Antibody-drug conjugates, methods for their preparation and uses

Abstract

Provided are antibody-drug conjugates, methods for preparing them, and uses thereof. The antibody-drug conjugates comprise an antibody, a cytotoxic payload, and a linker of Formula I, wherein the succinimidyl group of the linker of Formula I forms a thioether bond with a thiol group obtained by reduction of the interchain disulfide chain of the antibody, and the carbonyl group in the ester group of the linker is bonded to the amino group of the payload. The antibody-drug conjugates are used in the manufacture of therapeutic agents for the diagnosis, prevention, and treatment of neoplastic diseases.

Description

[Technical Field]

[0001] The present disclosure relates to antibody-drug conjugates in which an antibody is linked by a linker to a drug or toxin with anti-tumor activity to exert its anti-tumor role on tumor cells. [Background technology]

[0002] The statements in this section merely provide background information related to the present disclosure and may not necessarily constitute prior art. Antibody-drug conjugates (ADCs) are vectorized chemotherapy drugs that selectively deliver cytotoxic drugs to tumor / cancer cells (Antibody-Drug Conjugates: The Last Decade, Nicolas Joubert et al., Pharmaceuticals (Basel). September 14, 2020; 13(9):245). With the development of drug delivery technology, ADC-targeted delivery technology can effectively overcome the side effects caused by camptothecin's poor water solubility and insufficient tissue distribution. The commercially available ADC drugs Enhertu and sacituzumab govitecan have excellent efficacy in treating tumors, especially malignant tumors. Both Enhertu and sacituzumab govitecan use camptothecin-derived DNA topoisomerase inhibitors, which are more hydrophobic than tubulin inhibitors (e.g., MMAE and MMAF), as cytotoxic drugs. Sacituzumab govitecan uses MCC-triazole spacer-PEG7-lysine-PABC as a linker to degrade and release camptothecin SN38 in cell lysosomes (US 13 / 948,732). Enhertz, developed by AstraZeneca and Daiichi Sankyo, uses the cathepsin B-activating GGFG (an amino acid sequence consisting of glycine-glycine-phenylalanine-glycine linked by a peptide bond) tetrapeptide as a linker, introducing a self-cleaving structure to release the exatecan derivative Dxd (Yusuke Ogitani et al., Clin Cancer Res (2016) 22(20):5097-5108). However, the cytotoxic drugs MMAE, SN38, and Dxd are all substrates of P-glycoprotein (P-gp) (Front Pharmacol 2019;10:749), and some tumors with high P-gp expression may be resistant to these drugs. Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, there remains a need for new antibody-drug conjugates for delivering cytotoxic drugs into tumor / cancer cells. [Means for solving the problem]

[0004] The present disclosure provides a method for the preparation of a compound comprising administering to a subject an anti-MUC18 antibody or an anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof, a payload, and a linker of Formula I

[0005] [ka] wherein the succinimidyl group of the linker of Formula I forms a thioether bond with a thiol group obtained by reduction of the interchain disulfide chain of the antibody or antigen-binding fragment thereof; The carbonyl group in the ester group of the linker of formula I is bonded to the amino group of the payload; R1 and R2 are independently hydrogen, methyl, or isopropyl groups; R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is hydrogen or benzyl, and n 1 represents an integer from 0 to 2, and n 2 represents an integer between 0 and 2, R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 1 and 20) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof.

[0006] In some embodiments, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 8 to 15. In some embodiments, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 10 and 12.

[0007] In some embodiments, R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is benzyl, and n 1 represents 1 or 2, and n 2 represents 1 or 2, and R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 8 to 15.

[0008] In some embodiments, R3 represents a single bond and R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 8 to 15. In some embodiments, R3 represents a single bond and R4 represents a methylamino group.

[0009] In some embodiments, the linker is:

[0010] [ka] are selected from any one of the above or a combination thereof.

[0011] In some embodiments, the payload is at least one selected from the group consisting of a cytotoxic agent, a label, a nucleic acid, a radionuclide, a hormone, an immunomodulatory agent, a prodrug-converting enzyme, a ribonuclease, an agonist antibody, an antagonist antibody and fragments thereof, a fusion protein or derivative thereof.

[0012] In some embodiments, the cytotoxic agent comprises a tubulin inhibitor and / or a topoisomerase inhibitor, wherein the tubulin inhibitor comprises an auristatin or a derivative thereof, a maytansine or a derivative thereof, and the topoisomerase inhibitor comprises camptothecin and its derivatives.

[0013] In some embodiments, the payload is exatecan of Formula II

[0014] [ka] wherein exatecan of formula II is attached to the linker by the nitrogen atom of the amino group on its cyclohexane ring.

[0015] In some embodiments, the anti-MUC18 antibody or antigen-binding fragment thereof is selected from the group consisting of: HCDR1 having the amino acid sequence shown in SEQ ID NO: 4, HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, HCDR3 having the amino acid sequence shown in SEQ ID NO: 28, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 38, LCDR2 having the amino acid sequence STS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 52; HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, HCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 34, LCDR2 having the amino acid sequence LAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 46; HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, HCDR2 having the amino acid sequence shown in SEQ ID NO: 12, HCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 35, LCDR2 having the amino acid sequence LAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; HCDR1 having the amino acid sequence shown in SEQ ID NO: 2, HCDR2 having the amino acid sequence shown in SEQ ID NO: 13, HCDR3 having the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 36, LCDR2 having the amino acid sequence NAK, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 48; HCDR1 having the amino acid sequence shown in SEQ ID NO: 3, HCDR2 having the amino acid sequence shown in SEQ ID NO: 14, HCDR3 having the amino acid sequence shown in SEQ ID NO: 25, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 37, LCDR2 having the amino acid sequence FAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 49; HCDR1 having the amino acid sequence shown in SEQ ID NO: 4, HCDR2 having the amino acid sequence shown in SEQ ID NO: 15, HCDR3 having the amino acid sequence shown in SEQ ID NO: 26, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 38, LCDR2 having the amino acid sequence STS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 50; HCDR1 having the amino acid sequence shown in SEQ ID NO: 4, HCDR2 having the amino acid sequence shown in SEQ ID NO: 15, HCDR3 having the amino acid sequence shown in SEQ ID NO: 27, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 39, LCDR2 having the amino acid sequence STS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 51; HCDR1 having the amino acid sequence shown in SEQ ID NO: 5, HCDR2 having the amino acid sequence shown in SEQ ID NO: 17, HCDR3 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 40, LCDR2 having the amino acid sequence WAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 53; HCDR1 having the amino acid sequence shown in SEQ ID NO: 6, HCDR2 having the amino acid sequence shown in SEQ ID NO: 18, HCDR3 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 41, LCDR2 having the amino acid sequence WAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 53; HCDR1 having the amino acid sequence shown in SEQ ID NO: 7, HCDR2 having the amino acid sequence shown in SEQ ID NO: 19, HCDR3 having the amino acid sequence shown in SEQ ID NO: 30, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 42, LCDR2 having the amino acid sequence RTS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 54; HCDR1 having the amino acid sequence shown in SEQ ID NO: 8, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, HCDR3 having the amino acid sequence shown in SEQ ID NO: 31, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 43, LCDR2 having the amino acid sequence WAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 55; HCDR1 having the amino acid sequence set forth in SEQ ID NO: 9, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 21, HCDR3 having the amino acid sequence set forth in SEQ ID NO: 32, and LCDR1 having the amino acid sequence set forth in SEQ ID NO: 44, LCDR2 having the amino acid sequence WAS, LCDR3 having the amino acid sequence set forth in SEQ ID NO: 56, and HCDR1 having the amino acid sequence shown in SEQ ID NO: 10, HCDR2 having the amino acid sequence shown in SEQ ID NO: 22, HCDR3 having the amino acid sequence shown in SEQ ID NO: 33, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 45, LCDR2 having the amino acid sequence LMS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 57. Contains one of the following:

[0016] In some embodiments, the anti-MUC18 antibody or antigen-binding fragment thereof is a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 84, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 85; a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 86 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 87; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 88, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 89; or conservative variants thereof.

[0017] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof specifically binds to a binding peptide in human CD44 v7 / 8, wherein the binding peptide comprises the amino acid sequence set forth in SEQ ID NO:90.

[0018] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof does not bind to a binding peptide having the amino acid sequence set forth in SEQ ID NO:91 and / or SEQ ID NO:92 in human CD44 v7 / 8.

[0019] In some embodiments, the CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:93, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:94, an HCDR3 having the amino acid sequence set forth in SEQ ID NO:95, and an LCDR1 having the amino acid sequence set forth in SEQ ID NO:96, an LCDR2 having the amino acid sequence RAN, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:97.

[0020] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof is a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 100 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 101; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 100, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 104; or conservative variants thereof.

[0021] In some embodiments, the DAR is 1 to 10. In some embodiments, the DAR is 4 to 10. The present disclosure also provides a method for preparing the above-described antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, comprising the steps of: reducing an anti-MUC18 antibody or an anti-CD44 v7 / 8 antibody, or an antigen-binding fragment thereof, such that the interchain disulfide bond thereof is at least partially reduced; and linking a linker of Formula III

[0022] [ka] wherein the carbonyl group in the ester group of the linker of formula III is bonded to the amino group of the payload; R1 and R2 are independently selected from hydrogen, methyl, or isopropyl groups; R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is hydrogen or benzyl, and n 1 represents an integer from 0 to 2, and n 2 represents an integer between 0 and 2, R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 1 and 20) and reacting the carbon atom at the 3-position of the maleimide-N-yl of

[0023] In some aspects, the method comprises reacting an antibody or antigen-binding fragment thereof with a reducing agent in a buffer solution containing a chelating agent, followed by adding a solution of the linker-payload, wherein the linker has the structure of Formula III, and adjusting the pH of the reaction solution.

[0024] In some embodiments, the payload is exatecan of Formula II

[0025] [ka] wherein exatecan of formula II is bonded to the carbonyl group of the ester group in formula III by the nitrogen atom of the amino group on its cyclohexane ring.

[0026] In some embodiments, the DAR is 1-10, optionally 4-10. The present disclosure provides a pharmaceutical composition comprising the above-described antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient.

[0027] The present disclosure provides kits comprising the above-described antibody-drug conjugates, or isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates, or combinations thereof.

[0028] The present disclosure provides use of the antibody-drug conjugate, the antibody-drug conjugate prepared by the method, a pharmaceutical composition containing the antibody-drug conjugate, or the kit in the manufacture of a therapeutic agent for the diagnosis, prevention, and treatment of neoplastic diseases. In some embodiments, the neoplastic diseases include benign and malignant tumors that express MUC18 and / or CD44 v7 / 8.

[0029] In some embodiments, the neoplastic disease comprises melanoma, pharyngeal carcinoma, triple-negative breast cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, urothelial carcinoma, 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.

[0030] The present disclosure provides use of the antibody-drug conjugate, the antibody-drug conjugate prepared by the method, a pharmaceutical composition containing the antibody-drug conjugate, or the kit in the manufacture of a therapeutic agent targeting MUC18 and / or CD44 v7 / 8.

[0031] The present disclosure provides methods for diagnosing, preventing, and treating neoplastic diseases, comprising the step of administering to a subject a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent comprises the antibody-drug conjugate described above, an antibody-drug conjugate prepared by the method described above, or the pharmaceutical composition described above. [Brief explanation of the drawings]

[0032] [Figure 1A]FIG. 1A shows a graph of size exclusion chromatography of the 02-1 naked antibody, and FIG. 1B shows a graph of size exclusion chromatography of the Hu1H2-2 naked antibody. [Figure 1B] FIG. 1A shows a graph of size exclusion chromatography of the 02-1 naked antibody, and FIG. 1B shows a graph of size exclusion chromatography of the Hu1H2-2 naked antibody. [Figure 2A] FIG. 2A shows a graph of hydrophobic interaction chromatography of the 02-1 naked antibody, and FIG. 2B shows a graph of hydrophobic interaction chromatography of the Hu1H2-2 naked antibody. [Figure 2B] FIG. 2A shows a graph of hydrophobic interaction chromatography of the 02-1 naked antibody, and FIG. 2B shows a graph of hydrophobic interaction chromatography of the Hu1H2-2 naked antibody. [Figure 3A] FIG. 3A shows a graph of size exclusion chromatography of the antibody-drug conjugate 02-1-LP1 prepared in Example 5, and FIG. 3B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate 02-1-LP1. [Figure 3B] FIG. 3A shows a graph of size exclusion chromatography of the antibody-drug conjugate 02-1-LP1 prepared in Example 5, and FIG. 3B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate 02-1-LP1. [Figure 4A] FIG. 4A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-LP1 prepared in Example 6, and FIG. 4B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-LP1. [Figure 4B] FIG. 4A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-LP1 prepared in Example 6, and FIG. 4B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-LP1. [Figure 5A]FIG. 5A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-LP1 prepared in Example 7, and FIG. 5B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-LP1. [Figure 5B] FIG. 5A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-LP1 prepared in Example 7, and FIG. 5B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-LP1. [Figure 6A] FIG. 6A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-LP2 prepared in Example 8, and FIG. 6B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-LP2. [Figure 6B] FIG. 6A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-LP2 prepared in Example 8, and FIG. 6B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-LP2. [Figure 7A] FIG. 7A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-LP3 prepared in Example 9, and FIG. 7B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-LP3. [Figure 7B] FIG. 7A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-LP3 prepared in Example 9, and FIG. 7B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-LP3. [Figure 8A] FIG. 8A shows a size exclusion chromatography graph of the antibody-drug conjugate 02-1-vc-MMAE prepared in Comparative Example 1, and FIG. 8B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate 02-1-vc-MMAE. [Figure 8B]FIG. 8A shows a size exclusion chromatography graph of the antibody-drug conjugate 02-1-vc-MMAE prepared in Comparative Example 1, and FIG. 8B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate 02-1-vc-MMAE. [Figure 9A] FIG. 9A shows a size exclusion chromatography graph of the antibody-drug conjugate rituximab-vc-MMAE prepared in Comparative Example 2, and FIG. 9B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate rituximab-vc-MMAE. [Figure 9B] FIG. 9A shows a size exclusion chromatography graph of the antibody-drug conjugate rituximab-vc-MMAE prepared in Comparative Example 2, and FIG. 9B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate rituximab-vc-MMAE. [Figure 10A] FIG. 10A shows a graph of size exclusion chromatography of the antibody-drug conjugate Rituximab-LP1 prepared in Comparative Example 3, and FIG. 10B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Rituximab-LP1. [Figure 10B] FIG. 10A shows a graph of size exclusion chromatography of the antibody-drug conjugate Rituximab-LP1 prepared in Comparative Example 3, and FIG. 10B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Rituximab-LP1. [Figure 11A] FIG. 11A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-DXD prepared in Comparative Example 4, and FIG. 11B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-DXD. [Figure 11B] FIG. 11A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-DXD prepared in Comparative Example 4, and FIG. 11B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-DXD. [Figure 12A] FIG. 12A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-LP1 prepared in Comparative Example 5, and FIG. 12B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-LP1. [Figure 12B] FIG. 12A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-LP1 prepared in Comparative Example 5, and FIG. 12B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-LP1. [Figure 13A] FIG. 13A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-LP1 prepared in Comparative Example 6, and FIG. 13B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-LP1. [Figure 13B] FIG. 13A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-LP1 prepared in Comparative Example 6, and FIG. 13B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-LP1. [Figure 14A] FIG. 14A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-LP2 prepared in Comparative Example 7, and FIG. 14B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-LP2. [Figure 14B] FIG. 14A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-LP2 prepared in Comparative Example 7, and FIG. 14B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-LP2. [Figure 15A] Figure 15A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-LP3 prepared in Comparative Example 8, and Figure 15B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-LP3. [Figure 15B]Figure 15A shows a graph of size exclusion chromatography of the antibody-drug conjugate human IgG-LP3 prepared in Comparative Example 8, and Figure 15B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate human IgG-LP3. [Figure 16A] FIG. 16A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-DXD prepared in Comparative Example 9, and FIG. 16B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-DXD. [Figure 16B] FIG. 16A shows a graph of size exclusion chromatography of the antibody-drug conjugate Hu1H2-2-DXD prepared in Comparative Example 9, and FIG. 16B shows a graph of hydrophobic interaction chromatography detection of the antibody-drug conjugate Hu1H2-2-DXD. [Figure 17] FIG. 17 shows a time-fluorescence intensity statistical graph of endocytosis of A375 cells, HMVII cells, SK-MEL-2 cells, and GAK cells for antibody-drug conjugate 02-1-LP1. [Figure 18A1] Figure 18A1 shows the tumor volume-time curves of melanoma model mice treated with the antibody-drug conjugates rituximab-vc-MMAE, 02-1-vc-MMAE, rituximab-LP1, and 02-1-LP1, and Figure 18A2 is a partial view of Figure 18A1 showing the tumor volume-time curves of the 02-1-LP1, 02-1-vc-MMAE, and vehicle groups. [Figure 18A2] Figure 18A1 shows the tumor volume-time curves of melanoma model mice treated with the antibody-drug conjugates rituximab-vc-MMAE, 02-1-vc-MMAE, rituximab-LP1, and 02-1-LP1, and Figure 18A2 is a partial view of Figure 18A1 showing the tumor volume-time curves of the 02-1-LP1, 02-1-vc-MMAE, and vehicle groups. [Figure 18B1]Figure 18B1 shows the weight-time curves of melanoma model mice treated with the antibody-drug conjugates rituximab-vc-MMAE, 02-1-vc-MMAE, rituximab-LP1, and 02-1-LP1, and Figure 18B2 is a partial view of Figure 18B1 showing the tumor weight-time curves of the 02-1-LP1, 02-1-vc-MMAE, and vehicle groups. [Figure 18B2] Figure 18B1 shows the weight-time curves of melanoma model mice treated with the antibody-drug conjugates rituximab-vc-MMAE, 02-1-vc-MMAE, rituximab-LP1, and 02-1-LP1, and Figure 18B2 is a partial view of Figure 18B1 showing the tumor weight-time curves of the 02-1-LP1, 02-1-vc-MMAE, and vehicle groups. [Figure 19] Figure 19 shows in vitro killing curves of antibody-drug conjugates against the head and neck squamous cell carcinoma cell line Detroit 562, Figure 19A1 is a partial view of Figure 19 showing the in vitro killing curves of Hu1H2-2-LP2, Hu1H2-2-LP1, and Hu1H2-2-DXD, and Figure 19A2 is a partial view of Figure 19 showing the in vitro killing curves of human IgG-LP2, human IgG-LP1, and human IgG-DXD. [Figure 19A1] Figure 19 shows in vitro killing curves of antibody-drug conjugates against the head and neck squamous cell carcinoma cell line Detroit 562, Figure 19A1 is a partial view of Figure 19 showing the in vitro killing curves of Hu1H2-2-LP2, Hu1H2-2-LP1, and Hu1H2-2-DXD, and Figure 19A2 is a partial view of Figure 19 showing the in vitro killing curves of human IgG-LP2, human IgG-LP1, and human IgG-DXD. [Figure 19A2]Figure 19 shows in vitro killing curves of antibody-drug conjugates against the head and neck squamous cell carcinoma cell line Detroit 562, Figure 19A1 is a partial view of Figure 19 showing the in vitro killing curves of Hu1H2-2-LP2, Hu1H2-2-LP1, and Hu1H2-2-DXD, and Figure 19A2 is a partial view of Figure 19 showing the in vitro killing curves of human IgG-LP2, human IgG-LP1, and human IgG-DXD. [Figure 20] FIG. 20 shows tumor volume-time change curves in head and neck squamous cell carcinoma model mice treated with antibody-drug conjugates Hu1H2-2-LP1, Hu1H2-2-LP2, and human IgG-LP1. [Figure 21] FIG. 21 shows weight-time change curves of head and neck squamous cell carcinoma model mice treated with antibody-drug conjugates Hu1H2-2-LP1, Hu1H2-2-LP2, and human IgG-LP1. [Figure 22] Figure 22 shows tumor volume-time curves of lung cancer model mice treated with antibody-drug conjugates Hu1H2-2-LP1, Hu1H2-2-LP1(DAR4), Hu1H2-2-LP3, human IgG-LP1, human IgG-LP1(DAR4), and human IgG-LP3. [Figure 23] Figure 23 shows the weight-time curves of lung cancer model mice treated with antibody-drug conjugates Hu1H2-2-LP1, Hu1H2-2-LP1(DAR4), Hu1H2-2-LP3, human IgG-LP1, human IgG-LP1(DAR4), and human IgG-LP3. [Figure 24A] Figure 24A shows tumor volume-time curves for SCC-9 human head and neck squamous cell carcinoma CDX models treated with the antibody-drug conjugates rituximab-LP1 and 02-1-LP1. Figure 24B shows weight-time curves for SCC-9 human head and neck squamous cell carcinoma CDX models treated with the antibody-drug conjugates rituximab-LP1 and 02-1-LP1. [Figure 24B]Figure 24A shows tumor volume-time curves for SCC-9 human head and neck squamous cell carcinoma CDX models treated with the antibody-drug conjugates rituximab-LP1 and 02-1-LP1. Figure 24B shows weight-time curves for SCC-9 human head and neck squamous cell carcinoma CDX models treated with the antibody-drug conjugates rituximab-LP1 and 02-1-LP1. [Figure 25A] Figure 25A shows the tumor volume-time curves of a Huh-7 human liver cancer CDX model treated with the antibody-drug conjugates human IgG-LP1 and 02-1-LP1. Figure 25B shows the weight-time curves of a Huh-7 human liver cancer CDX model treated with the antibody-drug conjugates human IgG-LP1 and 02-1-LP1. [Figure 25B] Figure 25A shows the tumor volume-time curves of a Huh-7 human liver cancer CDX model treated with the antibody-drug conjugates human IgG-LP1 and 02-1-LP1. Figure 25B shows the weight-time curves of a Huh-7 human liver cancer CDX model treated with the antibody-drug conjugates human IgG-LP1 and 02-1-LP1. [Figure 26A] Figure 26A shows tumor volume-time curves for the LD1-0015-200617 human esophageal squamous cell carcinoma PDX model treated with antibody-drug conjugates human IgG-LP1 and 02-1-LP1. Figure 26B shows weight-time curves for the LD1-0015-200617 human esophageal squamous cell carcinoma PDX model treated with antibody-drug conjugates human IgG-LP1 and 02-1-LP1. [Figure 26B] Figure 26A shows tumor volume-time curves for the LD1-0015-200617 human esophageal squamous cell carcinoma PDX model treated with antibody-drug conjugates human IgG-LP1 and 02-1-LP1. Figure 26B shows weight-time curves for the LD1-0015-200617 human esophageal squamous cell carcinoma PDX model treated with antibody-drug conjugates human IgG-LP1 and 02-1-LP1. [Figure 27A]Figure 27A shows tumor volume versus time curves for the LD1-0016-390730 human esophageal adenocarcinoma PDX model treated with antibody-drug conjugate 02-1-LP1. Figure 27B shows weight versus time curves for the LD1-0016-390730 human esophageal adenocarcinoma PDX model treated with antibody-drug conjugate 02-1-LP1. [Figure 27B] Figure 27A shows tumor volume versus time curves for the LD1-0016-390730 human esophageal adenocarcinoma PDX model treated with antibody-drug conjugate 02-1-LP1. Figure 27B shows weight versus time curves for the LD1-0016-390730 human esophageal adenocarcinoma PDX model treated with antibody-drug conjugate 02-1-LP1. [Figure 28A] Figure 28A shows the tumor volume-time curves of the LD1-2025-362797 human small cell lung cancer PDX model treated with antibody-drug conjugate 02-1-LP1. Figure 28B shows the weight-time curves of the LD1-2025-362797 human small cell lung cancer PDX model treated with antibody-drug conjugate 02-1-LP1. [Figure 28B] Figure 28A shows the tumor volume-time curves of the LD1-2025-362797 human small cell lung cancer PDX model treated with antibody-drug conjugate 02-1-LP1. Figure 28B shows the weight-time curves of the LD1-2025-362797 human small cell lung cancer PDX model treated with antibody-drug conjugate 02-1-LP1. [Figure 29A] Figure 29A shows tumor volume versus time curves for the LD1-2009-362263 human triple-negative breast cancer PDX model treated with antibody-drug conjugate 02-1-LP1. Figure 29B shows weight versus time curves for the LD1-2009-362263 human triple-negative breast cancer PDX model treated with antibody-drug conjugate 02-1-LP1. [Figure 29B]Figure 29A shows tumor volume versus time curves for the LD1-2009-362263 human triple-negative breast cancer PDX model treated with antibody-drug conjugate 02-1-LP1. Figure 29B shows weight versus time curves for the LD1-2009-362263 human triple-negative breast cancer PDX model treated with antibody-drug conjugate 02-1-LP1. [Figure 30A] Figure 30A shows the tumor volume-time curves of the LD1-0060-200770 human cholangiocarcinoma PDX model treated with antibody-drug conjugate 02-1-LP1. Figure 30B shows the weight-time curves of the LD1-0060-200770 human cholangiocarcinoma PDX model treated with antibody-drug conjugate 02-1-LP1. [Figure 30B] Figure 30A shows the tumor volume-time curves of the LD1-0060-200770 human cholangiocarcinoma PDX model treated with antibody-drug conjugate 02-1-LP1. Figure 30B shows the weight-time curves of the LD1-0060-200770 human cholangiocarcinoma PDX model treated with antibody-drug conjugate 02-1-LP1. [Figure 31A] Figure 31A shows tumor volume-time curves for an OV-10-0073 human ovarian cancer PDX model treated with antibody-drug conjugates human IgG-LP1 and 02-1-LP1. Figure 31B shows weight-time curves for an OV-10-0073 human ovarian cancer PDX model treated with antibody-drug conjugates human IgG-LP1 and 02-1-LP1. [Figure 31B] Figure 31A shows tumor volume-time curves for an OV-10-0073 human ovarian cancer PDX model treated with antibody-drug conjugates human IgG-LP1 and 02-1-LP1. Figure 31B shows weight-time curves for an OV-10-0073 human ovarian cancer PDX model treated with antibody-drug conjugates human IgG-LP1 and 02-1-LP1. DETAILED DESCRIPTION OF THE INVENTION

[0033] Unless otherwise indicated, all numbers used in the specification and claims expressing content, concentration, ratio, mass, volume, time, temperature, thickness, technical effect, and the like, should be understood to be modified in all instances by the term "about" or "approximately." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and accompanying claims are approximations. For those of ordinary skill in the art, the numerical parameters may vary depending on the desired properties and effects sought to be obtained by the present disclosure, and each numerical parameter should be construed in accordance with the number of significant digits and conventional rounding techniques, or as understood by one of ordinary skill in the art.

[0034] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of this disclosure are approximations, the numerical values ​​set forth in the specific examples are provided as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification includes every narrower numerical range that falls within that broader numerical range, as if all such narrower numerical ranges were expressly written herein.

[0035] In ADC molecules, the linker, as the connecting structure connecting the antibody and payload, is a key factor for the successful construction of an ADC. Its molecular design and properties are important determinants of ADC efficacy in terms of pharmacokinetics (PK) / pharmacodynamics (PD) and therapeutic window. For optimal efficacy, an ideal linker should have the following properties: (1) The linker must possess sufficient stability in plasma to enable the ADC molecule to circulate in the bloodstream and localize at the tumor site without premature cleavage. Linker instability can cause premature release of the toxic payload and undesirable damage to non-target healthy cells, leading to systemic toxicity and adverse effects. (2) The linker must possess the ability to be rapidly cleaved once the ADC is internalized by the target tumor cells, releasing the free toxic payload. (3) Another property that should be considered in linker design is hydrophobicity. Hydrophobic linkers coupled with hydrophobic payloads often promote aggregation of ADC molecules. Such molecules are undesirable in the pursuit of therapeutically useful ADCs and may cause liver toxicity or elicit unwanted immune responses (Kyoji Tsuchikama et al., Antibody-drug conjugates: recent advances in conjugation and linker chemistry, Protein Cell. 2018 January;9(1):33-46).

[0036] Antibody-drug conjugates The present disclosure provides a method for producing a compound comprising administering to a subject an antibody or antigen-binding fragment thereof, a payload, and a linker of formula I

[0037] [ka] wherein the succinimidyl group of the linker of Formula I forms a thioether bond with a thiol group obtained by reduction of the interchain disulfide chain of the antibody or antigen-binding fragment thereof; The carbonyl in the ester group of the linker of formula I is attached to the amino group of the payload, R1 and R2 are independently hydrogen, methyl, or isopropyl groups; R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is hydrogen or benzyl, and n 1 represents an integer from 0 to 2, and n 2 represents an integer between 0 and 2, R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 1 and 20) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof.

[0038] The term "antibody-drug conjugate" or "ADC" refers to a conjugate of an anti-MUC18 / CD44 v7 / 8 antibody or antigen-binding fragment thereof described herein covalently attached to a payload. Generally, an antibody-drug conjugate may include an antibody or antigen-binding fragment thereof, a payload, and optionally a linker between the antibody or antigen-binding fragment thereof and the payload. An ADC may provide a therapeutic effect by delivering the payload to MUC18 and / or CD44 v7 / 8 cells, particularly MUC18 and / or CD44 v7 / 8 tumor cells, targeted by the antibody or antigen-binding fragment thereof. Antibody-drug conjugates can be prepared by various methods known in the art for preparing antibody-drug conjugates.

[0039] As used herein, "antibody" refers to a polypeptide of the immunoglobulin (Ig) family that binds to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer containing at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed 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 is composed 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). VH and VL each consist of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigens.

[0040] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including a diabody, Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv), bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain Fv (scFv), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody comprising 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 comprise the complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen as bound by the parent antibody or parent antibody fragment (e.g., parent scFv).

[0041] 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 differences in the bonding order or bonding properties of atoms in a molecule, preferably tautomers. Tautomers refer to functional isomers resulting from the rapid movement of atoms at two positions in a molecule. Stereoisomers refer to isomers caused by atoms or groups of atoms in a molecule that are bonded to each other in the same order and bond but have different spatial arrangements, preferably including optical isomers. Optical isomers refer to stereoisomers with different optical rotations due to the absence of antiaxial symmetry in a molecule, such as enantiomers, diastereomers, racemates, and mesoisomers.

[0042] The term "prodrug" refers to a compound obtained by modifying the chemical structure of a drug that is inactive or less active in vitro and that releases an active drug through enzymatic or non-enzymatic conversion in vivo, thereby exerting a pharmacological effect. In the present disclosure, the prodrug can be an ADC molecule or a payload.

[0043] The term "payload" encompasses compounds that are cytotoxic or capable of killing cells upon release from the antibody-drug conjugate, compounds having radiolabels, fluorophores, chromophores, imaging agents, and / or metal ions as detectable labels or with cell-killing effects, radionuclides or polypeptides, compounds that can modulate the body's immune activity (including activating or inhibitory effects), nucleic acids, polypeptides or proteins, enzymes, hormones, or nucleic acids.

[0044] In some ideal cases, in antibody-drug conjugates, the conjugated payload exhibits little or no cytotoxicity, or its cytotoxicity is so low that administration of a therapeutically effective amount of the ADC does not cause a systemic toxic response in the subject due to the conjugated payload. The payload can be a clinically validated drug for the treatment of a particular disease, or a compound, radionuclide, nucleic acid, protein, or polypeptide with acceptable pharmacological activity under conditions of clinical use.

[0045] In the present disclosure, the terminal succinimidyl group of Formula I and the thiol group obtained by reduction of the interchain disulfide chain of the antibody or antigen-binding fragment thereof are linked together to form a thioether bond.

[0046] [ka] which forms a thioether bond at position 3 with a thiol moiety after reduction of the interchain disulfide chain of the antibody or antigen-binding fragment thereof.

[0047] [ka] A bond with a represents a chemical bond that is attached to another group.

[0048] In the present disclosure, the disulfide bonds of an antibody or its antigen-binding fragment, including interchain disulfide bonds and intrachain disulfide bonds, preferably the interchain disulfide bonds, are treated, for example, activated to thiol, and then bound to a linker. The amino acid of the antibody or its antigen-binding fragment that is chemically bound to the succinimidyl group of the linker includes one or a combination of lysine, histidine, tyrosine, and cysteine. Optionally, the chemically bound amino acid of the antibody or its antigen-binding fragment is cysteine. In some embodiments, the linker of Formula I can be linked to the hinge, variable, and / or constant region of the antibody.

[0049] In some embodiments of the present disclosure, in the linker of Formula I, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 1 to 20. 3 can be any integer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0050] In some embodiments, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer of 8 to 15. In some embodiments, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer of 10 to 12. In some embodiments, R 4 represents a methylamino group.

[0051] In the present disclosure, the linker of Formula I contains a hydrophilic amino group R4, where R4 contains a polysarcosine group or a methylamino group, increasing the hydrophilicity of the antibody-drug conjugate. The introduction of a hydrophilic amino group is beneficial for improving the hydrophilicity of ADCs conjugated with hydrophobic payloads. Increasing the hydrophilicity of ADC molecules helps reduce aggregation of ADC molecules during the preparation process, thereby improving the stability, homogeneity, and purity of the antibody-drug conjugate.

[0052] In some embodiments, in the linker of Formula I, R3 represents a single bond. In some embodiments, R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 -, R5 is benzyl, and n 1 represents an integer of 1 or 2, and n 2 represents an integer of 1 or 2.

[0053] In some embodiments, R3 represents -CR5HCONH-, -CH2CONH-, -CR5HCONH-CH2CONH-, -(CR5HCONH)2-CH2CONH-, -CR5HCONH-(CH2CONH)2-, or -(CR5HCONH)2-(CH2CONH)2-, and R5 is benzyl.

[0054] In some embodiments, R1 is hydrogen. In some embodiments, R1 is isopropyl. In some embodiments, R2 is hydrogen. In some embodiments, R2 is methyl. In some embodiments, the linker in the antibody-drug conjugate is

[0055] [ka] is selected from the group consisting of:

[0056] In some embodiments, the payload in the antibody-drug conjugate is a label containing a radiolabel, a fluorophore, a chromophore, an imaging agent, and / or a metal ion as a detection label. Labels include, but are not limited to, chemically synthesized organic compounds, radionuclides, metal complexes, or polypeptides. Here, radiolabeling refers to a labeled compound in which one or more atoms of the compound molecule are replaced with a radionuclide so that the compound can be identified and used as a tracer. Radiolabels include amino acids, polypeptides, proteins, carbohydrates, nucleotides, nucleosides, purines, pyrimidines, steroids, lipid compounds, as well as tumor antigens, hormones, receptors, vitamins, and drugs used in medical research. Radionuclides are typically nuclei that spontaneously emit radiation, including, but not limited to, tritium, iodine-125, iodine-131, sulfur-35, phosphorus-32, and carbon-14. Fluorophores are typically groups containing conjugated double bonds, and fluoresce when the molecule returns from an excited state to a ground state. Chromophores refer to unsaturated groups and their associated chemical bonds that contain transitions in molecules that can absorb light radiation. Imaging agents typically refer to radiopharmaceuticals that, when introduced into the body, can image organs, tissues, or molecules in nuclear medicine.

[0057] In some embodiments, the payload in the antibody-drug conjugate is a nucleic acid, which can be a ribonucleic acid and / or a deoxyribonucleic acid. In some embodiments, the payload in the antibody-drug conjugate is a hormone, a growth factor, a clotting factor, or a plasminase (e.g., a prodrug converting enzyme capable of converting a prodrug into an active drug, a ribonuclease).

[0058] In some embodiments, the payload in the antibody-drug conjugate is an immunomodulatory agent (including cytokines and chemokines that can affect immunity), or a biologically active agonist or antagonist antibody.

[0059] 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 antitumor activity or is an antitumor drug.The payload is selected from a DNA topoisomerase inhibitor or a tubulin inhibitor.The DNA topoisomerase inhibitor can be a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0060] In this application, the term "topoisomerase inhibitor" generally refers to a compound that inhibits topoisomerase activity. Compounds known as topoisomerase I inhibitors have activity against topoisomerase I, and topoisomerase II inhibitors have activity against topoisomerase II. Some compounds have activity against both topoisomerase I and topoisomerase II and are known as topoisomerase I / II inhibitors.

[0061] The term "tubulin inhibitor" generally refers to compounds that inhibit the microtubule system of eukaryotic cells, preventing cell division and inhibiting cell proliferation. In some embodiments, the payload is camptothecin or its derivatives with topoisomerase inhibitory effects. The term "derivative" refers to a compound formed by replacing an atom or atomic group in the molecule of a parent compound with another atom or atomic group, and is called a derivative of the parent compound. The term "camptothecin and its derivatives" generally encompasses camptothecin and camptothecin derivatives. Camptothecin exerts its pharmacological effect by irreversibly inhibiting topoisomerase I. Camptothecin derivatives include exatecan, irinotecan, topotecan, lurtotecan, siratecan, 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), and 7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin, and stereoisomers, salts, and esters thereof. Methods for synthesizing camptothecin and its analogs or derivatives are known and are summarized and described in US Pat. No. 5,244,903, which is incorporated herein by reference in its entirety.

[0062] In some embodiments, the payload is an auristatin or a derivative thereof, or maytansine or a derivative thereof, which has a tubulin inhibitory effect. The term "auristatin or a derivative thereof" generally encompasses auristatin F and auristatin F derivatives. Auristatin F derivatives include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). The term "maytansine or a derivative thereof" generally encompasses maytansine and maytansine derivatives. Maytansine derivatives include maytansine DM1, maytansine DM2, and maytansine DM4.

[0063] In some embodiments, the payload is exatecan, a camptothecin derivative. As a topoisomerase inhibitor, exatecan can act throughout the cell cycle and has strong penetration and good therapeutic effect on slow-growing solid tumors. Furthermore, the number of intracellular targets is much smaller than that of tubulin inhibitors, so when an ADC molecule delivers the same number of payloads to cells, it can achieve a better killing effect. Exatecan molecules are not substrates of P-gp, which is beneficial for reducing or alleviating the problem of drug resistance.

[0064] In some embodiments, the payload is a camptothecin of Formula II

[0065] [ka] wherein the camptothecin of formula II is attached to the linker by the nitrogen atom of the amino group on its cyclohexane ring.

[0066] The structure of the exatecan molecule is rigid and has poor hydrophilicity. Therefore, when the exatecan molecule is linked to the GGFG tetrapeptide linker commonly used in the prior art to prepare ADCs, polymerization between ADC molecules is likely to occur, which does not meet the development requirements for ADC drugs (Bioorg. Med. Chem. Lett. 26 (2016) pp. 1542-1545). Therefore, the selection and compatibility of the linker and payload affect the safety and stability of ADC drugs.

[0067] Without being bound by any theory, in the antibody-drug conjugates provided by the present disclosure, the hydrophilicity of the linker-payload structure is improved due to the presence of multiple hydrophilic groups in the linker, which can reduce to some extent the aggregation and precipitation of ADC molecules caused by hydrophobic payloads.

[0068] After the ADC molecule is endocytosed into a cell, either the payload or the linker (or part of the linker)-payload structure is released, depending on whether the linker is degraded. In some embodiments, the amino group on the cyclohexane ring of exatecan of Formula II is bonded to a carbonyl group in the ester group of the linker of Formula I, forming a carbamate-containing linker-payload structure. Without being bound by any theory, in the linker-payload structures provided by the present disclosure, after the ADC molecule is endocytosed into a cell, the linker is cleaved by a cathepsin (e.g., cathepsin B) to release an intermediate or active metabolite of Formula IV.

[0069] [ka] (Wherein R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 1 and 20) Form.

[0070] The PABC (para-aminobenzyloxycarbonyl) group in the intermediate or active metabolite of formula IV then undergoes 1,6-elimination to release exatecan (see Angew. Chem. Int. Ed. 2015, 54, 7492-7509). Thus, the linker-payload structure in the ADC molecules provided by the present disclosure has good in vivo stability and biological activity.

[0071] Without being bound by any theory, the enzyme cleavage site in the linker-payload structure may be an amide bond in the linker, for example, an amide bond between the carbon atom on which the substituent represented by R2 is located and the group represented by R3, or an amide bond in the group represented by R3.

[0072] In some embodiments, the antibody-drug conjugate has a ratio of the number of conjugated payload molecules to each molecule of antibody or antigen-binding fragment thereof, i.e., drug-to-antibody ratio, or DAR, between 1 and 10. In some embodiments, the DAR is between 1 and 2, 2 and 4, 4 and 6, 2 and 8, 4 and 8, 4 and 10, 6 and 10, 7 and 10, or 8 and 10, with exemplary DAR values ​​being 4, 6, 7.8, 9.2, or 9.92.

[0073] DAR represents the average number of conjugated payload or drug molecules per antibody molecule, i.e., the average number of conjugated drug molecules. For antibody-drug conjugates, DAR is an important factor that affects their efficacy and safety. The production of antibody-drug conjugates is carried out by specifying reaction conditions, such as the amount of starting materials and reagents used in the reaction, to achieve a certain number of conjugated payload molecules. Mixtures containing various numbers of conjugated payload molecules are usually obtained when antibody-drug conjugates are prepared. Unless otherwise specified, in this disclosure, DAR is defined as the average value, i.e., the average number of conjugated payload or drug molecules.

[0074] In some embodiments, the antibody-drug conjugate has the following structure:

[0075] [ka] (Ab represents an antibody or an antigen-binding fragment thereof, and n is the same as DAR) Contains one of the following:

[0076] The antibody or antigen-binding fragment thereof is attached to the linker via a reduced reactive thiol group, and optionally, the disulfide bond in the hinge region of the antibody or antigen-binding fragment thereof is reduced to a reactive thiol group and subsequently attached to the linker.

[0077] 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. MUC18 is expressed at detectable levels in endothelial cells in vascular tissues, including vascular smooth muscle. In particular, MUC18 is overexpressed in human malignant melanoma, especially in metastatic lesions and advanced primary tumors.

[0078] In this disclosure, an anti-MUC18 antibody or antigen-binding fragment thereof comprises a heavy chain (H) and a light chain (L), and the CDRs of the heavy and light chains are shown in Table 1.

[0079] [Table 1-1]

[0080] [Table 1-2] As used herein, the term "CDR" or "complementarity-determining region" is intended to mean non-contiguous antigen-binding sites within the variable regions of the heavy and / or light chains. The numbering of CDR residues follows the nomenclature of IMGT, see Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003). Here, the definitions encompass overlapping or subsets of amino acid residues compared to each other. Nevertheless, application of any one of the definitions to refer to the CDRs of an antibody or antigen-binding fragment thereof, or a grafted antibody or variant thereof, is intended to be within the scope of the term as defined and used herein.

[0081] In some embodiments, the anti-MUC18 antibody or antigen-binding fragment thereof comprises the heavy chain and light chain CDRs of the antibody encoded as CL070336, CL070335, CL070333, CL070319, CL070321, CL070320, CL070324, CL070341, CL070350, CL070349, CL070348, CL070370, or J253.

[0082] In some embodiments, the anti-MUC18 antibody or antigen-binding fragment thereof comprises an HCDR1 (heavy chain CDR1) having the amino acid sequence set forth in SEQ ID NO: 4, an HCDR2 (heavy chain CDR2) having the amino acid sequence set forth in SEQ ID NO: 16, an HCDR3 (heavy chain CDR3) having the amino acid sequence set forth in SEQ ID NO: 28, and an LCDR1 (light chain CDR1) having the amino acid sequence set forth in SEQ ID NO: 38, an LCDR2 (light chain CDR2) having the amino acid sequence STS, and an LCDR3 (light chain CDR3) having the amino acid sequence set forth in SEQ ID NO: 52.

[0083] In some embodiments, the anti-MUC18 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) that is at least 85% or 90% identical to any of the VHs of the reference antibody sequences shown in Table 2, and / or a light chain variable region (VL) that is at least 85% or 90% identical to any of the VLs of the reference antibody sequences shown in Table 2.

[0084] Identity can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as found 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. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0085] In some embodiments, there are no mutations in the framework regions, or mutations that do not affect the binding of the antibody variable region to the antigen. Mutations that do not affect the binding of the antibody variable region to the antigen may increase the binding affinity of the antibody to the antigen or may maintain it substantially unchanged. In some embodiments, the anti-MUC18 or CD44 v7 / 8 antibody or antigen-binding fragment thereof further comprises conservatively modified variants. Conservatively modified variants include individual substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of amino acids with chemically similar amino acids. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles. The following eight groups contain amino acids that are conservative substitutions 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 the antibody containing the amino acid sequence.

[0086] [Table 2] In some embodiments, the anti-MUC18 antibody or antigen-binding fragment thereof comprises a VH that is at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identical to a VH shown in Table 3, and a VL that is at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identical to a VL shown in Table 3. The antibodies shown in Table 3 are humanized sequences of CL070324 and J253, respectively.

[0087] [Table 3] In some embodiments, the anti-MUC18 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:84, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:85.

[0088] In some embodiments, the anti-MUC18 antibody or antigen-binding fragment thereof is humanized CL070324, humanized hJ253-03-1, or humanized hJ253-03-7, or a conservative variant thereof.

[0089] Here, the CDR sequences and antibody variable region sequences of the anti-MUC18 antibodies shown in SEQ ID NOs: 1 to 89 are recorded in U.S. Patent No. US2022041749 (SEQ ID NOs: 8 to 90, SEQ ID NOs: 1 to 6), which are incorporated herein by reference in their entirety.

[0090] In some embodiments, the antibody or antigen-binding fragment thereof targets CD44 v7 / 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 primarily functions in cell adhesion and cell-cell interactions. Several splice variants of CD44 are known, including CD44 v7 / 8, which is a variant containing exons 7 and 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.

[0091] In some aspects, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof specifically binds to a binding peptide in human CD44 v7 / 8, wherein the binding peptide comprises the amino acid sequence set forth as QAGRRMDMDSSHSIT (SEQ ID NO: 90).

[0092] In some aspects, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof does not bind to a binding peptide having the amino acid sequence of PISHPMGRGHQAGRR (SEQ ID NO: 91) and / or SHSITLQPTANPNTG (SEQ ID NO: 92) in human CD44 v7 / 8.

[0093] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises a heavy chain containing an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 93, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 94, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 95, and a light chain containing an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 96, an LCDR2 having the amino acid sequence RAN, and an LCDR3 having the amino acid sequence set forth 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:

[0094] [Table 4] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises a VH that is at least 85% or 90% identical to the VH of a reference antibody sequence shown in Table 5, and / or a VL that is at least 85% or 90% identical to the VL of a reference antibody sequence shown in Table 5.

[0095] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises a VH that is at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identical to the VH of HIS1H2-2a or HIS1H2-2, and a VL that is at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% identical to the VL of HIS1H2-2a or HIS1H2-2, a humanized sequence of a reference antibody shown in Table 5.

[0096] [Table 5] In some embodiments, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 100, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 101 or SEQ ID NO: 104, or a conservative variant thereof.

[0097] The binding peptide sequences, non-binding peptide sequences, CDR sequences, and variable region sequences of the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof set forth in SEQ ID NOs: 90-101 are recorded in PCT Patent Application No. WO2020159754 (SEQ ID NOs: 2-14), which is incorporated by reference in its entirety.

[0098] The antibody-drug conjugates provided herein exhibit excellent killing activity against tumor cells, and extremely high killing activity against tumor cells with high expression of MUC18 or CD44 v7 / 8.

[0099] The antibody-drug conjugates provided by the present disclosure exhibit excellent antibody-tumor activity. In some embodiments, the antibody-drug conjugates exhibit excellent anti-tumor activity in subjects suffering from neoplastic diseases accompanied by various MUC18 expression levels (low, medium, and high). The MUC18 expression level can be measured by the H-Score approach known to those skilled in the art. The score ranges from 0 to 300, with higher scores indicating higher expression. For example, low (H-Score = 10-99), medium (H-Score = 100-199), and high (H-Score = 200-300). Furthermore, the antibody-drug conjugates do not exhibit any obvious side effects in vivo.

[0100] It should be noted that the antibody-drug conjugates of the present disclosure may absorb water, retain adsorbed water, or become hydrated when left in the atmosphere or recrystallized. Such hydrated compounds and their salts are also included in the present disclosure. Furthermore, isotopically variant compounds labeled with various radioactive or non-radioactive isotopes are also included in the present disclosure. Atoms constituting more than one antibody-drug conjugate of the present disclosure may also contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include deuterium (H), tritium (H), iodine-125 (I), and carbon-14 (C). Furthermore, compounds of the present disclosure may be radiolabeled with radioactive isotopes such as tritium (H), iodine-125 (I), or carbon-14 (C). Radiolabeled compounds can be used as therapeutic or preventative agents, research reagents such as diagnostic reagents, and diagnostic agents, such as in vivo imaging agents. All isotopic variants of the antibody-drug conjugates of the present disclosure, whether radioactive or not, are intended to be encompassed within the scope of the present disclosure.

[0101] Preparation method The present disclosure provides a method for preparing an antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient, comprising the steps of: reducing an antibody or antigen-binding fragment thereof such that the interchain disulfide bonds thereof are at least partially reduced; and

[0102] [ka] (In the linker-payload the carbonyl group in the ester group of the linker of formula III is attached to the amino group of the payload in the antibody-drug conjugate; R1 and R2 are independently selected from hydrogen, methyl, and isopropyl groups; R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is hydrogen or benzyl, and n 1 represents an integer from 0 to 2, and n 2 represents an integer between 0 and 2, R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 1 and 20) and reacting the carbon atom at the 3-position of the maleimide-N-yl of

[0103] In antibody-drug conjugates, the interchain disulfide bonds are reduced to thiol groups, which then react with the reactive groups of the linker represented by Formula III in the linker-payload. In many practical cases, the linker of Formula III bearing the payload is attached to the same antibody or antigen-binding fragment thereof bearing a reactive thiol group. In some embodiments, the antibody or antigen-binding fragment thereof is reacted with a reducing agent such as dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to induce disulfide bonds in the antibody or antigen-binding fragment thereof and form reactive thiol groups. The amount of reducing agent can be 0.3 to 10 molar equivalents of the antibody or antigen-binding fragment thereof, e.g., 1 to 10, 3 to 10, 5 to 10, or 7 to 10 molar equivalents of the antibody or antigen-binding fragment thereof.

[0104] In some embodiments, the method further comprises reacting the antibody or antigen-binding fragment thereof with a reducing agent in a buffer solution containing a chelator, followed by adding a linker-payload solution and carrying out the reaction. The linker-payload is particularly a compound formed by linking a linker of Formula III and a payload, wherein an amino group (primary amino group) in the payload is bonded to a carbonyl group in the ester group of the linker of Formula III. In the linker-payload, the payload is selected from the payloads described in the section [Antibody-Drug Conjugates]. The term "chelator" refers to a complex capable of forming a complex having a cyclic structure with a metal atom or ion through a coordinate bond.

[0105] In some embodiments, a reducing agent is reacted with an antibody or antigen-binding fragment thereof in a buffer solution containing a chelating agent to produce an antibody or antigen-binding fragment thereof in which interchain disulfide bonds have been partially or completely reduced. Chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). The chelating agent is used at a concentration of 1 mM to 20 mM, e.g., 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. Components of the buffer solution may be buffer salts commonly used in the art, such as sodium phosphate, sodium borate, sodium acetate, or similar buffer salts.

[0106] The reaction of the antibody or antigen-binding fragment thereof with the reducing agent is carried out at a controlled pH. In some embodiments, the antibody or antigen-binding fragment thereof is reacted with the reducing agent at a pH of 5 to 9, optionally 6 to 8, 6 to 7, 6.5 to 7.5, or 7 to 8. For example, the reaction is carried out at a pH of about 7. The pH of the reaction solution can be adjusted using either acidic or basic chemicals, exemplary acidic or basic chemicals including acetic acid, hydrochloric acid, phosphoric acid, sulfuric acid, sodium bicarbonate, sodium carbonate, sodium hydroxide, and triethylamine.

[0107] The reaction of the antibody or antigen-binding fragment thereof with the reducing agent is carried out at a controlled temperature. For example, 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, or 35 to 38°C, e.g., about 37°C.

[0108] The linker-payload may be dissolved in an organic solvent selected from any one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP), or a combination thereof.

[0109] In some embodiments, the linker-payload solution is added to a buffer solution of the antibody or antigen-binding fragment thereof that has been reduced or has a reactive thiol group in an amount of 1% to 20% by volume based on the volume of the buffer solution of the antibody or antigen-binding fragment thereof. In some embodiments, the volume ratio of the linker-payload solution added 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 antigen-binding fragment thereof.

[0110] In some embodiments, the molar ratio of linker-payload to antibody or antigen-binding fragment thereof is 4-20, optionally 8-20. In some embodiments, the molar ratio of linker-payload to antibody or antigen-binding fragment thereof is 10-20, 14-20, 16-20, or 18-20.

[0111] In some embodiments, the temperature at which the antibody or antigen-binding fragment thereof is reacted 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.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof reacts with the linker-payload for 0.5 to 2 hours, 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.

[0113] The reaction can be terminated by inactivating unreacted linker-payload using a thiol-containing reagent, such as, but not limited to, cysteine ​​or N-acetyl-(L)-cysteine ​​(NAC). More specifically, 1 to 2 molar equivalents of the thiol-containing reagent containing the linker-payload can be added to the reaction solution and incubated at room temperature (10 to 30°C) for 10 to 30 minutes, thereby terminating the reaction.

[0114] When an antibody or antigen-binding fragment thereof has a thiol group, the antibody-drug conjugate can also be obtained by reacting the compound using a known method (for example, by the method described in Patent Publication US2016 / 297890 (for example, by the method described in paragraphs

[0336] to

[0374] ). Antibodies or antigen-binding fragments thereof having a thiol group can be obtained by methods well known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)).

[0115] The antibody-drug conjugates provided by the present disclosure can be obtained by the above-mentioned preparation methods. In some embodiments, the prepared antibody-drug conjugates are subjected to a purification process, including, but not limited to, gel filtration, for example, purification using a gel column.

[0116] kit The present disclosure provides kits comprising the above-described antibody-drug conjugates, or isomers, isotopic variants, pharmaceutically acceptable salts, prodrugs, solvates, or combinations thereof. The kits may further include instructions for using the antibody-drug conjugates described herein in the methods of the disclosure, e.g., in methods for treating neoplastic diseases.

[0117] The kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., single- or dual-chamber syringes), and test tubes. The container may be made of various materials, such as glass or plastic. The container holds the formulation. The container holding the formulation may be a single-use vial or a multi-use vial that allows repeated administration of the reconstituted formulation.

[0118] The kit may further include a label or package insert present on or associated with the container, which may indicate instructions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous (e.g., intravenous infusion), or other mode of administration to treat a neoplastic disease (e.g., cancer) in a subject. The kit may further include other materials desirable from commercial, therapeutic, and user standpoints, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0119] Pharmaceutical Composition The present disclosure provides a pharmaceutical composition comprising the above-described antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient.

[0120] The antibody-drug conjugate or pharmaceutical composition of the present disclosure can be administered in an appropriate manner depending on the specific application form, physicochemical properties, etc. of the pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition may be formulated in the form of a lyophilized preparation or a liquid preparation, which may contain appropriate formulation additives known in the art. For example, the above-mentioned pharmaceutical compositions usually contain one or more pharmaceutical carriers, such as sterile liquids, such as water and oils (including oils of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)). For intravenous administration of the pharmaceutical composition, water is a more typical carrier. In addition, aqueous solutions of saline, glucose, and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The pharmaceutical composition may also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, as needed. The mode of administration of pharmaceutical compositions is usually parenteral administration, which may be, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous injection. For example, pharmaceutical compositions may be administered by infusion or bolus injection. See, for example, Handbook of Pharmaceutical Excipients, 3rd Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. See, for example, Remington's Pharmaceutical Sciences, 16th Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0121] The pharmaceutical composition of the present disclosure 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 (e.g., 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. The other anti-cancer agent used for this purpose may be administered to an individual simultaneously with the antibody-drug conjugate, separately, or sequentially, or may be administered at various intervals. Exemplary other anti-cancer agents may be, but are not limited to, paclitaxel, cisplatin, vinblastine, etc., as long as the other anti-cancer agent has anti-tumor activity.

[0122] In accordance with the present disclosure, the active agent or pharmaceutical composition comprising the same can be administered to a subject via any suitable route of administration. For example, the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following discussion of routes of administration is provided solely to illustrate various embodiments and should not be construed as limiting in any way.

[0123] In some embodiments, the antibody-drug conjugate of the present disclosure is administered to a subject.As used herein, the term "subject" refers to human and non-human animals.Non-human animals include all vertebrates such as mice, rats, rabbits, cats, dogs, pigs, monkeys, chimpanzees, gorillas, etc.In some embodiments, the subject is a human.

[0124] How to use The present disclosure provides the use of antibody-drug conjugates, antibody-drug conjugates prepared by the above methods, pharmaceutical compositions comprising the antibody-drug conjugates, and kits in the manufacture of therapeutic agents for the diagnosis, prevention, and treatment of neoplastic diseases.

[0125] Neoplastic diseases include benign and malignant tumors (e.g., cancer). In some embodiments, the benign and malignant tumors express MUC18 and / or CD44 v7 / 8. The type of neoplastic disease to which the antibody-drug conjugate is applied is not limited to the above-mentioned cancer cells, as long as the cancer cells express a protein that can be recognized by the antibody or antigen-binding fragment thereof in the antibody-drug conjugate. In some embodiments, the neoplastic disease is a solid tumor that expresses MUC18 and / or CD44 v7 / 8.

[0126] In some embodiments, the neoplastic disease comprises melanoma, pharyngeal carcinoma, triple-negative breast cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, urothelial carcinoma, 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.

[0127] The present disclosure provides for 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 manufacture of a therapeutic agent targeting MUC18 and / or CD44 v7 / 8.

[0128] The present disclosure provides methods for diagnosing, preventing, and treating neoplastic disease, comprising administering to a subject a therapeutically effective amount of the above-described therapeutic agent. As used herein, the term "therapeutically effective amount" refers to an amount of the active ingredient, i.e., an ADC described herein, sufficient to induce the intended effect, including but not limited to disease treatment, as defined herein. The therapeutic dose of an antibody-drug conjugate will vary depending on the particular condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, size, sex, and weight), the duration of treatment, the nature of concurrent therapy (if any), the particular route of administration, and the knowledge of a medical professional. In some embodiments, the dosage of an antibody-drug conjugate, such as those described above, can be empirically determined in individuals who have received one or more administrations of the antibody or antigen-binding fragment thereof. In some embodiments, acceptable therapeutic doses of an antibody-drug conjugate are 0.1-30 mg / kg, 0.5-30 mg / kg, 1-30 mg / kg, 1-25 mg / kg, 0.1-25 mg / kg, 0.1-20 mg / kg, 1-20 mg / kg, or 0.5-20 mg / kg. In some embodiments, the dosing frequency is once every 12 hours, once every day, once every 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 every month, once every 2 months, or once every 3 months or more. The treatment dose and frequency of administration can vary depending on the treatment regimen.

[0129] The various aspects and preferences of the present disclosure may be combined with one another, unless they are essentially contradictory to one another, and the various aspects formed by the combination are considered to be part of the disclosure of this application.

[0130] The technical solutions of the present disclosure will be described more clearly and specifically below with reference to the following embodiments as examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present disclosure in any way. The scope of protection of the present disclosure is limited only by the claims. [Example]

[0131] The present disclosure is illustrated by, but not limited to, the following examples. Furthermore, these examples should not be construed as limiting in any way. Furthermore, reagents, solvents, and starting materials not specifically described herein can be easily obtained from commercially available sources.

[0132] Example 1: Preparation of compound LP-1

[0133] [ka] Step 1: Synthesis of intermediate 11-1 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 to a mixed solution of compound 11-1A (Mc-Val-Ala-OH, purchased from MedChemExpress Shanghai, 2.4 g, 6.29 mmol) and 11-1B (3.18 g, 6.29 mmol) at room temperature. The reaction solution was reacted at room temperature for 24 hours, and the solvent therein was removed in vacuo. The crude residue was then further purified by flash chromatography to give compound 11-1 (3.9 g, 71%). LC-MS (ESI, m / z): 868.49 (M+H).

[0134] Step 2: Synthesis of intermediate 11-2 Compound 11-1 (2 g, 2.3 mmol) was dissolved in anhydrous THF (tetrahydrofuran) (50 mL), and hydrogen fluoride-pyridine (4.6 g, 46 mmol) was added thereto under an argon atmosphere at 0 °C. The reaction mixture was then stirred at 0 °C for 2 hours. The reaction solution was quenched by adding water. The resulting mixture was extracted with DCM, and the organic phase therein was dried and concentrated. The residue was purified by silica gel chromatography to give compound 11-2 (1.1 g, 76%). LC-MS (ESI, m / z): 630.31 (M+H).

[0135] Step 3: Synthesis of intermediate 11-3 Compound 11-2 (700 mg, 1.11 mmol) was dissolved in anhydrous DMF (N,N-dimethylformamide) (4 mL), and DIPEA (N,N-diisopropylethylamine) (0.39 mL, 2.23 mmol) and 4,4'-dinitrodiphenyl carbonate (406 mg, 1.33 mmol) were added thereto under an argon atmosphere at room temperature. The reaction mixture was then stirred overnight at ambient temperature. The solvent in the reaction mixture was removed by concentration, and the product thus obtained was precipitated using MTBE (tert-butyl ether). The yellow solid was collected by filtration, washed with diethyl ether, and dried to give compound 11-3. LC-MS (ESI, m / z): 795.41 (M+H).

[0136] Step 4: Synthesis of intermediate 11-4 Compound 11-3 (300 mg, 0.44 mmol) was dissolved in anhydrous DMF (4 mL), and dry pyridine (1 mL) was added thereto, followed by the addition of exatecan mesylate (purchased from MedChemExpress Shanghai, 234 mg, 0.44 mmol) and HOBt (1-hydroxybenzotriazole) (60 mg, 0.44 mmol). The reaction mixture was stirred overnight at room temperature under an argon atmosphere. The product thus obtained was purified by pre-HPLC to give intermediate 11-4 (230 mg, 48%). LC-MS (ESI, m / z): 1091.53 (M+H).

[0137] Step 5: Synthesis of intermediate 11-5 Compound 11-4 (200 mg, 0.183 mmol) was dissolved in 1 mL of anhydrous DCM, and 300 μL of TFA (trifluoroacetic acid) was added thereto at 0 ° C. The reaction mixture was stirred at room temperature for 30 minutes, and the solvent therein was removed by concentration to obtain the TFA salt of intermediate 11-5, which could be used in the next step without further purification. LC-MS (ESI, m / z): 991.47 (M+H).

[0138] Step 6: Synthesis of compound LP-1 Compound 11-5 (120 mg, 0.109 mmol) was dissolved in 1 mL of anhydrous DMF, and Ac-Sar10-COOH (N-acetyldecasarcosine, 84 mg, 0.109 mmol) was added thereto, followed by HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 50 mg, 0.130 mmol) and DIPEA (38 μL, 0.22 mmol). The reaction mixture was stirred overnight at room temperature, and the solvent therein was removed by concentration. The crude product was purified by preparative high-performance liquid chromatography (pre-HPLC) to give compound LP-1 (74 mg, 38%). LC-MS (ESI, m / z): 1743.85 (M+H).

[0139] Example 2: Preparation of compound LP-2

[0140] [ka] The synthesis of compound LP2 was carried out according to the synthesis steps of compound LP-1. Starting material 11-1A was replaced with Mc-GGFG-OH (purchased from MedChemExpress Shanghai, where GGFG represents the amino acid sequence consisting of glycine-glycine-phenylalanine-glycine linked by a peptide bond), to obtain compound LP-2, which was a beige amorphous solid. LC-MS (ESI, m / z): 1891.90 (M+H).

[0141] Example 3: Preparation of compound LP-3

[0142] [ka] Compound LP-3 is an intermediate of LP-1. Step 6 was removed, and intermediate 11-5 was LP-3.

[0143] Example 4: Method for detecting antibody-drug conjugates The antibody-drug conjugates were concentrated, medium exchanged, purified, antibody concentrations were measured, the average number of drug molecules carried by each antibody was calculated, and the antibody-drug conjugates were identified according to the methods described below.

[0144] Procedure A: Concentration of antibodies or antibody-drug conjugates An ultrafiltration tube (Amicon Ultra, 50,000 MWCO, Millipore Corporation) was taken out, and the antibody or antibody-drug conjugate solution to be concentrated was added thereto. The ultrafiltration tube was centrifuged until the antibody or antibody-drug conjugate solution therein reached the desired volume, and then taken out.

[0145] Procedure B: Measurement of antibody concentration The absorbance of the antibody was measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific) according to the method specified by the manufacturer. The antibody concentration is the ratio of the antibody absorbance value at the detection wavelength to the absorption coefficient.

[0146] Procedure C: Antibody medium exchange According to the instructions provided by the manufacturer (Thermo Fisher Scientific), Zeba spin desalting columns (5 mL, 40K MWCO) were pre-equilibrated with phosphate-buffered saline containing sodium chloride (50 mM) and EDTA (2 mM) (referred to as "PBS7.0 / EDTA," 50 mM, pH 7.0). Two mL of sample was loaded onto each Zeba spin desalting column and centrifuged (1000 g, 4 minutes). The flow-through fraction was then collected and concentrated as per procedure A, the antibody concentration was determined as per procedure B, and the antibody concentration was adjusted with PBS7.0 / EDTA.

[0147] Procedure D: Purification of antibody-drug conjugates A Zeba spin desalting column (5 mL, 40K MWCO) was pre-equilibrated with storage buffer according to the manufacturer's instructions (Thermo Fisher Scientific). Histidine-acetate buffer (20 mM histidine, pH 5.5) containing 150 mM NaCl or phosphate buffer (50 mM, pH 7.0) containing 50 mM NaCl was used as the storage buffer. The reaction solution (approximately 2 mL) containing the antibody-drug conjugate was applied to the Zeba spin desalting column and centrifuged (1000 g, 4 min). The flow-through fraction (approximately 2 mL) was then collected, and the elution process was repeated twice to remove low molecular weight compounds, including unbound linker-payload and reducing agent.

[0148] Procedure E: Determination of antibody concentration and average number of drug molecules bound to each antibody (DAR value) in antibody-drug conjugates (1) The concentration of the conjugated drug in the antibody-drug conjugate can be obtained by measuring the UV absorption values ​​at 280 nm and 370 nm of an aqueous solution of the antibody-drug conjugate and calculating according to the following formula:

[0149] At any given wavelength, the total absorbance of a system is equal to the sum of the absorbances of all light-absorbing chemicals present in the system (additivity of absorbance). Therefore, assuming that the molar absorption coefficients of the antibody and drug remain unchanged before and after conjugation of the antibody and drug, the concentrations of antibody and drug in the antibody-drug conjugate can be expressed as follows:

[0150] A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A Equation (1) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C AEquation (2) A 280 represents the total absorbance value at 280 nm of the antibody-drug conjugate aqueous solution, and A 370 represents the total absorbance value at 370 nm of an aqueous solution of the antibody-drug conjugate. A,280 represents the absorbance value of the antibody at 280 nm, and A A,370 represents the absorbance value of the antibody at 370 nm, and A D,280 represents the absorption value of the conjugate precursor (drug) at 280 nm, and A D,370 represents the absorption value of the conjugate precursor at 370 nm, and ε A,280 represents the molar extinction coefficient of the antibody at 280 nm, and ε A,370 represents the molar extinction coefficient of the antibody at 370 nm, and ε D,280 represents the molar extinction coefficient of the conjugate precursor at 280 nm, and ε D,370 represents the molar extinction coefficient at 370 nm of the conjugate precursor, and C A represents the concentration of antibody in the antibody-drug conjugate, and C D represents the concentration of drug molecules in the antibody-drug conjugate.

[0151] In this case, ε A,280 , ε A,370 , ε D,280 , and ε D,370 are all known values ​​(calculated from the antibody sequence or measured by UV absorption of the compound). For example, ε A,280 can be calculated from the amino acid sequence of the antibody using known methods (Protein Science, 1995, Vol. 4, pp. 2411-2423). Antibodies usually have no absorbance at 370 nm, therefore, ε A,370 is usually 0. D,280 and εD,370 The value of C can be calculated by measuring the change in absorbance of the conjugate precursor using the Lambert-Beer law (absorbance = molar concentration × molar extinction coefficient × path length) at 280 nm and 370 nm, respectively. A and C Dis the absorbance value A of the antibody-drug conjugate at 280 nm and 370 nm 280 and A 370 and then simultaneously solving equations (1) and (2). Furthermore, the average number of drug molecules bound to each antibody (DAR value) can be obtained by measuring C D C A can be obtained by dividing by

[0152] Procedure F: Average number of drug molecules conjugated to each antibody (DAR value) - (2) The average number of drug molecules conjugated to each antibody molecule in an antibody-drug conjugate can be determined by liquid chromatography-mass spectrometry (LC-MS) analytical method, in addition to "Procedure E" above, as described below.

[0153] Preparation of Samples for High-Performance Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis (Reduction of Antibody-Drug Conjugates) Three microliters of 100 mM dithiothreitol (DTT) and 21 microliters of deionized water were added to the antibody-drug conjugate (approximately 5 mg / mL, 6 μL). The reaction solution was incubated in a 37°C water bath for 30 minutes, after which the disulfide bonds between the light and heavy chains and between the heavy chains in the antibody-drug conjugate were completely cleaved, and the resulting sample was ready for LC-MS detection and analysis.

[0154] High-Performance Liquid Chromatography Parameters Column type: Agilent PLRP-S, 1000Å, 50×2.1mm, 8μm Detection wavelength: 280 nm Bandwidth: 4nm Column temperature: 80℃ Autosampler thermostat: 5℃ Flow rate: 0.5mL / min Injection volume: 5μL Mobile phase A: 0.05% TFA, H2O Mobile phase B: 0.05% TFA, ACN 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) MS parameters Atomization gas temperature: 350℃ Atomization gas flow rate: 13 L / min Atomizer: 45psig Capillary voltage: 5000V Fragmentation voltage: 350V Mass-to-charge ratio range: 500~8000 m / z Acquisition speed: 1 spectrum / second Data analysis The light chain conjugated to the i drug molecule is denoted as Li and the heavy chain conjugated to the i small molecule is denoted as Hi, which can be determined by ESI scan of the mass spectrometer.

[0155] The corrected peak area ratio can be obtained by substituting the peak area percentage (%) of each strand into the following formula: i Peak area ratio of light chain conjugated to drug molecule = 100% × A Li / (A L0 +A L1 ) i Peak area ratio of heavy chain conjugated to drug molecule = 100% × A Hi / (A H0 +A H1 +A H2 +A H3 ) The average number of drug molecules conjugated to each antibody can be calculated by the following formula:

[0156] Average number of conjugated drug molecules = (L0 peak area ratio × 0 + L1 peak area ratio × 1 + H0 peak area ratio × 0 + H1 peak area ratio × 1 + H2 peak area ratio × 2 + H3 peak area ratio × 3) × 2 Procedure G: Average number of drug molecules bound to each antibody (DAR value) - (3) In addition to the above-described "Procedure E" and "Procedure F," the average number of drug molecules conjugated to each antibody molecule in an antibody-drug conjugate can also be determined using a hydrophobic interaction chromatography (HIC) analytical method described below.

[0157] The elution of antibody-drug conjugates from a hydrophobic interaction chromatography column is based on the difference in salt ion concentration in the eluate. As the salt ion concentration decreases, the number of small molecule drugs in the eluted antibody-drug conjugates increases, i.e., antibody-drug conjugates with low DAR values ​​are preferentially eluted. The peak order of each component is D0 (antibody not conjugated to any linker-payload), D2 (antibody conjugated to two linker-payloads), D4 (antibody conjugated to four linker-payloads), D6 (antibody conjugated to six linker-payloads), and D8 (antibody conjugated to eight linker-payloads). The content percentage of each component can be obtained by measuring the peak area ratio of each peak. The HIC-DAR of the corresponding sample is then calculated as follows: Average number of conjugated drug molecules = D0 peak area ratio × 0 + D2 peak area ratio × 2 + D4 peak area ratio × 4 + D6 peak area ratio × 6 + D8 peak area ratio × 8 Procedure H: Measurement of Aggregates in Antibody-Drug Conjugates Aggregates in the antibody-drug conjugates were detected using high performance liquid chromatography (HPLC) size exclusion chromatography, as follows.

[0158] High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system Detector: UV absorption spectrometer (detection wavelength: 280 nm) Column type: TOSOH TSKgel G3000SWXL (7.8 x 300 mm, 5 μm) Mobile phase: 200 mmol / L KHPO4, 150 mmol / L NaCl, 15% (v / v) isopropanol, pH 7.0 Flow rate: 0.75mL / min Analysis time: 18 minutes Column temperature: room temperature Injection volume: 50μg Data Analysis: The size-exclusion chromatogram of the quality control material (QC, 02-1 naked antibody, i.e., the antibody numbered 02-1 not conjugated to a linker-payload) is shown in Figure 1A. The size-exclusion chromatogram of the quality control material (QC, Hu1H2-2 naked antibody, i.e., the antibody numbered hu1H2-2 not conjugated to a linker-payload) is shown in Figure 1B. The aggregate content of the Hu1H2-2 naked antibody was 1.17%. The retention time of the major peak (single peak) of the 150 kDa quality control material was 9.5-10.5 min. The retention time of the aggregates should be earlier than the retention time of the monomers mentioned above.

[0159] Procedure I: Comparison of the hydrophobicity of antibody-drug conjugates The hydrophobicity of the antibody-drug conjugates was analyzed using high performance liquid chromatography hydrophobic interaction chromatography (HIC). The method was as follows: High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system Detector: ultraviolet absorption photometer (detection wavelength: 280 nm) Column type: TOSOH TSKgel Butyl-NPR (inner diameter 4.6 mm x 3.5 cm, 2.5 μm) Mobile phase A: 500 mol / L (NH4)2SO4, 50 mmol / L KHPO4, pH 7.0 Mobile phase B: 50 mmol / L KHPO4, 25% (v / v) isopropanol, pH 7.0 Analysis time: 25 minutes Column temperature: room temperature Elution procedure (B%): 0% to 25% (0 to 1 min), 25% (1 to 3 min), 25% to 80% (3 to 13 min), 80% (13 to 17 min), 80% to 0% (17 to 17.10 min), 0% (17.10 to 25 min) Injection volume: 10μL Data Analysis: The hydrophobic interaction chromatography chromatogram of the quality control material (QC, 02-1 naked antibody) is shown in Figure 2A. The hydrophobicity chromatogram of the quality control material (QC, Hu1H2-2 naked antibody) is shown in Figure 2B, and its retention time was 3.387 minutes. Samples with shorter retention times were less hydrophobic. The antibody-drug conjugate was more hydrophobic than the unconjugated naked antibody, and therefore its retention time was longer.

[0160] Example 5: Preparation of antibody-drug conjugate 02-1-LP1(DAR8) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.523 mL mg -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C. The antibody concentration after the medium replacement was 23.2 mg / mL. 0.482 mL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added to 2.155 mL of 02-1 antibody aqueous solution, and 4.31 mL of 50 mM phosphate buffer (pH 7.0, PBS 7.0) and 69.47 μL of 200 mM EDTA solution were simultaneously added. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0161] The VH sequence of the 02-1 antibody was the amino acid sequence shown in SEQ ID NO: 84, and its VL sequence was the amino acid sequence shown in SEQ ID NO: 85. For the purposes of preparation and / or detection in the Examples, the constant region sequence of human IgG1 was used as the constant region of the 02-1 antibody, the amino acid sequence shown in SEQ ID NO: 102 was selected as the heavy chain constant region sequence of the 02-1 antibody, and the amino acid sequence shown in SEQ ID NO: 103 was selected as the light chain constant region sequence of the 02-1 antibody.

[0162] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload LP-1 prepared in Example 1 was dissolved in N,N-dimethylacetamide (DMA), and 0.489 mL of the solution (corresponding to 14.2 times the equivalent amount of the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0163] Purification of antibody-drug conjugate: The reaction solution was purified by the method of operation D in Example 4 to obtain antibody-drug conjugate 02-1-LP1. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 4. D,280 = 6384 and ε D,370 =16180), and were characterized using Procedure F, Procedure H, and Procedure I.

[0164] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 12.13 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 7.78. The average number of conjugated payloads per antibody measured by procedure F was 7.80. Figure 3A shows an aggregate detection graph, and the aggregate content of antibody-drug conjugate 02-1-LP1 measured by procedure H was 1.18%. Figure 3B shows a hydrophobic interaction chromatography detection graph of antibody-drug conjugate 02-1-LP1, and the retention time of antibody-drug conjugate 02-1-LP1 measured by procedure I was 6.499 minutes.

[0165] Example 6: Preparation of antibody-drug conjugate Hu1H2-2-LP1(DAR8) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.54 mL mg -1 cm -1The medium was then replaced with PBS 7.0 / EDTA according to procedure C. The antibody concentration after the medium replacement was 9.37 mg / mL. 80 μL of 5 mM TCEP solution (equivalent to 10 times the antibody content) was added to 640.34 μL of Hu1H2-2 (HIS1H2-2) antibody aqueous solution, and 0.2 mL of 50 mM PBS 7.0 and 79.66 μL of deionized water were simultaneously added. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0166] The VH sequence of the Hu1H2-2 antibody was the amino acid sequence shown in SEQ ID NO: 100, and its VL sequence was the amino acid sequence shown in SEQ ID NO: 104. For the purposes of preparation and / or detection in the Examples, the amino acid sequence shown in SEQ ID NO: 102 was selected as the heavy chain constant region sequence of the Hu1H2-2 antibody, and the amino acid sequence shown in SEQ ID NO: 103 was selected as the light chain constant region sequence of the Hu1H2-2 antibody.

[0167] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload LP-1 prepared in Example 1 was dissolved in DMA, and then 72 μL of the solution (corresponding to 18 times the equivalent amount of antibody) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0168] Purification of antibody-drug conjugate: The reaction solution was purified by the method of procedure D in Example 4 to obtain antibody-drug conjugate Hu1H2-2-LP1. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 4. D,280 = 6384 and ε D,370 =16180), procedure H, and procedure I were used to characterize the samples.

[0169] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 6.74 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 9.20. Figure 4A shows an aggregate detection graph, and the aggregate content of antibody-drug conjugate Hu1H2-2-LP1 measured by procedure H was 1.24%. Figure 4B shows a hydrophobic interaction chromatography detection graph of antibody-drug conjugate Hu1H2-2-LP1, and the retention time of antibody-drug conjugate Hu1H2-2-LP1 measured by procedure I was 6.239 minutes.

[0170] Example 7: Preparation of antibody-drug conjugate Hu1H2-2-LP1(DAR4) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.54 mL mg -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C. The antibody concentration after the medium replacement was 23 mg / mL. 23.52 μL of 5 mM TCEP solution was added to 456.52 μL of Hu1H2-2 antibody aqueous solution, and 0.28 mL of 50 mM PBS 7.0 and 639.96 μL of deionized water were simultaneously added. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0171] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload LP-1 prepared in Example 1 was dissolved in DMA, and then 112 μL of the solution (equivalent to 8 times the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0172] Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 4. D,280 = 6384 and ε D,370 =16180) and characterized using procedure H.

[0173] For the prepared antibody-drug conjugate Hu1H2-2-LP1(DAR4), the concentration of the antibody-drug conjugate measured and calculated by procedure E was 6.14 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 3.59. Figure 5A shows an aggregate detection graph, and the aggregate content in antibody-drug conjugate Hu1H2-2-LP1(DAR4) measured by procedure H was 0.44%. Figure 5B shows a hydrophobic interaction chromatography detection graph for antibody-drug conjugate Hu1H2-2-LP1.

[0174] Example 8: Preparation of antibody-drug conjugate Hu1H2-2-LP2 (DAR8) Antibody reduction: The reduction method in Example 8 is the same as the antibody reduction method in Example 6. The amino acid sequence of the Hu1H2-2 antibody in Example 8 is the same as the amino acid sequence of the Hu1H2-2 antibody in Example 6.

[0175] Conjugation of antibody and linker-payload: The mixed solution in the antibody reduction step was incubated at 4°C for 10 minutes. The linker-payload LP-2 prepared in Example 2 was dissolved in DMA, and then 72 μL of the solution (corresponding to 18 times the equivalent amount of the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0176] Purification of antibody-drug conjugate: The reaction solution was purified by the method of procedure D in Example 4 to obtain antibody-drug conjugate Hu1H2-2-LP2. Characterization of antibody-drug conjugates: The obtained antibody-drug conjugates were analyzed by the above procedure E(ε D,280 =5814 and ε D,370 =14742), and were characterized using Procedure F, Procedure H, and Procedure I.

[0177] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 6.64 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 9.92. Figure 6A shows an aggregate detection graph, and the aggregate content in antibody-drug conjugate Hu1H2-2-LP2 measured by procedure H was 1.37%. Figure 6B shows a hydrophobic interaction chromatography detection graph of antibody-drug conjugate Hu1H2-2-LP2, and the retention time of antibody-drug conjugate Hu1H2-2-LP1 measured by procedure I was 6.561 minutes.

[0178] Example 9: Preparation of antibody-drug conjugate Hu1H2-2-LP3(DAR8) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.54 mL mg -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C. The antibody concentration after the medium replacement was 23.07 mg / mL. 70 μL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added to 325.10 μL of Hu1H2-2 antibody aqueous solution, and 0.3 mL of 50 mM PBS 7.0 and 804.90 μL of deionized water were simultaneously added. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0179] The amino acid sequence of the Hu1H2-2 antibody is the same as the amino acid sequence of the Hu1H2-2 antibody in Example 6. Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload LP-3 prepared in Example 3 was dissolved in DMA, and then 90 μL of the solution (corresponding to 18 times the equivalent amount of antibody) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0180] Purification of antibody-drug conjugate: The reaction solution was purified by the method of procedure D to finally obtain antibody-drug conjugate Hu1H2-2-LP3. Characterization of antibody-drug conjugates: The obtained antibody-drug conjugates were analyzed by the above procedure E(ε D,280 =5186 and ε D,370 =13688), and were characterized using Procedure F, Procedure H, and Procedure I.

[0181] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 4.10 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 8.50. Figure 7A shows an aggregate detection graph, and the aggregate content of antibody-drug conjugate Hu1H2-2-LP3 measured by procedure H was 3.37%. Figure 7B shows a hydrophobic interaction chromatography detection graph of antibody-drug conjugate Hu1H2-2-LP3, and the retention time of antibody-drug conjugate Hu1H2-2-LP3 measured by procedure I was 5.878 minutes.

[0182] Comparative Example 1: Preparation of antibody-drug conjugate 02-1-vc-MMAE(DAR4) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.523 mL mg -1 cm -1 The medium was then replaced with PBS7.0 / EDTA according to procedure C, and the antibody concentration after medium replacement was 31.6 mg / mL. 0.076 mL of 5 mM TCEP solution (equivalent to 2.3 times the antibody content) was added to 0.759 mL of 02-1 antibody aqueous solution, and 0.48 mL of 50 mM PBS7.0 and 1.084 mL of deionized water were simultaneously added thereto. After confirming that the pH of the solution was 7.0±0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours. The sequence of the 02-1 antibody was the same as that of the 02-1 antibody in Example 5.

[0183] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. Linker-payload vc-MMAE (DC Chemicals, DC7556) was dissolved in DMA, and then 0.322 mL of the solution (corresponding to 7 times the equivalent of the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0184] Purification of antibody-drug conjugate: The reaction solution was purified by the method of operation D in Example 4 to obtain antibody-drug conjugate 02-1-vc-MMAE. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were characterized using Procedure B, Procedure G, Procedure H and Procedure I of Example 4.

[0185] The concentration of the antibody-drug conjugate calculated by procedure B was 9.71 mg / mL. Figure 8A shows an aggregate detection graph, and the aggregate content in antibody-drug conjugate 02-1-vc-MMAE measured by procedure H was 2.74%. Figure 8B shows a hydrophobic interaction chromatography detection graph of antibody-drug conjugate 02-1-vc-MMAE. The average number of conjugated payloads per antibody in antibody-drug conjugate 02-1-vc-MMAE measured and calculated by procedures G and I was 3.99 (HIC-DAR).

[0186] Comparative Example 2: Preparation of antibody-drug conjugate rituximab-vc-MMAE(DAR4) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.5 mL mg -1 cm -1The medium was then replaced with PBS 7.0 / EDTA according to procedure C, resulting in an antibody concentration of 12 mg / mL. 0.015 mL of 5 mM TCEP solution (equivalent to 2.2 times the antibody content) was added to 0.438 mL of an aqueous solution of rituximab antibody (purchased from Shanghai Minbiotech Co., Ltd.), and 0.14 mL of 50 mM PBS 7.0 and 1.107 mL of deionized water were simultaneously added. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0187] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload vc-MMAE was dissolved in DMA, and then 0.049 mL of the solution (corresponding to 7 times the equivalent of the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0188] Purification of antibody-drug conjugate: The reaction solution was purified by the method of operation D in Example 4 to obtain the antibody-drug conjugate rituximab-vc-MMAE. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were characterized using Procedure B, Procedure G, Procedure H, and Procedure I of Example 4.

[0189] The concentration of the antibody-drug conjugate measured and calculated by procedure B was 3.78 mg / mL. Figure 9A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate rituximab-vc-MMAE measured by procedure H was 3.78%. Figure 9B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate rituximab-vc-MMAE, and the average number of conjugated payloads per antibody in the antibody-drug conjugate rituximab-vc-MMAE measured and calculated by procedure G was 4.39 (HIC-DAR).

[0190] Comparative Example 3: Preparation of antibody-drug conjugate Rituximab-LP1(DAR8) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.5 mL mg -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C, and the antibody concentration after the medium replacement was 11.8 mg / mL. 0.667 mL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added to 6.05 mL of rituximab antibody aqueous solution, and 1.02 mL of 100 mM PBS 7.0 and 2.46 mL of deionized water were simultaneously added thereto. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0191] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. Linker-payload LP-1 was dissolved in DMA, and then 0.667mL of the solution (corresponding to 14 times the equivalent of the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0192] Purification of antibody-drug conjugate: The reaction solution was purified by the method of Procedure D in Example 4 to obtain antibody-drug conjugate rituximab-LP-1. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 4. D,280 = 6384 and ε D,370 =16180), procedure H, and procedure I were used to characterize the samples.

[0193] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 6.23 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 7.30. Figure 10A shows an aggregate detection graph, and the aggregate content of the antibody-drug conjugate Rituximab-LP-1 measured by procedure H was 4.70%. Figure 10B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate Rituximab-LP1, and the retention time of the antibody-drug conjugate Rituximab-LP1 measured by procedure I was 7.094 minutes.

[0194] Comparative Example 4: Preparation of antibody-drug conjugate human IgG-GGFG-DXd (DAR8) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.35 mL mg -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C, resulting in an antibody concentration of 10 mg / mL. 0.327 mL of 5 mM TCEP solution (equivalent to 10 times the antibody content) was added to 2.45 mL of human IgG protein aqueous solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., Product No. SP001), followed by simultaneous addition of 0.7 mL of 50 mM PBS 7.0 and 23 μL of deionized water. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0195] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. Linker-payload GGFG-DXd (purchased from DC Chemicals, DC50025) was dissolved in DMA, and then 0.294 mL of the solution (corresponding to 18 times the equivalent of the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0196] Purification of antibody-drug conjugate: The reaction solution was purified by the method of Procedure D in Example 4 to obtain antibody-drug conjugate human IgG-GGFG-DXd (referred to as "human IgG-DXD"). Human IgG and HuIgG are used interchangeably in this disclosure.

[0197] Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 4. D,280 =5178 and ε D,370 =20217), procedure H, and procedure I were used to characterize the samples.

[0198] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 8.20 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 7.08. Figure 11A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate human IgG-DXD measured by procedure H was 4.25%. Figure 11B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate human IgG-DXD, and the retention time of the antibody-drug conjugate human IgG-DXD measured by procedure I was 8.222 minutes.

[0199] Comparative Example 5: Preparation of antibody-drug conjugate human IgG-LP1 (DAR8) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.35 mL mg -1 cm -1The medium was then replaced with PBS 7.0 / EDTA according to procedure C, resulting in an antibody concentration of 10 mg / mL. 0.267 mL of 5 mM TCEP solution (equivalent to 10 times the antibody content) was added to 1.33 mL of human IgG protein aqueous solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., Product No. SP001), and 0.4 mL of 50 mM PBS 7.0 was simultaneously added. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0200] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. Linker-payload LP-1 was dissolved in DMA, and then 0.2 mL of the solution (corresponding to 15 times the equivalent amount of antibody) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0201] Purification of antibody-drug conjugate: The reaction solution was purified by the method of Procedure D in Example 4 to obtain antibody-drug conjugate human IgG-LP1. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 4. D,280 = 6384 and ε D,370 =16180), procedure H, and procedure I were used to characterize the samples.

[0202] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 11.08 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 9.15. Figure 12A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate human IgG-LP1 measured by procedure H was 4.38%. Figure 12B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate human IgG-LP1, and the retention time of the antibody-drug conjugate human IgG-LP1 measured by procedure I was 6.213 minutes.

[0203] Comparative Example 6: Preparation of antibody-drug conjugate human IgG-LP1 (DAR4) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.35 mL mg -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C. The antibody concentration after the medium replacement was 15 mg / mL. 34.02 μL of 5 mM TCEP solution (equivalent to 2.43 times the antibody content) was added to 0.7 mL of human IgG protein aqueous solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., Product No. SP001), and 0.28 mL of 50 mM PBS 7.0 was simultaneously added. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0204] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. Linker-payload LP-1 was dissolved in DMA, and then 0.112 mL of the solution (equivalent to 8 times the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0205] Purification of antibody-drug conjugate: The reaction solution was purified by the method of Procedure D in Example 4 to obtain antibody-drug conjugate human IgG-LP1 (DAR4). Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 4. D,280 = 6384 and ε D,370 =16180), procedure H, and procedure I were used to characterize the samples.

[0206] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 5.75 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 3.53. Figure 13A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate human IgG-LP1(DAR4) measured by procedure H was 1.90%. Figure 13B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate human IgG-LP1.

[0207] Comparative Example 7: Preparation of antibody-drug conjugate human IgG-LP2 (DAR8) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.35 mL mg -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C, resulting in an antibody concentration of 10 mg / mL. 0.093 mL of 5 mM TCEP solution (equivalent to 10 times the antibody content) was added to 0.467 mL of human IgG protein aqueous solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., Product No. SP001), followed by simultaneous addition of 0.2 mL of 50 mM PBS 7.0 and 0.24 mL of deionized water. After confirming that the pH of the solution was 7.0 ± 0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours.

[0208] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. Linker-payload LP-2 was dissolved in DMA, and then 0.07mL of the solution (corresponding to 15 times the equivalent amount of antibody) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0209] Purification of antibody-drug conjugate: The reaction solution was purified by the method of Procedure D in Example 4 to obtain antibody-drug conjugate human IgG-LP2. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 4. D,280 =5814 and ε D,370 =14742), procedure H, and procedure I were used to characterize the product.

[0210] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 4.68 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 9.18. Figure 14A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate human IgG-LP2 measured by procedure H was 2.92%. Figure 14B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate human IgG-LP2, and the retention time of the antibody-drug conjugate human IgG-LP2 measured by procedure I was 6.506 minutes.

[0211] Comparative Example 8: Preparation of antibody-drug conjugate human IgG-LP3 (DAR8) Antibody reduction: The antibody medium was diluted with the same solution as in procedure B of Example 4 (the extinction coefficient of the antibody at 280 nm is 1.35 mL mg -1 cm -1 The medium was then replaced with PBS 7.0 / EDTA according to procedure C, resulting in an antibody concentration of 18 mg / mL. 0.672 mL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added to 4 mL of human IgG protein aqueous solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., Product No. SP001), and 1.44 mL of 50 mM PBS 7.0 and 1.088 mL of deionized water were simultaneously added. After confirming that the pH of the solution was 7.0±0.1, the mixture was placed in a 37°C environment and allowed to react for 2 hours. The source of human IgG protein was the same as that of human IgG protein in Comparative Example 4.

[0212] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. Linker-payload LP-3 was dissolved in DMA, and then 0.672 mL of the solution (corresponding to 14 times the equivalent of the antibody content) was added to the mixed solution. The reaction of the mixed solution was continued at 22°C for 30 minutes.

[0213] Purification of antibody-drug conjugate: The reaction solution was purified by the method of Procedure D in Example 4 to obtain antibody-drug conjugate human IgG-LP3. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 4. D,280 =5186 and ε D,370 =13688), procedure H, and procedure I.

[0214] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 7.42 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 7.28. Figure 15A shows an aggregate detection graph, and the aggregate content in the antibody-drug conjugate human IgG-LP3 measured by procedure H was 5.37%. Figure 15B shows a hydrophobic interaction chromatography detection graph of the antibody-drug conjugate human IgG-LP3, and the retention time of the antibody-drug conjugate human IgG-LP3 measured by procedure I was 6,450 minutes.

[0215] Comparative Example 9: Preparation of antibody-drug conjugate Hu1H2-2-GGFG-DXD(DAR8) Reduction of antibody: The reduction in Comparative Example 9 was the same as that in Example 6. The sequence of the Hu1H2-2 antibody was the same as that of the Hu1H2-2 antibody in Example 6.

[0216] Conjugation of antibody and linker-payload: The above mixed solution was incubated at 4°C for 10 minutes. The linker-payload GGFG-DXd was dissolved in DMA, and then 72 μL of the solution (corresponding to 18 times the equivalent of the antibody content) was added to the mixed solution. The reaction of the mixed solution continued at 22°C for 30 minutes.

[0217] Purification of antibody-drug conjugate: The reaction solution was purified by the method of procedure D to obtain antibody-drug conjugate Hu1H2-2-GGFG-DXd (referred to as "Hu1H2-2-DXD").

[0218] Characterization of antibody-drug conjugates: The obtained antibody-drug conjugates were analyzed by the above procedure E(ε D,280 =5178 and ε D,370 =20217), procedure H, and procedure I were used to characterize the samples.

[0219] The concentration of the antibody-drug conjugate measured and calculated by procedure E was 6.53 mg / mL, and the average number of conjugated payloads per antibody measured and calculated by procedure E was 6.75. As shown in Figure 16A, the aggregate content of antibody-drug conjugate Hu1H2-2-DXD measured by procedure H was 1.70%. As shown in Figure 16B, the retention time of antibody-drug conjugate Hu1H2-2-DXD measured by procedure I was 7.764 minutes.

[0220] Experimental Example 1: Endocytosis of 02-1-LP1 by MUC18-overexpressing tumor cells 50 μg / mL of 02-1-LP1 was combined with A375 cells (purchased from the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences), HMVII cells (purchased from the Center for Type Culture Collection, Biovector NTCC), SK-MEL-2 cells (purchased from Shanghai Xunqing Biotechnology Co., Ltd.), and GAK cells (purchased from the Center for Type Culture Collection, Biovector NTCC). After washing away excess ADC, the cells were cultured in a 37°C incubator. Median fluorescence intensity (MFI) was measured by flow cytometry at 0, 0.5, 1, 2, 4, and 8 hours. As shown in Figure 17, the 02-1-LP1 antibody could be endocytosed by A375 (A), HMVII (B), SK-MEL-2 (C), and GAK (D) cells.

[0221] Experimental Example 2: In vivo inhibitory effect of A375 xenograft tumor and its effect on body weight change in mice Six-week-old Balb / c mice were purchased from Jiangsu GemPharmatech Co. Ltd., and each mouse was subcutaneously inoculated with 4 million A375 cells to establish a MUC18-positive A375 melanoma mouse xenograft model. 24 days after inoculation (average tumor volume was approximately 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 Comparative Example 5 were intravenously injected, and the vehicle group was set as a negative control. After administration, 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. Because A375 cells express little CD20, rituximab was used as an isotype control for the 02-1 antibody at that time.

[0222] Figures 18A1 and 18A2 show the growth inhibitory effects of the MUC18-targeting antibody-drug conjugates 02-1-LP1 and 02-1-vc-MMAE on A375 xenografts. Figure 18A1 shows that 02-1-vc-MMAE reduces tumor growth rate, while 02-1-LP1 significantly reduces tumor volume. Figure 18A2 is a partial view of Figure 18A1 showing the curves of 02-1-LP1, 02-1-vc-MMAE, and vehicle groups to further clarify the trend graph of the active ingredients.

[0223] Figures 18B1 and 18B2 showed that the MUC18-targeting antibody-drug conjugates 02-1-LP1 and 02-1-vc-MMAE did not significantly affect the body weight of mice. Figure 18B2 was a subplot of 18B1 showing the curves of the 02-1-LP1, 02-1-vc-MMAE, and vehicle groups to further clarify the trend graph of the active ingredient.

[0224] Experimental Example 3: Toxicological study of 02-1-LP1 and 02-1-vc-MMAE in cynomolgus monkeys Experiment on repeated administration of 02-1-LP1 (prepared in Example 5) to cynomolgus monkeys: Six cynomolgus monkeys (purchased from Guangxi Frontier Biotechnology Co., Ltd.) were randomly divided into three groups, with one male and one female in each group. Vehicle, 10 mg / kg of 02-1-LP1, and 30 mg / kg of 02-1-LP1 were intravenously injected on days 1, 22, and 43, respectively. During the experiment, the animals were observed for any abnormalities, and their blood samples were collected for hematological and blood biochemistry index analysis. On day 50, the animals were euthanized and sampled for pathological analysis. As shown in Table 6, in this experiment, none of the test substances at any dose caused animal death. The target organs for the test substances were reticulocytes, gastrointestinal tract, kidney, and spleen. The HNSTD (highest dose without severe toxicity) was 30 mg / kg.

[0225] Repeated administration study of 02-1-vc-MMAE (prepared in Comparative Example 1) to cynomolgus monkeys: Six cynomolgus monkeys were randomly divided into three groups, each containing one male and one female. 3 mg / kg of 02-1-vc-MMAE, 6 mg / kg of 02-1-vc-MMAE, and 10 mg / kg of 02-1-vc-MMAE were intravenously injected on days 1 and 22, respectively. During the experiment, the animals were observed for any abnormalities, and their blood samples were collected for hematological and blood biochemistry index analysis. On day 43, the animals were euthanized and samples were collected for pathological analysis. As shown in Table 6, in this study, two animals in the high-dose group (10 mg / kg) died 10 days after the first administration, and no deaths were observed in the 3 mg / kg and 6 mg / kg groups. The target organs for the test substance were reticulocytes, leukocytes, and skin. The HNSTD (highest non-severely toxic dose) was 6 mg / kg.

[0226] [Table 6] 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.

[0227] Combined with Experimental Example 2, 02-1-LP1 has a higher tolerable dose and better safety than 02-1-vc-MMAE, and at the same time, a lower dose of 02-1-LP1 can achieve better antitumor effects.

[0228] Experimental Example 4: In vitro killing assay of ADC against Detroit 562 cells Detroit 562 cells (purchased from Nan Jing Cobioer Biosciences Co., Ltd.) were cultured to a cell density of 80%. The cells were harvested and plated in a 96-well plate at a cell density of 2–5 × 10. 4 The concentration was adjusted to 1 / mL. 100 μL of cells were seeded into each well, and the ADC molecule was serially diluted 3-fold using an initial concentration of 300 nM. After dilution was complete, the ADC molecule was added to the cell culture medium and cultured for 5 days, during which time cell apoptosis was monitored periodically. After 5 days, 15 μL of CCK-8 kit stock solution was added to the 96-well plate and incubated for 0.5 to 2 hours in a 37°C incubator. The absorbance at 450 nm was measured, and a cell viability curve was plotted based on the OD (optical density) reading and the ADC dilution gradient.

[0229] 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 were selected as ADC molecules, where human IgG was used as an isotype control for the Hu1H2-2 antibody, and DXD was used as a positive drug.

[0230] Figure 19 shows the in vitro killing effects of the above ADC molecules against the head and neck squamous cell carcinoma cell line Detroit562, and Figures 19A1 and 19A2 are partial views of Figure 19. Figure 19A1 shows that Hu1H2-2-LP2 and Hu1H2-2-LP1 of the present disclosure have superior killing effects against Detroit562 in vitro compared to the positive control Hu1H2-2-DXD. Figure 19A2 shows that human IgG-LP2 and human IgG-LP1 have better killing effects than human IgG-DXD in vitro, and that linker-payload LP-1 and linker-payload LP-2 have superior killing effects against Detroit562 in vitro compared to GGF-DXd.

[0231] Experimental Example 5: In vivo efficacy of Detriot562 in a CDX mouse model and the effect of the ADC on body weight change in mice Six-week-old Balb / c nude mice were purchased from Jiangsu GemPharmatech Co., Ltd. Five million Detriot 562 cells were subcutaneously inoculated into each mouse to establish a CDX (cell-derived xenograft) mouse model. Ten days after inoculation (average tumor volume was approximately 150 mm), 3 The tumors were intravenously injected with 10 mg / kg of human IgG-LP1 prepared in Comparative Example 5, 10 mg / kg of Hu1H2-2-LP1 prepared in Example 6, and 10 mg / kg of Hu1H2-2-LP2 prepared in Example 8. After administration, tumor volumes were measured twice a week using Vernier calipers, and calculated according to the following formula: TV = (length × width). 2 / 2.

[0232] Figure 20 shows the in vivo efficacy data of Hu1H2-2-LP1, Hu1H2-2-LP2, and human IgG-LP1 in a mouse model of head and neck squamous cell carcinoma established using Detroit 562 cells. The results showed that Hu1H2-2-LP1 and Hu1H2-2-LP2 could effectively inhibit tumor growth in mice.

[0233] Figure 21 shows the effects of Hu1H2-2-LP1, Hu1H2-2-LP2, and human IgG-LP1 on mouse body weight. During the experimental period, the body weight of mice in the Hu1H2-2-LP1 and Hu1H2-2-LP2 groups continued to increase, while the body weight of the control group (human IgG-LP1) fluctuated. The results showed that Hu1H2-2-LP1 and Hu1H2-2-LP2 had little effect on the body weight changes of mice.

[0234] Experimental Example 6: In vivo efficacy of the PC-9 CDX mouse model and the effect of ADC on mouse body weight change Six-week-old Balb / c nude mice were purchased from Jiangsu GemPharmatech Co. Ltd. Five million PC-9 cells (purchased from Meisen Chinese Tissue Culture Collections) were subcutaneously inoculated into each mouse to obtain CDX models. Thirteen days after inoculation (average tumor volume was approximately 200 mm), 3 The tumor volumes were measured twice a week using a Vernier caliper, and calculated according to the following formula: TV = (length x width). 2 / 2.

[0235] Figure 22 shows the in vivo efficacy data of the above ADCs in a lung cancer model mouse established with PC-9 cells. The results showed that Hu1H2-2-LP1, Hu1H2-2-LP1(DAR4), and Hu1H2-2-LP3 could effectively inhibit tumor growth in mice.

[0236] Figure 23 shows the effect of the above ADCs on the body weight of lung cancer model mice established with PC-9. The results showed that Hu1H2-2-LP1, Hu1H2-2-LP1(DAR4), and Hu1H2-2-LP3 had little effect on the body weight of the mice and no obvious gastrointestinal toxicity.

[0237] Experimental Example 7: In vivo inhibitory effect on SCC-9 human head and neck squamous cell carcinoma CDX model and its effect on body weight change in mice Six-week-old Balb / c nude mice were purchased from Shanghai BK / KY Biotechnology Co., Ltd. Eight million SCC-9 cells (purchased from ATCC) were subcutaneously inoculated into each mouse to obtain CDX models. 29 days after inoculation (average tumor volume was approximately 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's Phosphate Buffered Saline) was the negative control. The administration was once a week for two times (QW x 2). After administration, 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 x width). 2 / 2.

[0238] Figure 24A shows that 02-1-LP1 can effectively inhibit tumor growth in the SCC-9 CDX model, and Figure 24B shows that 02-1-LP1 has little effect on body weight in SCC-9 model mice.

[0239] Experimental Example 8: In vivo inhibitory effect on Huh-7 human liver cancer CDX model and its effect on body weight change in mice Six-week-old Balb / c nude mice were purchased from Shanghai BK / KY Biotechnology Co., Ltd. Five million Huh-7 cells (purchased from Shanghai BioGene Biotech Co., Ltd.) were subcutaneously inoculated into each mouse to obtain CDX models. Nine days after inoculation (average tumor volume was approximately 230 mm), 3 The tumor volumes were measured twice a week using a Vernier caliper, and calculated according to the following formula: TV = (length x width). 2 / 2.

[0240] Figure 25A shows that 02-1-LP1 can effectively inhibit tumor growth in the Huh-7 CDX model, and Figure 25B shows that 02-1-LP1 has little effect on mouse body weight in the Huh-7 model.

[0241] Experimental Example 9: In vivo inhibitory effect of LD1-0015-200617 on human esophageal squamous cell carcinoma PDX model and its effect on body weight change in mice LD1-0015-200617 (LideBiotech CO., LTD) is a human esophageal squamous cell carcinoma patient-derived xenograft model (H-Score = 75) expressing human MUC18. Six- to eight-week-old Nu / Nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. LD1-0015-200617 tumors were dissected into approximately 3 mm x 3 mm x 3 mm pieces and subcutaneously implanted into the flanks of the mice. Thirty-nine days after implantation (average tumor size approximately 171 mm) 3), human IgG-LP1 prepared in Comparative Example 5, and 02-1-LP1 prepared in Example 5 were each intravenously injected. The dose for all was 10 mpk, once a week for three times (QW x 3). After administration, tumor volume was measured twice a week using a Vernier caliper, and tumor volume was calculated according to the following formula: TV = (length x width). 2 / 2.

[0242] Figure 26A shows that 02-1-LP1 can effectively inhibit tumor growth in the LD1-0015-200617 PDX model, and Figure 26B shows that 02-1-LP1 has little effect on mouse body weight in the LD1-0015-200617 PDX model.

[0243] Experimental Example 10: In vivo inhibitory effect of LD1-0016-390730 on human esophageal adenocarcinoma PDX model and its effect on body weight change in mice LD1-0016-390730 (LideBiotech CO., LTD) is a human esophageal adenocarcinoma patient-derived xenograft model (H-Score = 115) expressing human MUC18. Six- to eight-week-old Nu / Nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. LD1-0016-390730 tumors were dissected into approximately 3 mm x 3 mm x 3 mm pieces and subcutaneously implanted into the flanks of the mice. Forty-five days after implantation (average tumor size approximately 191 mm), tumor size was approximately 191 mm. 3 ), 02-1-LP1 prepared in Example 5 was intravenously injected. The administration dose was 10 mpk, once a week for two times (QW x 2). After administration, the tumor volume was measured twice a week using a Vernier caliper, and the tumor volume was calculated according to the following formula: TV = (length x width). 2 / 2.

[0244] Figure 27A shows that 02-1-LP1 can effectively inhibit tumor growth in the LD1-0016-390730 PDX model, and Figure 27B shows that 02-1-LP1 has little effect on mouse body weight in the LD1-0016-390730 PDX model.

[0245] Experimental Example 11: In vivo inhibitory effect of LD1-2025-362797 on human small cell lung cancer PDX model and its effect on body weight change in mice LD1-2025-362797 (LideBiotech CO., LTD) is a human small cell lung cancer patient-derived xenograft model (H-Score = 110) expressing human MUC18. Six- to eight-week-old Nu / Nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. LD1-2025-362797 tumors were dissected into approximately 3 mm x 3 mm x 3 mm pieces and subcutaneously implanted into the flanks of the mice. 21 days after implantation (average tumor size approximately 144 mm) 3 ), and the first dose of 10 mpk of 02-1-LP1 prepared in Example 5 was intravenously injected. 3 Once tumors re-grew (49 days after the first dose), a second dose of 10mpk 02-1-LP1 was also intravenously injected. After dosing, tumor volumes were measured twice a week with Vernier calipers, and tumor volume was calculated according to the formula: TV = (length x width). 2 / 2.

[0246] Figure 28A shows that 02-1-LP1 can effectively inhibit tumor growth in the LD1-2025-362797 PDX model. Notably, tumors decreased again after a second dose of 02-1-LP1, indicating that 02-1-LP1 remains active against re-grown tumors. Figure 28B shows that 02-1-LP1 had little effect on mouse body weight in the LD1-2025-362797 PDX model.

[0247] Experimental Example 12: In vivo inhibitory effect of LD1-2009-362263 on human triple-negative breast cancer PDX model and its effect on mouse body weight change LD1-2009-362263 (LideBiotech CO., LTD) is a human triple-negative breast cancer patient-derived xenograft model (H-Score = 240) expressing human MUC18. Six- to eight-week-old Nu / Nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. LD1-2009-362263 tumors were dissected into approximately 3 mm x 3 mm x 3 mm pieces and subcutaneously implanted into the flanks of the mice. Forty-nine days after implantation (average tumor size approximately 152 mm) 3 ), 02-1-LP1 prepared in Example 5 was intravenously injected. The administration dose was 10 mpk, once a week for two times (QW x 2). After administration, the tumor volume was measured twice a week using a Vernier caliper, and the tumor volume was calculated according to the following formula: TV = (length x width). 2 / 2.

[0248] Figure 29A shows that 02-1-LP1 can effectively inhibit tumor growth in the LD1-2009-362263 PDX model, and Figure 29B shows that 02-1-LP1 has little effect on mouse body weight in the LD1-2009-362263 PDX model.

[0249] Experimental Example 13: In vivo inhibitory effect of LD1-0060-200770 on human cholangiocarcinoma PDX model and its effect on body weight change in mice LD1-0060-200770 (LideBiotech CO., LTD) is a human cholangiocarcinoma patient-derived xenograft model (H-Score = 15) expressing human MUC18. Six- to eight-week-old Nu / Nu mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. LD1-0060-200770 tumors were sectioned into approximately 3 mm x 3 mm x 3 mm pieces and subcutaneously implanted into the flanks of the mice. Forty-two days after implantation (average tumor size approximately 155 mm) 3 ), 02-1-LP1 prepared in Example 5 was intravenously injected. The administration dose was 10 mpk, once a week for two times (QW x 2). After administration, the tumor volume was measured twice a week using a Vernier caliper, and the tumor volume was calculated according to the following formula: TV = (length x width). 2 / 2.

[0250] Figure 30A shows that 02-1-LP1 can effectively inhibit tumor growth in the LD1-0060-200770 PDX model, and Figure 30B shows that 02-1-LP1 has little effect on mouse body weight in the LD1-0060-200770 PDX model.

[0251] Experimental Example 14: In vivo inhibitory effect on OV-10-0073 human ovarian cancer PDX model and its effect on body weight change in mice OV-10-0073 (WuXi AppTech (Shanghai) Co., Ltd.) is a human ovarian cancer patient-derived xenograft model (H-Score = 40) expressing human MUC18. 6- to 8-week-old BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. OV-10-0073 tumors were grown to approximately 30 mm. 3 The tumors were then dissected into small pieces and implanted subcutaneously into the flanks of mice. 34 days after implantation (average tumor size approximately 185 mm 3), 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 in single doses. After administration, tumor volumes were measured twice a week with Vernier calipers, and tumor volumes were calculated according to the following formula: TV = (length × width). 2 / 2.

[0252] Figure 31A shows that 02-1-LP1 can effectively inhibit tumor growth in the OV-10-0073PDX model, and Figure 31B shows that 02-1-LP1 has little effect on mouse body weight in the OV-10-0073PDX model.

[0253] The above experimental results demonstrate that in the ADCs provided by the present disclosure, after conjugation with exatecan, the linker can be coupled to antibodies targeting MUC18 or CD44 v7 / 8 using a simple chemical method. Compared with conventional random conjugation methods, the DAR values ​​(DAR8) of the anti-MUC18 antibody-drug conjugates or anti-CD44 v7 / 8 antibody-drug conjugates obtained using such linkers are higher. HIC detection showed that the chromatographic peaks of the ADCs provided by the present disclosure were narrower than those of the linker-payloads of vc-MMAE or GGF-DXd, suggesting that the prepared products were highly homogeneous and hydrophilic. Compared with conventional vc-MMAE and GGF-DXd conjugates, the in vitro tumor cell growth inhibitory activity of the conjugates of the present disclosure was improved or maintained in terms of biological activity and safety.

[0254] The ADCs prepared in the present disclosure possess specific MUC18- or CD44 v7 / 8-dependent antitumor activity and exhibit extremely high killing activity against tumor cells with high expression of MUC18 or CD44 v7 / 8. The MUC18-specific ADCs prepared in the present disclosure exhibited increased HNSTD, improved safety, and reduced toxic and side effects. Surprisingly, the results of Experimental Examples 9 to 14 also demonstrated that the MUC18-specific ADCs prepared in the present disclosure exhibited excellent killing activity against tumor cells with various expression levels (low, medium, and high) of MUC18.

[0255] The ADCs prepared in this disclosure have superior in vivo antitumor efficacy compared to ADCs conjugated with vc-MMAE or GGFG-DXd, and at the same time, exhibit significantly better safety than ADCs conjugated with vc-MMAE and have a similar MTD (maximum tolerated dose) to the GGFG-DXd ADC (Yusuke Ogitani, et al., Cancer Res. 2016 Oct. 15; 22(20):5097-5108). This drug conjugate is expected to have a wider therapeutic window than the existing clinical molecules vc-MMAE or GGFG-DXd.

[0256] The ADCs provided by the present disclosure have better in vitro stability and reduced aggregation during the preparation process. It is expected that ADCs having a linker of Formula I will have better stability in plasma, and the ADCs may have better in vivo drug metabolism properties, such as a longer half-life, a smaller amount of free small molecule toxins, etc.

Claims

1. Anti-MUC18 antibody or anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof, a payload, and a linker of formula I 【Chemistry 1】 (In the formula, the succinimidyl group of the linker of Formula I forms a thioether bond with a thiol group obtained by reduction of the interchain disulfide chain of the antibody or antigen-binding fragment thereof; The carbonyl group in the ester group of the linker of formula I is bonded to the amino group of the payload; R １ and R ２ are independently selected from hydrogen, methyl, and isopropyl groups; R ３ Ha-(CR ５ HCONH)n １ - (CH ２ CONH)n ２ represents - or a single bond, R ５ is hydrogen or benzyl, and n １ represents an integer of 0 to 2, and n ２ represents an integer from 0 to 2, R ４ is a methylamino group or -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer from 1 to 20) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof.

2. R ４ - (NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer of 8 to 15; R ４ - (NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer from 10 to 12; and / or R ３ But-(CR ５ HCONH)n １ - (CH ２ CONH)n ２ represents - or a single bond, R ５ is benzyl, and n １ represents 1 or 2, and n ２ represents 1 or 2; R ４ - (NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer from 8 to 15; and / or R ３ represents a single bond, R ４ - (NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer from 8 to 15; and / or R ３ represents a single bond, R ４ represents a methylamino group, The antibody-drug conjugate of claim 1.

3. The linker is: 【Chemistry 2】 The antibody-drug conjugate of claim 1, wherein the conjugate is selected from any one of the following:

4. the payload is at least one selected from the group consisting of a cytotoxic agent, a label, a nucleic acid, a radionuclide, a hormone, an immunomodulator, a prodrug-converting enzyme, a ribonuclease, an agonist antibody, an antagonist antibody and fragments thereof, a fusion protein or derivatives thereof; and / or the cytotoxic agent comprises a tubulin inhibitor and / or a topoisomerase inhibitor, the tubulin inhibitor comprising an auristatin or a derivative thereof, a maytansine or a derivative thereof, and the topoisomerase inhibitor comprising camptothecin and its derivatives; and / or The payload is exatecan of formula II 【Transformation 3】 2. The antibody-drug conjugate of claim 1, wherein exatecan of formula II is attached to the linker through the nitrogen atom of the amino group on its cyclohexane ring.

5. the anti-MUC18 antibody or antigen-binding fragment thereof HCDR1 having the amino acid sequence shown in SEQ ID NO:4, HCDR2 having the amino acid sequence shown in SEQ ID NO:16, HCDR3 having the amino acid sequence shown in SEQ ID NO:28, and LCDR1 having the amino acid sequence shown in SEQ ID NO:38, LCDR2 having the amino acid sequence STS, and LCDR3 having the amino acid sequence shown in SEQ ID NO:52; HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, HCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 34, LCDR2 having the amino acid sequence LAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 46; HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, HCDR2 having the amino acid sequence shown in SEQ ID NO: 12, HCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 35, LCDR2 having the amino acid sequence LAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; HCDR1 having the amino acid sequence shown in SEQ ID NO: 2, HCDR2 having the amino acid sequence shown in SEQ ID NO: 13, HCDR3 having the amino acid sequence shown in SEQ ID NO: 24, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 36, LCDR2 having the amino acid sequence NAK, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 48; HCDR1 having the amino acid sequence shown in SEQ ID NO:3, HCDR2 having the amino acid sequence shown in SEQ ID NO:14, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, and LCDR1 having the amino acid sequence shown in SEQ ID NO:37, LCDR2 having the amino acid sequence FAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO:49; HCDR1 having the amino acid sequence shown in SEQ ID NO:4, HCDR2 having the amino acid sequence shown in SEQ ID NO:15, HCDR3 having the amino acid sequence shown in SEQ ID NO:26, and LCDR1 having the amino acid sequence shown in SEQ ID NO:38, LCDR2 having the amino acid sequence STS, and LCDR3 having the amino acid sequence shown in SEQ ID NO:50; HCDR1 having the amino acid sequence shown in SEQ ID NO:4, HCDR2 having the amino acid sequence shown in SEQ ID NO:15, HCDR3 having the amino acid sequence shown in SEQ ID NO:27, and LCDR1 having the amino acid sequence shown in SEQ ID NO:39, LCDR2 having the amino acid sequence STS, and LCDR3 having the amino acid sequence shown in SEQ ID NO:51; HCDR1 having the amino acid sequence shown in SEQ ID NO:5, HCDR2 having the amino acid sequence shown in SEQ ID NO:17, HCDR3 having the amino acid sequence shown in SEQ ID NO:29, and LCDR1 having the amino acid sequence shown in SEQ ID NO:40, LCDR2 having the amino acid sequence WAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO:53; HCDR1 having the amino acid sequence shown in SEQ ID NO:6, HCDR2 having the amino acid sequence shown in SEQ ID NO:18, HCDR3 having the amino acid sequence shown in SEQ ID NO:29, and LCDR1 having the amino acid sequence shown in SEQ ID NO:41, LCDR2 having the amino acid sequence WAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO:53; HCDR1 having the amino acid sequence shown in SEQ ID NO:7, HCDR2 having the amino acid sequence shown in SEQ ID NO:19, HCDR3 having the amino acid sequence shown in SEQ ID NO:30, and LCDR1 having the amino acid sequence shown in SEQ ID NO:42, LCDR2 having the amino acid sequence RTS, and LCDR3 having the amino acid sequence shown in SEQ ID NO:54; HCDR1 having the amino acid sequence shown in SEQ ID NO:8, HCDR2 having the amino acid sequence shown in SEQ ID NO:20, HCDR3 having the amino acid sequence shown in SEQ ID NO:31, and LCDR1 having the amino acid sequence shown in SEQ ID NO:43, LCDR2 having the amino acid sequence WAS, and LCDR3 having the amino acid sequence shown in SEQ ID NO:55; HCDR1 having the amino acid sequence set forth in SEQ ID NO:9, HCDR2 having the amino acid sequence set forth in SEQ ID NO:21, HCDR3 having the amino acid sequence set forth in SEQ ID NO:32, and LCDR1 having the amino acid sequence set forth in SEQ ID NO:44, amino acid sequence WAS an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 56; HCDR1 having the amino acid sequence shown in SEQ ID NO: 10, HCDR2 having the amino acid sequence shown in SEQ ID NO: 22, HCDR3 having the amino acid sequence shown in SEQ ID NO: 33, and LCDR1 having the amino acid sequence shown in SEQ ID NO: 45, LCDR2 having the amino acid sequence LMS, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

57. The antibody-drug conjugate of claim 1, comprising:

6. the anti-MUC18 antibody or antigen-binding fragment thereof a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 84, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 85; a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 86 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 87; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 88, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 89; The antibody-drug conjugate according to any one of claims 1 to 5, comprising:

7. the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof specifically binds to a binding peptide in human CD44 v7 / 8, wherein the binding peptide comprises the amino acid sequence set forth in SEQ ID NO:90; and / or the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof does not bind to a binding peptide having the amino acid sequence set forth in SEQ ID NO:91 and / or SEQ ID NO:92 in human CD44 v7 / 8; Optionally, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:93, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:94, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:95, and an LCDR1 having the amino acid sequence set forth in SEQ ID NO:96, an LCDR2 having the amino acid sequence RAN, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:97; and / or the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof is a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 100, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 101; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 100, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 104; The antibody-drug conjugate according to any one of claims 1 to 4, comprising:

8. DAR is 1 to 10; and / or The antibody-drug conjugate of any one of claims 1 to 4, wherein the DAR is 4 to 10.

9. 1. A method for preparing an antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient, comprising the steps of: reducing an anti-MUC18 antibody or an anti-CD44 v7 / 8 antibody, or antigen-binding fragment thereof, such that disulfide bonds therebetween are at least partially reduced; and 【Chemistry 4】 (In the formula, In the linker-payload, the carbonyl group in the ester group of the linker of formula III is bonded to the amino group of the payload; R １ and R ２ are independently selected from hydrogen, methyl, and isopropyl groups; R ３ Ha-(CR ５ HCONH)n １ - (CH ２ CONH)n ２ represents - or a single bond, R ５ is hydrogen or benzyl, and n １ represents an integer of 0 to 2, and n ２ represents an integer from 0 to 2, and R ４ is a methylamino group or -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer from 1 to 20) with the carbon atom at the 3-position of maleimido-N-yl; Optionally, the method of preparing includes reacting the antibody or antigen-binding fragment thereof with a reducing agent in a buffer solution containing a chelating agent, followed by adding a solution of the linker-payload, wherein the linker has the structure of Formula III, and adjusting the pH of the reaction solution.

10. The payload is exatecan of formula II 【Transformation 5】 and exatecan of formula II is linked to the carbonyl group of the ester group in formula III through the nitrogen atom of the amino group on its cyclohexane ring, and / or the antibody-drug conjugate has a DAR of 1 to 10.

11. the anti-MUC18 antibody or antigen-binding fragment thereof a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 84, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 85; a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 86, and a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 87; a light chain variable region having an amino acid sequence as set forth in a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 88, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 89; or a conservative variant thereof, the anti-CD44 v7 / 8 antibody or antigen-binding fragment thereof is a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 100, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 101; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 100, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 104; 11. The method of claim 9 or 10, wherein the method comprises:

12. A pharmaceutical composition comprising the antibody-drug conjugate of claim 1, or the antibody-drug conjugate prepared by the method of claim 9, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof, and a pharmaceutically acceptable excipient.

13. A kit comprising the antibody-drug conjugate of claim 1, or the antibody-drug conjugate prepared by the method of claim 9, the pharmaceutical composition of claim 12, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, solvate, or combination thereof.

14. A pharmaceutical composition according to claim 12 for use in the diagnosis, prevention and treatment of neoplastic diseases, comprising: The neoplastic disease includes benign and malignant tumors that express MUC18 and / or CD44 v7 / 8. Preferably, the neoplastic disease comprises melanoma, pharyngeal carcinoma, triple-negative breast cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, urothelial carcinoma, bladder neuroendocrine tumor, small cell lung cancer, non-small cell lung cancer, cutaneous squamous cell carcinoma, bile duct cancer, metastatic pancreatic cancer, lung squamous cell carcinoma, head and neck squamous cell carcinoma and / or esophageal squamous cell carcinoma.

15. A kit according to claim 13 for use in the diagnosis, prevention and treatment of neoplastic diseases, comprising: The neoplastic disease includes benign and malignant tumors that express MUC18 and / or CD44 v7 / 8. Preferably, the neoplastic disease comprises melanoma, pharyngeal cancer, triple-negative breast cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, urothelial carcinoma, bladder neuroendocrine tumor, small cell lung cancer, non-small cell lung cancer, cutaneous squamous cell carcinoma, bile duct cancer, metastatic pancreatic cancer, lung squamous cell carcinoma, head and neck squamous cell carcinoma and / or esophageal squamous cell carcinoma.

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