 was dissolved in DMA and then added to the mixture. The added volume was 0.667 mL (equivalent to 14-fold equivalent of the antibody content). The mixture was further reacted at 22 °C for 30 minutes.

[0374] Purification of antibody-drug conjugate: The above reaction solution was purified by the method of Operation D in Example 4 to obtain the antibody-drug conjugate Rituximab-LP1.

[0375] Characterization of antibody-drug conjugate: The obtained antibody-drug conjugate was characterized by Operation E (ε D,280 = 6384 and ε D,370 = 16180), Operation H, and Operation I in Example 4.

[0376] The concentration of the antibody-drug conjugate was calculated to be 6.23 mg / mL by measurement through operation E, and the average number of payloads conjugated to each antibody was calculated to be 7.30 by measurement through operation E. Figure 10A The detection spectrum of aggregates is shown, and the aggregate content of the antibody-drug conjugate Rituximab-LP1 was measured to be 4.70% by operation H. Figure 10B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate Rituximab-LP1 is shown, and the retention time of the antibody-drug conjugate Rituximab-LP1 measured by operation I was 7.094 min.

[0377] Comparative Example 4: Preparation of the antibody-drug conjugate Human IgG-GGFG-DXd (DAR 8)

[0378] Reduction of the antibody: The antibody was exchanged with PBS7.0 / EDTA by operation B (the extinction coefficient of the antibody at 280 nm was 1.35 mL mg - 1 cm -1 ) and operation C in Example 4, and the concentration of the antibody after exchange was 10 mg / mL. 0.327 mL of a 5 mM TCEP solution (equivalent to 10-fold equivalent of the antibody content) was added to 2.45 mL of an aqueous solution of human IgG protein (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: SP001), and at the same time, 0.7 mL of 50 mM PBS7.0 and 23 μL of deionized water were added. After confirming that the solution pH was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours.

[0379] Conjugation of the antibody with the linker-payload: The above mixture was incubated at 4 °C for 10 minutes, and the linker-payload GGFG-DXd (purchased from DC Chemicals, DC50025) was dissolved in DMA and then added to the mixture. The added volume was 0.294 mL (equivalent to 18-fold equivalent of the antibody content), and the mixture was continuously reacted at 22 °C for 30 minutes.

[0380] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 4 to obtain the antibody-drug conjugate Human IgG-GGFG-DXd (abbreviation: "Human IgG-DXD"). Human IgG and HuIgG can be used interchangeably in this disclosure.

[0381] Characterization of the antibody-drug conjugate: Operation E in Example 4 was adopted (ε D,280 = 5178 and ε D,370= 20217), operations H and I are used to characterize the obtained antibody-drug conjugate.

[0382] The concentration of the antibody-drug conjugate calculated by measurement in operation E is 8.20 mg / mL, and the average number of payloads conjugated to each antibody calculated by measurement in operation E is 7.08. Figure 11A The detection spectrum of aggregates is shown. The aggregate content of the antibody-drug conjugate Human IgG-DXD measured by operation H is 4.25%. Figure 11B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate Human IgG-DXD is shown. The retention time of the antibody-drug conjugate Human IgG-DXD measured by operation I is 8.222 min.

[0383] Comparative Example 5: Preparation of antibody-drug conjugate Human IgG-LP1 (DAR 8)

[0384] Reduction of the antibody: By operations B (the extinction coefficient of the antibody at 280 nm is 1.35 mL / mg - 1 cm -1 ) and C in Example 4, the antibody is exchanged with PBS 7.0 / EDTA. The concentration of the antibody after exchange is 10 mg / mL. In 1.33 mL of an aqueous solution of human IgG protein (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: SP001), 0.267 mL of a 5 mM TCEP solution (equivalent to 10-fold equivalent of the antibody content) is added, and at the same time, 0.4 mL of 50 mM PBS 7.0 is added. After confirming that the solution pH is 7.0 ± 0.1, the mixture is placed in an environment at 37 °C and reacted for 2 hours.

[0385] Conjugation of the antibody with the linker-payload: The above mixture is incubated at 4 °C for 10 minutes. After the linker-payload LP-1 is dissolved in DMA, it is added to the mixture, and the added volume is 0.2 mL (equivalent to 15-fold equivalent of the antibody content). The mixture is continued to react at 22 °C for 30 minutes.

[0386] Purification of the antibody-drug conjugate: The above reaction solution is purified by the method of operation D in Example 4 to obtain the antibody-drug conjugate Human IgG-LP1.

[0387] Characterization of the antibody-drug conjugate: Operations E (ε D,280 = 6384 and ε D,370 = 16180), H, and I in Example 4 are used to characterize the obtained antibody-drug conjugate.

[0388] The concentration of the antibody-drug conjugate calculated by measurement through operation E was 11.08 mg / mL, and the average number of payloads conjugated to each antibody calculated by measurement through operation E was 9.15. Figure 12A The detection spectrum of aggregates is shown. The aggregate content of the antibody-drug conjugate Human IgG-LP1 measured through operation H was 4.38%. Figure 12B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate Human IgG-LP1 is shown. The retention time of the antibody-drug conjugate Human IgG-LP1 measured through operation I was 6.213 min.

[0389] Comparative Example 6: Preparation of the antibody-drug conjugate Human IgG-LP1 (DAR 4)

[0390] Reduction of the antibody: Reduction of the antibody: Through operation B in Example 4 (the extinction coefficient of the antibody at 280 nm was 1.35 mL mg -1 cm -1 ), and operation C, the antibody was exchanged with PBS 7.0 / EDTA. The concentration of the antibody after the exchange was 15 mg / mL. 34.02 μL of a 5 mM TCEP solution (equivalent to 2.43 times the equivalent amount of the antibody content) was added to 0.7 mL of an aqueous solution of human IgG protein (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: SP001), and at the same time, 0.28 mL of 50 mM PBS 7.0 was added. After confirming that the solution pH was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours.

[0391] Conjugation of the antibody with the linker-payload: The above mixture was incubated at 4 °C for 10 minutes. After the linker-payload LP-1 was dissolved in DMA, it was added to the mixture, and the added volume was 0.112 mL (equivalent to 8 times the equivalent amount of the antibody content). The mixture was continuously reacted at 22 °C for 30 minutes.

[0392] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 4 to obtain the antibody-drug conjugate Human IgG-LP1 (DAR4).

[0393] Characterization of the antibody-drug conjugate: The obtained antibody-drug conjugate was characterized by operation E (ε D,280 = 6384 and ε D,370 = 16180), operation H, and operation I in Example 4.

[0394] The concentration of the antibody-drug conjugate calculated by measurement through operation E was 5.75 mg / mL, and the average number of payloads conjugated to each antibody calculated by measurement through operation E was 3.53. Figure 13A The detection spectrum showing aggregates was presented, and the aggregate content of the antibody-drug conjugate Human IgG-LP1 (DAR4) measured through operation H was 1.90%. Figure 13B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate Human IgG-LP1 (DAR4) was presented.

[0395] Comparative Example 7: Preparation of the antibody-drug conjugate Human IgG-LP2 (DAR 8)

[0396] Reduction of the antibody: By operations B (the extinction coefficient of the antibody at 280 nm was taken as 1.35 mL mg - 1 cm -1 ) and C in Example 4, the antibody was exchanged with PBS 7.0 / EDTA, and the concentration of the antibody after the exchange was 10 mg / mL. 0.093 mL of 5 mM TCEP solution (equivalent to 10-fold equivalent of the antibody content) was added to 0.467 mL of the aqueous solution of human IgG protein (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: SP001), and at the same time, 0.2 mL of 50 mM PBS 7.0 and 0.24 mL of deionized water were added. After confirming that the solution pH was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours.

[0397] Conjugation of the antibody with the linker-payload: The above mixture was incubated at 4 °C for 10 minutes, and the linker-payload LP-2 was dissolved in DMA and then added to the mixture, with the added volume being 0.07 mL (equivalent to 15-fold equivalent of the antibody content), and the mixture was continuously reacted at 22 °C for 30 minutes.

[0398] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 4 to obtain the antibody-drug conjugate Human IgG-LP2.

[0399] Characterization of the antibody-drug conjugate: Operations E (ε D,280 = 5814 and ε D,370 = 14742), operation H and operation I in Example 4 were used to characterize the obtained antibody-drug conjugate.

[0400] The concentration of the antibody-drug conjugate calculated by measurement through operation E was 4.68 mg / mL, and the average number of payloads conjugated to each antibody calculated by measurement through operation E was 9.18. Figure 14AThe detection chromatogram of the aggregates is shown. The aggregate content of the antibody-drug conjugate Human IgG-LP2 measured by operation H is 2.92%. Figure 14B The hydrophobic chromatography detection chromatogram of the antibody-drug conjugate Human IgG-LP2 is shown. The retention time of the antibody-drug conjugate Human IgG-LP2 measured by operation I is 6.506 min.

[0401] Comparative Example 8: Preparation of antibody-drug conjugate Human IgG-LP3 (DAR 8)

[0402] Reduction of the antibody: By operation B (the extinction coefficient of the antibody at 280 nm is 1.35 mL / mg - 1 cm -1 ) and operation C in Example 4, the antibody was exchanged with PBS 7.0 / EDTA. The concentration of the antibody after the exchange was 18 mg / mL. 0.672 mL of a 5 mM TCEP solution (equivalent to 7-fold equivalent of the antibody content) was added to 4 mL of an aqueous solution of human IgG protein (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: SP001), and at the same time, 1.44 mL of 50 mM PBS 7.0 and 1.088 mL of deionized water were added. After confirming that the solution pH was 7.0 ± 0.1, the mixture was placed in an environment at 37 °C and reacted for 2 hours. The source of the human IgG1 protein was the same as that of the human IgG protein in Comparative Example 4.

[0403] Conjugation of the antibody with the linker-payload: The above mixture was incubated at 4 °C for 10 minutes. After the linker-payload LP-3 was dissolved in DMA, it was added to the mixture, and the added volume was 0.672 mL (equivalent to 14-fold equivalent of the antibody content). The mixture was continued to react at 22 °C for 30 minutes.

[0404] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 4 to obtain the antibody-drug conjugate Human IgG-LP3.

[0405] Characterization of the antibody-drug conjugate: Operations E (ε D,280 = 5186 and ε D,370 = 13688), operation H and operation I in Example 4 were used to characterize the obtained antibody-drug conjugate.

[0406] The concentration of the antibody-drug conjugate calculated by measurement with operation E was 7.42 mg / mL, and the average number of payloads conjugated to each antibody calculated by measurement with operation E was 7.28. Figure 15AThe detection spectrum of aggregates is shown. The aggregate content of the antibody-drug conjugate Human IgG-LP3 measured by operation H is 5.37%. Figure 15B The hydrophobic chromatography detection spectrum of the antibody-drug conjugate Human IgG-LP3 is shown. The retention time of the antibody-drug conjugate Human IgG-LP3 measured by operation I is 6.450 min.

[0407] Comparative Example 9: Preparation of the antibody-drug conjugate Hu1H2-2-GGFG-DXD (DAR 8)

[0408] Reduction of the antibody: The reduction method of the antibody in Comparative Example 9 is the same as that in Example 6. The sequence of the Hu1H2-2 antibody is the same as that of the Hu1H2-2 antibody in Example 6.

[0409] Coupling between the antibody and the drug linker: The above mixture was incubated at 4 °C for 10 minutes. The linker-payload GGFG-DXd was dissolved in DMA and then added to the mixture, and the added volume was 72 μL (equivalent to 18-fold equivalents of the antibody content). The mixture was placed at 22 °C and reacted for another 30 minutes.

[0410] Purification of the antibody-drug conjugate: The above reaction solution was purified by the method described in operation D, and finally the antibody-drug conjugate Hu1H2-2-GGFG-DXd (abbreviation "Hu1H2-2-DXD") was obtained.

[0411] Characterization of the antibody-drug conjugate: The above operation E (ε D,280 = 5178 and ε D,370 = 20217) and operations H and I were used to characterize the obtained antibody-drug conjugate.

[0412] The concentration of the antibody-drug conjugate calculated by measurement using operation E is 6.53 mg / mL, and the average number of payloads conjugated to each antibody calculated by measurement using operation E is 6.75. The aggregate content of the antibody-drug conjugate Hu1H2-2-DXD measured by operation H is 1.70%, as Figure 16A shown. The retention time of the antibody-drug conjugate Hu1H2-2-DXD measured by operation I is 7.764 min, as Figure 16B shown.

[0413] Test Example 1: Endocytosis of 02-1-LP1 by tumor cells with high expression of MUC18

[0414] 50 μg / mL of 02-1-LP1 was respectively combined with A375 cells (purchased from Shanghai Institute of Life Sciences, Chinese Academy of Sciences), HMVII cells (purchased from Biovector NTCC Type Culture Collection Center), SK-MEL-2 cells (purchased from Shanghai Xunqing Biotechnology Co., Ltd.) and GAK cells (purchased from Biovector NTCC Type Culture Collection Center). After washing away the excess ADC, the cells were cultured in an incubator at 37 °C. At 0, 0.5, 1, 2, 4, and 8 hours, the median fluorescence intensity (MFI) was detected by flow cytometry. As Figure 17 shown, 02-1-LP1 can be endocytosed by A375 (A), HMVII (B), SK-MEL-2 (C), and GAK (D) cells.

[0415] Test Example 2: Inhibitory effect of 02-1-LP1 on A375 xenograft tumors in vivo and its effect on the body weight change of mice

[0416] Six-week-old Balb / c mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. Four million A375 cells were subcutaneously inoculated into each mouse to construct a MUC18-positive A375 melanoma mouse xenograft tumor model. On the 24th day after inoculation (the average tumor volume was about 230 mm 3 ), Rituximab-vc-MMAE (6 mpk (mg / kg)) prepared in Comparative Example 2; 02-1-vc-MMAE (1.5, 3, 6 mpk) prepared in Comparative Example 1; Rituximab-LP1 (2.5, 5 mpk) prepared in Comparative Example 3; and 02-1-LP1 (2.5, 5 mpk) prepared in Example 5 were respectively intravenously injected; and a solvent group was set as a negative control. After administration, the tumor volume was measured twice a week using vernier calipers, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2. Since A375 cells hardly express CD20, Rituximab was used as an isotype control for the 02-1 antibody at this time.

[0417] Figure 18A1 and 18A2 showed the growth inhibitory effects of the antibody-drug conjugate 02-1-LP1 and 02-1-vc-MMAE targeting MUC18 on A375 xenograft tumors. Figure 18A1 showed that 02-1-vc-MMAE reduced the tumor growth rate; while 02-1-LP1 caused a significant reduction in tumor volume. Figure 18A2 is Figure 18A1 a local view showing the curves of 02-1-LP1, 02-1-vc-MMAE, and the solvent group to make the trend graph of the active ingredient clearer.

[0418] Figure 18B1 and18B2 It was shown that the antibody-drug conjugates 02-1-LP1 and 02-1-vc-MMAE targeting MUC18 had no significant effect on the body weight of mice. Figure 18B2 This is a partial view of 18B1, showing the curves of 02-1-LP1, 02-1-vc-MMAE and the vehicle group to make the trend graph of the active ingredient clearer.

[0419] Test Example 3: Toxicity experiments of 02-1-LP1 and 02-1-vc-MMAE on cynomolgus monkeys

[0420] Repeated-dose experiment of 02-1-LP1 (prepared in Example 5) on cynomolgus monkeys: Six cynomolgus monkeys (purchased from Guangxi Frontier Biotechnology Co., Ltd.) were randomly divided into 3 groups, with one male and one female in each group. On the 1st day, 22nd day and 43rd day, the vehicle, 10 mg / kg 02-1-LP1 and 30 mg / kg 02-1-LP1 were intravenously infused respectively. During the experiment, the animals were observed for any abnormalities; and blood samples were collected for hematological and blood biochemical index analysis. On the 50th day, the animals were euthanized and samples were taken for pathological analysis. As shown in Table 6, in this experiment, none of the test substances at all doses caused animal death, and the test substance-related target organs were reticulocytes, digestive tract, kidney and spleen. The HNSTD (highest non-severe toxicity dose) was 30 mg / kg.

[0421] Repeated-dose experiment of 02-1-vc-MMAE (prepared in Comparative Example 1) on cynomolgus monkeys: Six cynomolgus monkeys were randomly divided into 3 groups, with one male and one female in each group. On the 1st day and 22nd day, 3 mg / kg 02-1-vc-MMAE, 6 mg / kg 02-1-vc-MMAE and 10 mg / kg 02-1-vc-MMAE were intravenously infused respectively. During the experiment, the animals were observed for any abnormalities; and blood samples were collected for hematological and blood biochemical index analysis. On the 43rd day, the animals were euthanized and samples were taken for pathological analysis. As shown in Table 6, in this experiment, two animals in the high-dose group (10 mg / kg) died on the 10th day after the first administration, and no deaths occurred in the 3 mg / kg and 6 mg / kg groups. The test substance-related target organs were reticulocytes, white blood cells and skin. The HNSTD (highest non-severe toxicity dose) was 6 mg / kg.

[0422] Table 6. Summary of the results of repeated-dose toxicity experiments of 02-1-LP1 and 02-1-vc-MMAE in cynomolgus monkeys

[0423]

[0424]

[0425] HNSTD*: Maximum dose without severe toxicity

[0426] The results showed that the safety of 02-1-LP1 prepared in Example 5 was significantly better than that of 02-1-vc-MMAE prepared in Comparative Example 1.

[0427] Combined with Test Example 2, it can be seen that 02-1-LP1 has a higher tolerance dose and better safety than 02-1-vc-MMAE; at the same time, a lower dose of 02-1-LP1 can achieve a better anti-tumor effect.

[0428] Test Example 4: In vitro killing experiment of ADC on Detroit562 cells

[0429] Culture Detroit562 cells (purchased from Nankebai Biotechnology Co., Ltd.) to make the cell density reach 80%, collect the cells, seed them in 96-well plates, and adjust the cell density to 2-5×10 4 / ml. Seed 100 μL in each well, and perform 3-fold serial dilution of the ADC molecule starting from a concentration of 300 nM. After dilution, add the ADC molecule to the cell culture medium and incubate statically for 5 days. During this period, regularly observe the apoptosis of the cells. After 5 days, add 15 μL of the CCK-8 kit stock solution to the 96-well plates, react in a 37°C incubator for 0.5-2 h, measure the absorbance at 450 nm, and draw a cell survival curve based on the optical density (OD) reading value and the ADC dilution gradient.

[0430] The ADC molecules selected were Human IgG-DXD prepared in Comparative Example 4, Human IgG-LP1 prepared in Comparative Example 5, Human IgG-LP2 prepared in Comparative Example 7, Hu1H2-2-LP1 prepared in Example 6, Hu1H2-2-LP2 prepared in Example 8, and Hu1H2-2-DXD prepared in Comparative Example 9. Among them, Human IgG was used as the isotype control of the Hu1H2-2 antibody; DXD was used as the positive drug.

[0431] Figure 19 Show the in vitro killing effect of the above ADC molecules on the head and neck squamous cell carcinoma cell line Detroit562, Figure 19A1 and Figure 19A2 is Figure 19 a partial view of. Figure 19A1 It shows that compared with the positive control Hu1H2-2-DXD, the in vitro killing effect of the disclosed Hu1H2-2-LP2 and Hu1H2-2-LP1 on Detroit562 is more excellent. Figure 19A2It is shown that Human IgG-LP2 and Human IgG-LP1 have better in vitro killing effects than Human IgG-DXD, indicating that compared with GGFG-DXd, the linkers-payloads LP-1 and LP-2 have better in vitro killing effects on Detroit562.

[0432] Test Example 5: Effects of ADC on in vivo pharmacodynamics and changes in mouse body weight in a CDX mouse model constructed with Detroit562

[0433] Six-week-old Balb / c nude mice were purchased from Jiangsu Genscript Biotech Co., Ltd. Five million Detroit562 cells were subcutaneously inoculated into each mouse to construct a CDX (cell derived xenograft) mouse model. On the 10th day after inoculation (average tumor volume was about 150 mm 3 ), Human IgG-LP1 (10 mg / kg) prepared in Comparative Example 5; Hu1H2-2-LP1 (10 mg / kg) prepared in Example 6; Hu1H2-2-LP2 (10 mg / kg) prepared in Example 8 were respectively intravenously injected. After administration, the tumor volume was measured twice a week using vernier calipers, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0434] Figure 20 It shows the in vivo pharmacodynamic data of Hu1H2-2-LP1, Hu1H2-2-LP2 and Human IgG-LP1 in a mouse model of head and neck squamous cell carcinoma constructed with Detroit562 cells. The results show that Hu1H2-2-LP1 and Hu1H2-2-LP2 can effectively inhibit the growth of tumors in mice.

[0435] Figure 21 It shows the effects of Hu1H2-2-LP1, Hu1H2-2-LP2 and Human IgG-LP1 on the body weight of mice. During the experimental period, the body weights of the mice in the Hu1H2-2-LP1 group and the Hu1H2-2-LP2 group continued to increase; while the body weight of the control group Human IgG-LP1 fluctuated. The results show that the effects of Hu1H2-2-LP1 and Hu1H2-2-LP2 on the changes in mouse body weight are relatively small.

[0436] Test Example 6: Effects of ADC on in vivo pharmacodynamics and changes in mouse body weight in a CDX mouse model constructed with PC-9

[0437] Six-week-old Balb / c nude mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. Five million PC-9 cells (purchased from Zhejiang Meisen Cell Technology Co., Ltd.) were subcutaneously inoculated into each mouse to obtain a CDX model. On the 13th day after inoculation (average tumor volume was about 200 mm 3 ), Human IgG-LP1 prepared in Comparative Example 5, Human IgG-LP1 (DAR4) prepared in Comparative Example 6, Human IgG-LP3 prepared in Comparative Example 8, and Hu1H2-2-LP1 prepared in Example 6 were intravenously injected respectively; Hu1H2-2-LP1 (DAR4) prepared in Example 7, and Hu1H2-2-LP3 prepared in Example 9. The dosing dose was 10 mpk (mg / kg) once a week for 2 times (QW×2). After dosing, the tumor volume was measured twice a week using vernier calipers, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0438] Figure 22 Show the in vivo efficacy data of the above ADCs on mice with a lung cancer model constructed by PC-9 cells. The results showed that Hu1H2-2-LP1, Hu1H2-2-LP1 (DAR4), and Hu1H2-2-LP3 could effectively inhibit the growth of tumors in mice.

[0439] Figure 23 Show the effect of the above ADCs on the body weight of mice with a lung cancer model constructed by PC-9 cells. The results showed that Hu1H2-2-LP1, Hu1H2-2-LP1 (DAR4), and Hu1H2-2-LP3 had little effect on the body weight of mice and had no obvious gastrointestinal toxicity.

[0440] Test Example 7: In vivo inhibitory effect on the SCC-9 human head and neck squamous cell carcinoma CDX model and its effect on the change of body weight of mice

[0441] Six-week-old Balb / c nude mice were purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd. Eight million SCC-9 cells (purchased from ATCC) were subcutaneously inoculated into each mouse to obtain a CDX model. On the 29th day after inoculation (average tumor volume was about 200 mm 3 ), Rituximab-LP1 (10 mpk) prepared in Comparative Example 3 and 02-1-LP1 (2.5, 5, 10 mpk) prepared in Example 5 were intravenously injected respectively. DPBS (Dulbecco phosphate buffered saline) was used as a negative control. Dosing was once a week for 2 times (QW×2). After dosing, the tumor volume was measured twice a week using vernier calipers, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0442] Figure 24A It is shown that 02-1-LP1 can effectively inhibit tumor growth in the SCC-9CDX model. Figure 24B It is shown that 02-1-LP1 has little effect on the body weight of mice in the SCC-9 model.

[0443] Test Example 8: In vivo inhibitory effect on the Huh-7 human cancer CDX model and its effect on changes in mouse body weight

[0444] Six-week-old Balb / c nude mice were purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd. Each mouse was subcutaneously inoculated with 5 million Huh-7 cells (purchased from Shanghai Bio-Gene Biotechnology Co., Ltd.) to obtain a CDX model. On the 9th day after inoculation (average tumor volume was approximately 230 mm 3 ), human IgG-LP1 prepared in Comparative Example 5 and 02-1-LP1 prepared in Example 5 were respectively intravenously injected. The dosing dose was 10 mpk, once a week for a total of 2 times (QW×2). After dosing, the tumor volume was measured twice a week with vernier calipers, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0445] Figure 25A It is shown that 02-1-LP1 can effectively inhibit tumor growth in the Huh-7 CDX model. Figure 25B It is shown that 02-1-LP1 has little effect on the body weight of mice in the Huh-7 model.

[0446] Test Example 9: In vivo inhibitory effect on the LD1-0015-200617 human esophageal squamous cell carcinoma PDX model and its effect on changes in mouse body weight

[0447] LD1-0015-200617 (LideBiotech CO., LTD) is a patient-derived xenograft model of human esophageal squamous cell carcinoma expressing human MUC18 (H-Score = 75). Six- to eight-week-old Nu / Nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The tumor of LD1-0015-200617 was cut into fragments of approximately 3 mm×3 mm×3 mm and subcutaneously implanted into the flanks of the mice. On the 39th day after implantation (average tumor size ~171 mm 3 ), human IgG-LP1 prepared in Comparative Example 5 and 02-1-LP1 prepared in Example 5 were respectively intravenously injected. The dosing dose was 10 mpk, once a week for a total of 3 times (QW×3). After dosing, the tumor volume was measured twice a week with vernier calipers, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0448] Figure 26AIt is shown that 02-1-LP1 can effectively inhibit tumor growth in the LD1-0015-200617 PDX model. Figure 26B It is shown that in the LD1-0015-200617 PDX model, 02-1-LP1 has little effect on the body weight of mice.

[0449] Test Example 10: In vivo inhibitory effect on the LD1-0016-390730 human esophageal adenocarcinoma PDX model and its effect on the change of body weight of mice

[0450] LD1-0016-390730 (LideBiotech CO., LTD) is a patient-derived xenograft model of human esophageal adenocarcinoma expressing human MUC18 (H-Score = 115). 6-8-week-old Nu / Nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The tumors of LD1-0016-390730 were cut into fragments of about 3 mm × 3 mm × 3 mm and subcutaneously implanted into the flanks of the mice. On the 45th day after implantation (average tumor size ~191 mm 3 ), 02-1-LP1 prepared in Example 5 was intravenously injected. The dosing dose was 10 mpk, once a week for a total of 2 times (QW×2). After dosing, the tumor volume was measured twice a week with a vernier caliper, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0451] Figure 27A It is shown that 02-1-LP1 can effectively inhibit tumor growth in the LD1-0016-390730 PDX model. Figure 27B It is shown that in the LD1-0016-390730 PDX model, 02-1-LP1 has little effect on the body weight of mice.

[0452] Test Example 11: In vivo inhibitory effect on the LD1-2025-362797 human small cell lung cancer PDX model and its effect on the change of body weight of mice

[0453] LD1-2025-362797 (LideBiotech CO., LTD) is a patient-derived xenograft model of human small cell lung cancer expressing human MUC18 (H-Score = 110). 6-8-week-old Nu / Nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The tumors of LD1-2025-362797 were cut into fragments of about 3 mm × 3 mm × 3 mm and subcutaneously implanted into the flanks of the mice. On the 21st day after implantation (average tumor size ~144 mm 3 ), the first dose of 10 mpk 02-1-LP1 prepared in Example 5 was intravenously injected. When the tumor volume recovered to about 700 mm 3At time point 0 (49 days after the first administration), a second administration of 10 mpk of 02-1-LP1 was given by intravenous injection. After administration, the tumor volume was measured twice a week with vernier calipers, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0454] Figure 28A It was shown that in the LD1-2025-362797 PDX model, 02-1-LP1 could effectively inhibit tumor growth. Notably, after the second dose of 02-1-LP1, the tumor decreased again, indicating that 02-1-LPI remained active against the regrown tumor. Figure 28B It was shown that in the LD1-2025-362797 PDX model, 02-1-LP1 had little effect on the body weight of mice.

[0455] Test Example 12: In vivo inhibitory effect on the LD1-2009-362263 human triple-negative breast cancer PDX model and its effect on changes in body weight of mice

[0456] LD1-2009-362263 (Lide Biotech CO., LTD) is a patient-derived xenograft model of human triple-negative breast cancer expressing human MUC18 (H-Score = 240). 6-8-week-old Nu / Nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The tumors of LD1-2025-362797 were cut into fragments of about 3 mm × 3 mm × 3 mm and subcutaneously implanted into the flanks of the mice. On the 49th day after implantation (average tumor size ~152 mm 3 ), 02-1-LP1 prepared in Example 5 was given by intravenous injection. The administration dose was 10 mpk, once a week for a total of 2 times (QW×2). After administration, the tumor volume was measured twice a week with vernier calipers, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0457] Figure 29A It was shown that 02-1-LP1 could effectively inhibit tumor growth in the LD1-2009-362263 PDX model. Figure 29B It was shown that in the LD1-2009-362263 PDX model, 02-1-LP1 had little effect on the body weight of mice.

[0458] Test Example 13: In vivo inhibitory effect on the LD1-0060-200770 human cholangiocarcinoma PDX model and its effect on changes in body weight of mice

[0459] LD1-0060-200770 (LideBiotech CO., LTD) is a patient-derived xenograft model of human cholangiocarcinoma that expresses human MUC18 (H-Score = 15). Nu / Nu mice at 6-8 weeks old were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The tumors of LD1-0060-200770 were cut into fragments of about 3 mm × 3 mm × 3 mm and subcutaneously implanted into the flanks of the mice. On the 42nd day after implantation (average tumor size ~155 mm 3 ), 02-1-LP1 prepared in Example 5 was intravenously injected. The dosing dose was 10 mpk, once a week for a total of 2 times (QW×2). After dosing, the tumor volume was measured twice a week with a vernier caliper, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0460] Figure 30A It shows that 02-1-LP1 can effectively inhibit tumor growth in the LD1-0060-200770 PDX model. Figure 30B It shows that 02-1-LP1 has little effect on the body weight of mice in the LD1-0060-200770 PDX model.

[0461] Test Example 14: In vivo inhibitory effect on the OV-10-0073 human ovarian cancer PDX model and its effect on the change of mouse body weight

[0462] OV-10-0073 (WuXi AppTec (Shanghai) Co., Ltd.) is a patient-derived xenograft model of human ovarian cancer that expresses human MUC18 (H-Score = 40). Mice at 6-8 weeks old were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The tumors of OV-10-0073 were cut into fragments of about 30 mm 3 , and subcutaneously implanted into the flanks of the mice. On the 34th day after implantation (average tumor size ~185 mm 3 ), single doses of Human IgG-LP1 (2.5, 5 mpk) prepared in Comparative Example 5 and 02-1-LP1 (1.25, 2.5, 5 mpk) prepared in Example 5 were intravenously injected respectively. After dosing, the tumor volume was measured twice a week with a vernier caliper, and the tumor volume was calculated according to the following formula: TV = (length × width) 2 / 2.

[0463] Figure 31A It shows that 02-1-LP1 can effectively inhibit tumor growth in the OV-10-0073 PDX model. Figure 31B It shows that 02-1-LP1 has little effect on the body weight of mice in the OV-10-0073 PDX model.

[0464] As can be seen from the above experimental results, in the ADC provided by the present disclosure, after the linker is conjugated with irinotecan, it is conjugated with an antibody targeting MUC18 or CD44v7 / 8 by a simple chemical method. Compared with the traditional random conjugation method, the DAR value of the anti-MUC18 or CD44v7 / 8 antibody-drug conjugate obtained by applying this linker is higher (DAR8). The HIC detection shows that the chromatographic peak of the ADC of the present disclosure is narrower than that of the linker-payload of vc-MMAE or GGFG-DXd, indicating that the generated product has high homogeneity and hydrophilicity. Compared with the traditional vc-MMAE and GGFG-DXd conjugates, the in vitro tumor cell proliferation inhibitory activity of the conjugate of the present disclosure is improved or maintained in terms of biological activity and safety.

[0465] The ADC prepared in the present disclosure has specific MUC18 or CD44v7 / 8-dependent anti-tumor activity and extremely high killing activity against tumor cells with high expression of MUC18 or CD44v7 / 8. The ADC with MUC18 specificity prepared in the present disclosure has increased HNSTD, indicating improved safety and reduced toxicity and side effects. Surprisingly, the results of Test Examples 9-14 also show that the ADC with MUC18 specificity prepared in the present disclosure shows excellent killing activity against tumor cells with different MUC18 expression levels (low, medium, high).

[0466] The ADC prepared in the present disclosure has more excellent in vivo anti-tumor efficacy than the ADC conjugated with vc-MMAE or GGFG-DXd, and at the same time shows significantly better safety than the ADC of vc-MMAE and has a similar MTD (maximum tolerated dose) to the GGFG-DXd ADC (Yusuke Ogitani Clin, et al., Cancer Res. 2016 Oct 15; 22(20):5097-5108.). It is expected that this drug conjugate will have a higher therapeutic window than the existing clinical molecules vc-MMAE or GGFG-DXd.

[0467] The ADC provided by the present disclosure has better stability in vitro and reduced aggregates during the preparation process. The ADC with the linker of Formula I has better stability in plasma, and it is expected that the ADC may have better drug metabolism characteristics in vivo, such as a longer half-life, a lower amount of free small molecule toxins, etc.

Claims

1. An antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, comprising an anti-MUC18 or anti-CD44v7 / 8 antibody or an antigen-binding fragment thereof, a payload, and a linker represented by Formula I, wherein: the linker represented by Formula I forms a thioether bond connection with the thiol moiety after reduction of the interchain disulfide bond of the antibody or its antigen-binding fragment through a succinimidyl group; the carbonyl group in the ester group of the linker represented by Formula I is connected to the amino group in the payload; R 1 、R 2 are each independently selected from hydrogen, methyl or isopropyl; R 3 represents -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 - or a single bond, R 5 is selected from hydrogen or benzyl, n 1 represents an integer from 0 to 2, n 2 represents an integer from 0 to 2; R 4 represents methylamino or -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 1 to 20.

2. The antibody-drug conjugate according to claim 1, R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , where n 3 represents an integer from 8 to 15.

3. The antibody-drug conjugate according to claim 1 or 2, R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , where n 3 represents an integer from 10 to 12.

4. The antibody-drug conjugate according to any one of claims 1-3, R 3 represents -(CR 5 H 2 CONH)n 1 -(CH 2 CONH)n 2 - or a single bond, R 5 is selected from benzyl, n 1 represents 1 or 2, n 2 represents 1 or 2; R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 8 to 15.

5. The antibody-drug conjugate according to any one of claims 1-4, R 3 represents a single bond; R 4 represents -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 8 to 15.

6. The antibody-drug conjugate according to any one of claims 1-5, R 3 represents a single bond; R 4 represents methylamino.

7. The antibody-drug conjugate according to any one of claims 1-6, wherein the linker is selected from any one of the following groups: and or a combination thereof.

8. The antibody-drug conjugate according to any one of claims 1-7, wherein the payload is selected from at least one of the group consisting of cytotoxins, markers, nucleic acids, radionuclides, hormones, immunomodulators, prodrug converting enzymes, ribonucleases, agonistic antibodies, antagonistic antibodies and fragments thereof, fusion proteins or derivatives.

9. The antibody-drug conjugate according to claim 8, wherein the cytotoxin comprises a tubulin inhibitor and / or a topoisomerase inhibitor; the tubulin inhibitor comprises auristatin or a derivative thereof, maytansine or a derivative thereof; the topoisomerase inhibitor comprises camptothecin and its derivatives.

10. The antibody-drug conjugate according to any one of claims 1-9, wherein the payload is irinotecan represented by Formula II, and is connected to the linker through the nitrogen atom of the amino group on the cyclohexane ring, 11. The antibody-drug conjugate according to any one of claims 1-10, wherein the anti-MUC18 antibody or its antigen-binding fragment comprises: Heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO:4, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO:16, heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO:28, and light chain CDR1 with the amino acid sequence shown in SEQ ID NO:38, light chain CDR2 with the amino acid sequence STS, light chain CDR3 with the amino acid sequence shown in SEQ ID NO:52; Heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO:1, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO:11, heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO:23, and light chain CDR1 with the amino acid sequence shown in SEQ ID NO:34, light chain CDR2 with the amino acid sequence LAS, light chain CDR3 with the amino acid sequence shown in SEQ ID NO:46; The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:1, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:12, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:23, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:35, the light chain CDR2 with the amino acid sequence LAS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:47; The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:2, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:13, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:24, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:36, the light chain CDR2 with the amino acid sequence NAK, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:48; The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:3, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:14, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:25, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:37, the light chain CDR2 with the amino acid sequence FAS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:49; The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:4, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:15, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:26, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:38, the light chain CDR2 with the amino acid sequence STS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:50; The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:4, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:15, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:27, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:39, the light chain CDR2 with the amino acid sequence STS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:51; The heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:5, the heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:17, the heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:29, and the light chain CDR1 of the amino acid sequence shown in SEQ ID NO:40, the light chain CDR2 with the amino acid sequence WAS, and the light chain CDR3 of the amino acid sequence shown in SEQ ID NO:53; Heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:6, heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:18, heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:29, and light chain CDR1 of the amino acid sequence shown in SEQ ID NO:41, light chain CDR2 with the amino acid sequence WAS, and light chain CDR3 of the amino acid sequence shown in SEQ ID NO:53; Heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:7, heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:19, heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:30, and light chain CDR1 of the amino acid sequence shown in SEQ ID NO:42, light chain CDR2 with the amino acid sequence RTS, and light chain CDR3 of the amino acid sequence shown in SEQ ID NO:54; Heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:8, heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:20, heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:31, and light chain CDR1 of the amino acid sequence shown in SEQ ID NO:43, light chain CDR2 with the amino acid sequence WAS, and light chain CDR3 of the amino acid sequence shown in SEQ ID NO:55; Heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:9, heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:21, heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:32, and light chain CDR1 of the amino acid sequence shown in SEQ ID NO:44, light chain CDR2 with the amino acid sequence WAS, and light chain CDR3 of the amino acid sequence shown in SEQ ID NO:56; or, Heavy chain CDR1 of the amino acid sequence shown in SEQ ID NO:10, heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO:22, heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO:33, and light chain CDR1 of the amino acid sequence shown in SEQ ID NO:45, light chain CDR2 with the amino acid sequence LMS, and light chain CDR3 of the amino acid sequence shown in SEQ ID NO:

57.

12. The antibody-drug conjugate according to any one of claims 1-11, wherein the anti-MUC18 antibody or its antigen-binding fragment comprises: The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:84, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:85; The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:86, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:87; or The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:89, or a conservative variant thereof.

13. The antibody-drug conjugate according to any one of claims 1-10, wherein the anti-CD44 v7 / 8 antibody or its antigen-binding fragment specifically binds to a human CD44 v7 / 8 binding peptide, and the binding peptide comprises the amino acid sequence shown in SEQ ID NO:

90.

14. The antibody-drug conjugate according to any one of claims 1-10 and 13, wherein the CD44 v7 / 8 antibody or its antigen-binding fragment does not bind to human CD44 v7 / 8 binding peptides with the amino acid sequences shown in SEQ ID NO:91 and / or SEQ ID NO:

92.

15. The antibody-drug conjugate according to claim 13 or 14, wherein the anti-CD44 v7 / 8 antibody or its antigen-binding fragment comprises: Heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO:93, heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO:94, heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO:95; and light chain CDR1 with the amino acid sequence shown in SEQ ID NO:96, CDR2 with the amino acid sequence RAN, and light chain CDR3 with the amino acid sequence shown in SEQ ID NO:

97.

16. The antibody-drug conjugate according to any one of claims 13-15, wherein the anti-CD44 v7 / 8 antibody or its antigen-binding fragment comprises: The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:100, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:101; or The heavy chain variable region sequence of the amino acid sequence shown in SEQ ID NO:100, and the light chain variable region sequence of the amino acid sequence shown in SEQ ID NO:104, or a conservative variant thereof.

17. The antibody-drug conjugate according to any one of claims 1-16, wherein the DAR is 1 to 10.

18. The antibody-drug conjugate according to any one of claims 1-17, wherein the DAR is 4 to 10.

19. A method for preparing the antibody-drug conjugate according to any one of claims 1-18, or an isomer, isotope variant, pharmaceutically acceptable salt, prodrug, solvate or combination thereof, and a pharmaceutically acceptable excipient, comprises the following steps: The disulfide bonds of the anti-MUC18 or anti-CD44 v7 / 8 antibody or its antigen-binding fragment are at least partially reduced after reduction treatment, and react with the 3-position carbon atom of the maleimide-N-group of the linker shown in Formula III, wherein in the linker-payload, the carbonyl group in the ester group of the linker shown in Formula III is connected to the amino group of the payload; R 1 、R 2 are each independently selected from hydrogen, methyl or isopropyl; R 3 represents -(CR 5 HCONH)n 1 -(CH 2 CONH)n 2 - or a single bond, R 5 is selected from hydrogen or benzyl, n 1 represents an integer from 0 to 2, n 2 represents an integer from 0 to 2; and R 4 represents methylamino or -(NCH 3 COCH 2 )n 3 -NCH 3 COCH 3 , n 3 represents an integer from 1 to 20.

20. According to the preparation method of claim 19, the method further comprises: An antibody or its antigen-binding fragment reacts with a reducing agent in a buffer solution containing a chelating agent, a solution of a linker-payload is added, the linker has the structure of Formula III, and the pH value of the reaction solution is adjusted.

21. The preparation method according to claim 19 or 20, the method further comprises: The payload is irinotecan shown in Formula II, and the nitrogen atom of the amino group on its cyclohexane ring is connected to the carbonyl group of the ester group in Formula III.

22. The preparation method according to any one of claims 19-21, the DAR is 1 to 10.

23. The preparation method according to any one of claims 19-22, The anti-MUC18 antibody or its antigen-binding fragment comprises a heavy chain variable region sequence with the amino acid sequence shown in SEQ ID NO:84, and a light chain variable region sequence with the amino acid sequence shown in SEQ ID NO:85; a heavy chain variable region sequence with the amino acid sequence shown in SEQ ID NO:86, and a light chain variable region sequence with the amino acid sequence shown in SEQ ID NO:87; or a heavy chain variable region sequence with the amino acid sequence shown in SEQ ID NO:88, and a light chain variable region sequence with the amino acid sequence shown in SEQ ID NO:89, or its conservative variant; The anti-CD44v7 / 8 antibody or its antigen-binding fragment comprises a heavy chain variable region sequence with the amino acid sequence shown in SEQ ID NO:100, and a light chain variable region sequence with the amino acid sequence shown in SEQ ID NO:101; or a heavy chain variable region sequence with the amino acid sequence shown in SEQ ID NO:100, and a light chain variable region sequence with the amino acid sequence shown in SEQ ID NO:104, or its conservative variant.

24. A pharmaceutical composition, which comprises the antibody-drug conjugate according to any one of claims 1-18, or the antibody-drug conjugate prepared by the method according to any one of claims 19-23, or its isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates or combinations thereof, and a pharmaceutically acceptable excipient.

25. A kit, which comprises the antibody-drug conjugate according to any one of claims 1-18, or the antibody-drug conjugate prepared by the method according to any one of claims 19-23, the pharmaceutical composition according to claim 24, or its isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates or combinations thereof.

26. Use of the antibody-drug conjugate according to any one of claims 1-18, the antibody-drug conjugate prepared by the method according to any one of claims 19-23, the pharmaceutical composition according to claim 24, or the kit according to claim 25 in the preparation of a therapeutic agent for diagnosing, preventing and treating neoplastic diseases.

27. The use according to claim 26, wherein the neoplastic diseases include benign tumors and malignant tumors expressing MUC18 and / or CD44v7 / 8.

28. The use according to claim 26 or 27, wherein the neoplastic disease includes melanoma, pharyngeal cancer, triple-negative cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, urothelial cancer, bladder neuroendocrine tumor, small cell lung cancer, non-small cell lung cancer, cutaneous squamous cell carcinoma, cholangiocarcinoma, metastatic pancreatic cancer, squamous cell carcinoma of the lung, squamous cell carcinoma of the head and neck, and / or esophageal squamous cell carcinoma.

29. The use of the antibody-drug conjugate according to any one of claims 1-18, the antibody-drug conjugate prepared by the method according to any one of claims 19-23, the pharmaceutical composition according to claim 24, or the kit according to claim 25 in the preparation of a therapeutic agent targeting MUC18 and / or CD44v7 / 8.

30. A method for diagnosing, preventing, and treating a neoplastic disease, comprising administering to a subject a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent comprises the antibody-drug conjugate according to any one of claims 1-18, the antibody-drug conjugate prepared by the method according to any one of claims 19-23, or the pharmaceutical composition according to claim 24.

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