Linker drugs, antibody-drug conjugates thereof, and methods for preparing and using the same

Abstract

The present invention relates to the pharmaceutical field, specifically to linker-drug conjugates, antibody-drug conjugates thereof, and methods for preparing and using them. Specifically, the present invention relates to a linker-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt. The linker-drug conjugate has a structure shown in Formula I, where M is a chemical structure containing a maleimide (M) fragment or a cyclooctyne fragment, X is a linker fragment consisting of 1 to 5 amino acids or derivatives thereof, and D is a cytotoxin. The linker-drug conjugate of the present invention can release the payload by enzymatic degradation by removing the hydrophobic structure PAB, thereby reducing the hydrophobicity of the linker-drug, reducing the content of polymer generated during ADC preparation, and exhibiting corresponding cytotoxic activity. MXD (Formula I)

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from and includes CN Application No. 202310093383.0, filed January 31, 2023, and CN Application No. 202310947390.2, filed July 28, 2023, the entire contents of all of which applications are incorporated herein by reference.

[0002] Technical Field The present invention relates to the field of drugs, in particular to linker drug conjugates, antibody drug conjugates, their preparation methods and uses. [Background technology]

[0003] Research into antibody-drug conjugates (ADCs) dates back to the 1980s, but it wasn't until 2000 that the first antibody-drug conjugate (Mylotarg, developed by Pfizer) was approved by the FDA for the treatment of acute myeloid leukemia. However, due to limitations in conjugation technology, target specificity, and efficacy, the complete antibody-drug conjugate was unstable in the blood and caused fatal toxicity, leading to its withdrawal from the market in 2010. This further cast a shadow over the already murky ADC drug research.

[0004] However, Seagen improved on existing technology and used its new antibody conjugation technology to develop a new antibody-drug conjugate, brentuximab vedotin (SGN-35, trade name Adcetris), which was approved by the FDA in 2011 for the treatment of Hodgkin's lymphoma and systemic anaplastic large cell lymphoma. In 2013, antibody-drug conjugates achieved another breakthrough, with the first solid tumor-targeting drug, ado-trastuzumab emtansine (T-DM1, trade name Kadcyla), co-developed by Genentech and ImmunoGen, receiving FDA approval for HER2-positive breast cancer. The successful development of these two drugs once again drew attention to ADC drugs as a subject of active research.

[0005] In recent years, ADCs have developed rapidly, with 15 ADC drugs currently approved for market launch and over 200 ADCs currently in clinical trials, and ADCs are playing an increasingly important role in the field of tumor targeting therapy.

[0006] Antibody-drug conjugates consist of three parts: an antibody (Ab), a linker, and a small molecule cytotoxic drug (payload / warhead). The antibody portion of an ADC is required to have tumor specificity, good intracellular uptake efficiency, good antigen affinity, and low or no immunogenicity. The most commonly used antibody moiety is IgG1. The warhead moiety is usually a cytotoxic drug that acts on microtubules, DNA, or RNA, such as camptothecins, maytansinoids, and auristatins. It must have sufficiently high toxicity, sufficient water solubility, and its target of action must be intracellular. The linker connecting the antibody and the small molecule cytotoxic drug must be stable in the blood circulation and rapidly decompose within cells to efficiently release the toxin. Summary of the Invention

[0007] The inventors of the present application have creatively designed a novel linker structure, thereby providing the present invention.

[0008] Linker-drug conjugates A first aspect of the present invention relates to a linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein said linker drug conjugate has the structure shown in Formula I: MXD Formula I During the ceremony, M is a chemical structure containing a maleimide (m) fragment or a chemical structure containing cyclooctyne and its derivative fragments (e.g., BCN, DIBO, DIBAC, BARAC, etc.); X is a linker fragment consisting of 1 to 5 amino acids or derivatives thereof, and is preferably Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), Ala-(3-cyan o-alanine), 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys- Pro, Ala-Ala-Ala-Ala-Gln, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Ala-Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-(2-amino-4-cyanobutanoic acid), 2-amino-4-cyanobutanoic acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln and deuterated versions thereof; D is a cytotoxin.

[0009] In the linker-drug conjugate of the present invention, the linker is composed of a chemical structure containing a maleimide (m) fragment, or a chemical structure containing cyclooctyne and its derivative fragments (e.g., BCN, DIBO, DIBAC, BARAC, etc.), and a linker fragment consisting of 1 to 5 amino acids or their derivatives, and has the following properties: it is stable in the blood circulation, can be rapidly degraded within cells or the tumor microenvironment, and enables efficient release of toxins.

[0010] Chemical structures containing maleimide (m) fragments may be selected from 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC), 6-maleimidohexanoyl (MC), M-(PEG)n, MC-(PEG)n, MCC-(PEG)n, etc., whose structures are as follows: [Table 1] In the formula, n represents the degree of polymerization of PEG and is preferably an integer of 2 to 12.

[0011] In one embodiment, the chemical structure containing the maleimide (m) fragment is 6-maleimidohexanoyl (MC). In one embodiment, the chemical structure containing the maleimide (m) fragment is MC-(PEG)n, where n is selected from 4, 8, or 12. The inventors have found that a low degree of polymerization of PEG is somewhat beneficial in order to reduce the polymer content (HMW%) in ADCs prepared from linker-drug conjugates. Therefore, the degree of polymerization of PEG, n, is preferably 4.

[0012] The chemical structure containing the cyclooctyne fragment can be selected from BCN, DIBO, DIBAC, BARAC, etc., and their structures are as follows: [Table 2]

[0013] In most ADCs currently under investigation, the PAB (p-aminobenzylcarbamoyl) structure, a self-cleaving fragment within the linker, is essential for effective payload release. However, due to the hydrophobic nature of the PAB structure, a relatively large amount of polymer is likely to be generated during ADC preparation. Therefore, further research and development of novel linker structures is still required to overcome the above issues.

[0014] For this reason, the inventors of the present application have found through experimental studies that, in the case of a cytotoxin containing a D-hydroxy group (-OH), a primary amino group (-NH2), or a secondary amino group (-NHR), the amino acid fragment contained in the linker-drug conjugate of the present application can be bound to a payload having a hydroxy group (-OH), a primary amino group (-NH2), or a secondary amino group (-NHR), thereby releasing the payload through an enzymatic degradation reaction while removing the hydrophobic PAB structure, thereby reducing the hydrophobicity of the linker-drug, reducing the content of polymer generated during ADC preparation, and exhibiting corresponding cytotoxic activity.

[0015] Thus, in certain embodiments, D is a cytotoxin that contains a hydroxy group (-OH), a primary amino group (-NH2), or a secondary amino group (-NHR).

[0016] In certain embodiments, the cytotoxin containing a hydroxy group (-OH), a primary amino group (-NH), or a secondary amino group (-NHR) may be selected from the following: topoisomerase I inhibitors such as camptothecins and their derivatives (e.g., exatecan, 14-aminocamptothecin, 9-aminocamptothecin, belotecan, etc.); topoisomerase II inhibitors such as anthracyclines and their derivatives (e.g., daunorubicin); maytansinoids and their derivatives, calicheamicins and their derivatives, docamycins and their derivatives (e.g., duocarmycin derivatives), DNA alkylating agents such as pyrrolobenzodiazepains and their derivatives (e.g., PBD); microtubule inhibitors such as monomethylauristatins and their derivatives (e.g., MMAE and its derivatives, or MMAF and its derivatives); immunosuppressants such as Protac compounds and molecular glue compounds (e.g., lenalidomide), and rapamycin and its derivatives; and various other small molecule compounds with cytotoxic activity. In some embodiments, the cytotoxin containing a hydroxy group (-OH), a primary amino group (-NH2), or a secondary amino group (-NHR) is selected from exatecan, belotecan, and rapamycin. In some embodiments, the cytotoxin containing a hydroxy group (-OH) is rapamycin. In some embodiments, the cytotoxin containing a secondary amino group (-NHR) is belotecan. In some embodiments, the cytotoxin containing a primary amino group (-NH2) is exatecan.

[0017] In one embodiment, the linker fragment consisting of 1 to 5 amino acids or derivatives thereof in Formula I is selected from the group consisting of Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3- cyano-alanine), 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Ly s-Pro,Ala-Ala-Ala-Ala-Gln,Ala-Ala-Ala-Gln,Ala-Ala-Gln,Ala-Gln,Ala-Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-(2-amino-4-cyanobutanoic acid), 2-amino-4-cyanobutanoic acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln and deuterated versions thereof.

[0018] The amino acid monomers or derivatives thereof in Formula I, or fragments consisting of multiple amino acids or derivatives thereof, are recognized by enzymes in the lysosomes of tumor cells, which can cleave ester or amide bonds (peptide bonds) formed between carboxyl groups in these compounds and compounds containing hydroxyl groups (-OH), primary amines (-NH), or secondary amines (-NHR) without the need for additional self-cleaving moieties.

[0019] In one embodiment, the linker fragment consisting of 1 to 5 amino acids or derivatives thereof in Formula I is Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-L-alanine), Ala-Ala-Ala-(3-cyano-L-alanine), Ala-Ala-(3-cyano-L-alanine), Ala-(3-cyano-L-alanine), Ala-(3-cyano- L-alanine), 3-cyano-L-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-P ro, Ala-Ala-Ala-Ala-Gln, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Ala-Ala-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid), Ala-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid), Ala-Ala-((S)-2-amino-4-cyanobutanoic acid), Ala-((S)-2-amino-4-cyanobutanoic acid), (S)-2-amino-4-cyanobutanoic acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln, Gln.

[0020] In some embodiments, the linker fragment is selected from linker fragments containing asparagine (Asn), such as Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Pro-Asn, and Lys-Asn. In some embodiments, the linker fragment is Ala-Ala-Asn (AAN).

[0021] In one embodiment, the linker drug conjugate of the invention is MCC-AAQ-Exatecan, having the structure shown below. [ka]

[0022] Antibody-drug conjugates A second aspect of the present invention relates to an antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, wherein the antibody drug conjugate is formed by conjugating a linker drug conjugate according to the first aspect of the invention to an antibody.

[0023] In one embodiment, the antibody drug conjugate has the structure shown in Formula II: Ab-(LD)p Formula II

[0024] During the ceremony, Ab is antibody; L is a linker, the structure of which is M'-X, where M' binds to Ab and X binds to D; M' is a chemical structure formed by binding M, as defined above, to Ab via a maleimide group; X is a linker fragment consisting of 1 to 5 amino acids or derivatives thereof as defined above; D is a cytotoxin as defined above; p is any number between 2 and 8 (e.g., 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.21, 6.22, 6.23, 6.24, 6.25, 6.26, 6.27, 6.28, 6.29 ... 3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8).

[0025] In Formula II, LD indicates that the linker and cytotoxin are covalently linked to form the LD molecule, and Ab-(LD)p indicates that p LD molecules are covalently conjugated to Ab.

[0026] In the present invention, the drug-antibody ratio (DAR) refers to the number of drug molecules conjugated to an antibody (e.g., p in Formula II). The number of drug molecules contained in the antibody-drug conjugates described herein may be an integer or a decimal, and in either case refers to the average number of drug molecules conjugated per antibody molecule. The phrase "p is any number between 2 and 8" means that p can be any integer between 2 and 8 (including the endpoints 2 and 8) or any decimal between 2 and 8, such as 2.3, 3.9, 4.0, or 4.2. Furthermore, those skilled in the art will understand that even when using the same preparation method, the DAR values ​​of antibody-drug conjugates prepared in different batches are not necessarily identical and may vary, for example, within a range of ±0.5.

[0027] The drug-antibody ratio (DAR) can be verified by conventional analytical means such as mass spectrometry, ELISA assay, HIC, and HPLC. Additionally, the quantitative distribution of the ADC with respect to p may also be measured. In some cases, separation, purification, and verification of a homogeneous ADC where p is a particular value from ADCs with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0028] In the present invention, one method for covalently conjugating a linker-drug conjugate to an antibody (Ab) is to conjugate a maleimide with a thiol group in the antibody to form an -S-succinimide structure. For example, when MCC-AAQ-Exatecan is covalently conjugated to an Ab, the resulting ADC has the following structural formula: [ka]

[0029] In the ADCs formed with the above-mentioned maleimide linker, -S- may be an exogenous sulfhydryl group introduced by engineering, or may be a sulfhydryl group derived from the antibody itself, generated as a result of reduction of the antibody Ab and subsequent cleavage of disulfide bonds.

[0030] Another method is to conjugate cyclooctyne with an azide group (-N3) introduced into the engineered antibody to form a triazole structure. For example, when BCN-AAQ-Exatecan is covalently conjugated to Ab, the resulting ADC has the following structural formula: [ka]

[0031] There are no particular limitations on the antibodies that can be used in the present invention, and they can be selected from mouse-derived antibodies, rabbit-derived antibodies, phage display-derived antibodies, yeast display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies.

[0032] In certain embodiments, the antibody is a monoclonal antibody, selected from, but not limited to, a HER2 antibody and an EGFR antibody.

[0033] In one embodiment, the antibody is a HER2 antibody. In one embodiment, the antibody consists of a light chain and a heavy chain, and the light chain comprises CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In one embodiment, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0034] In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence SEQ ID NO: 7. In one embodiment, the light chain further comprises a light chain constant region having the amino acid sequence SEQ ID NO: 8. In one embodiment, the amino acid sequence of the light chain is SEQ ID NO: 9.

[0035] In some embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence SEQ ID NO: 10. In some embodiments, the heavy chain further comprises a heavy chain constant region having the amino acid sequence SEQ ID NO: 11. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 12.

[0036] In some embodiments, the antibody is an EGFR antibody. In some embodiments, the antibody consists of a light chain and a heavy chain, and the light chain comprises CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0037] In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence SEQ ID NO: 19. In one embodiment, the light chain further comprises a light chain constant region having the amino acid sequence SEQ ID NO: 20. In one embodiment, the amino acid sequence of the light chain is SEQ ID NO: 21.

[0038] In some embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence SEQ ID NO: 22. In some embodiments, the heavy chain further comprises a heavy chain constant region having the amino acid sequence SEQ ID NO: 23. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 24.

[0039] Information regarding some of the sequences relevant to the present invention is provided in Table 1.

[0040] [Table 3]

[0041] [Table 4]

[0042] Pharmaceutical compositions and uses for producing drugs A third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of said salt, or the above-mentioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of said salt.

[0043] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic agent, an immunotherapeutic agent, and an immunosuppressant agent used in the treatment of tumors.

[0044] In certain embodiments, the chemotherapeutic agent is, for example, adriamycin, cyclophosphamide, taxanes (e.g., paclitaxel (Taxol), docetaxel (Taxotere)), capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (letrozole, forvestrol, anastrozole), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, semustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisolone, or the like, or a combination thereof.

[0045] In one embodiment, the immunotherapeutic agent is a PD-1 antibody, a PD-L1 antibody, an EGFR monoclonal antibody, a CD20 monoclonal antibody, a HER2 monoclonal antibody (e.g., trastuzumab, trastuzumab biosimilar, trastuzumab-dkst), or the like, or a combination thereof.

[0046] In certain embodiments, the immunosuppressant is (1) a glucocorticoid, such as cortisone and prednisone; (2) a microbial metabolite, such as cyclosporine and tacrolimus; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal antilymphocyte antibodies, such as antilymphocyte globulin and OKT3; or (5) an alkylating agent, such as cyclophosphamide. In specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, Prograf, Zenapax, Simulect, cyclosporine, tacrolimus, rapamycin, mycophenolic acid, mizoribine, cyclophosphamide, fingolimod, or the like.

[0047] In certain embodiments, the pharmaceutical composition further comprises at least one pharmaceutical adjuvant.

[0048] A fourth aspect of the present invention relates to the use of a linker drug conjugate as defined above, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof, or an antibody drug conjugate as defined above, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof, in the manufacture of a medicament, wherein said medicament is for use in the prevention and / or treatment of tumors.

[0049] A fifth aspect of the present invention provides a method of treating and / or preventing tumors, the method comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of a linker drug conjugate as described above, or a pharmaceutically acceptable salt, solvate or solvate of said salt, or an antibody drug conjugate as described above, or a pharmaceutically acceptable salt, solvate or solvate of said salt.

[0050] In the present invention, the term "tumor" refers to a lesion formed by localized cell hyperproliferation in an organ or tissue, including hematological tumors and solid tumors, and encompassing benign and malignant tumors. In one embodiment, the tumor is selected from tumors that are positive for or highly express the following markers: HER2 and EGFR.

[0051] In certain embodiments, the tumor is selected from breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, intraspinal tumor, mediastinal tumor, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumor, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumor, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal cancer, laryngeal cancer, oral cancer, esophageal cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), kidney cancer, cervical cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, Kaposi's sarcoma, non-melanoma skin cancer (including squamous cell carcinoma and basal cell carcinoma), hemangioma, glioma, and glioblastoma cerebral.

[0052] Preparation method A sixth aspect of the present invention provides a process for preparing a linker drug conjugate as described above, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof, which comprises the steps of: 1. Preparing a linker fragment X consisting of 1 to 5 amino acids or derivatives thereof; 2. X is coupled to a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to give MX; 3. Conjugating the cytotoxin D to MX to give the linker-drug conjugate MXD.

[0053] Next, the above preparation method will be further explained using MCC-AAQ-Exatecan as an example. A typical preparation process for MCC-AAQ-Exatecan is as follows: 1. Fmoc-Gln is reacted with Exatecan to obtain intermediate 1. The structural formula of intermediate 1 is as follows: [ka] 2. Intermediate 1 is deprotected with DEA ​​to remove the Fmoc group, yielding intermediate 2, whose structural formula is as follows: [ka] 3. Intermediate 2 is reacted with Fmoc-Ala-Ala to obtain intermediate 3. The structural formula of intermediate 3 is as follows: [ka] 4. The Fmoc protecting group of intermediate 3 is deprotected to obtain intermediate 4. The structural formula of intermediate 4 is as follows: [ka] 5. Condensation of intermediate 4 with MCC gives the target product, whose structural formula is as follows: [ka]

[0054] A seventh aspect of the present invention provides a method for preparing the above-mentioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of the above-mentioned salt, comprising the steps of: 1. Dilute a fixed weight of monoclonal antibody with PBS buffer and adjust the pH of the antibody to approximately 7.5. Measure the protein concentration, weigh the net weight of the antibody solution, and calculate the total protein content. Add the TCEP solution to the antibody and allow to react at room temperature with continuous stirring. 2. Add an excess amount of cytotoxic drug solution to the reduced antibody solution, mix evenly, and then react at room temperature while continuing to stir; after the reaction is complete, add an excess amount of N-acetylcysteine ​​solution to the reaction solution, and react at room temperature while continuing to stir; 3. Purify the conjugate product using an ultrafiltration centrifuge tube, exchange it into a storage solution, and then filter it through a sterile filter to obtain the antibody-drug conjugate.

[0055] Additionally, the present invention provides: Linker-drug conjugates An eighth aspect of the present invention relates to a linker drug conjugate, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof, wherein said linker drug conjugate has the structure shown in Formula I: MXD Formula I During the ceremony, M is a chemical structure containing a maleimide (m) fragment or a chemical structure containing cyclooctyne and its derivative fragments (e.g., BCN, DIBO, DIBAC, BARAC, etc.); X is a linker fragment consisting of 1 to 4 amino acids selected from Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, and Gln; D is a target cytotoxin containing a primary amine (-NH2) group.

[0056] In the linker-drug conjugate of the present invention, the linker is composed of a chemical structure containing a maleimide (m) fragment, or a chemical structure containing cyclooctyne and its derivative fragments (e.g., BCN, DIBO, DIBAC, BARAC, etc.), and a linker fragment consisting of 1 to 4 amino acids, and has the following properties: it is stable in the blood circulation, can be rapidly degraded within cells or the tumor microenvironment, and enables efficient release of the toxin.

[0057] Chemical structures containing maleimide (m) fragments may be selected from 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC), 6-maleimidohexanoyl (MC), M-(PEG)n, MC-(PEG)n, MCC-(PEG)n, etc., whose structures are as follows: [Table 5] In the formula, n represents the degree of polymerization of PEG and is preferably an integer of 2 to 12.

[0058] In one embodiment, the chemical structure containing the maleimide (m) fragment is 6-maleimidohexanoyl (MC). In one embodiment, the chemical structure containing the maleimide (m) fragment is MC-(PEG)n, where n is selected from 4, 8, or 12. The inventors have found that a low degree of polymerization of PEG is somewhat beneficial in order to reduce the polymer content (HMW%) in ADCs prepared from linker-drug conjugates. Therefore, the degree of polymerization of PEG, n, is preferably 4.

[0059] The chemical structure containing the cyclooctyne fragment can be selected from BCN, DIBO, DIBAC, BARAC, etc., and their structures are as follows: [Table 6]

[0060] The linker fragment of one to four amino acids in Formula I is selected from Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pro, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Gly-Gly-Gly-Gln, Gly-Gly-Gln, and Gln. These amino acid monomers or fragments of multiple amino acids are recognized by an enzyme in the lysosomes of tumor cells. This enzyme can cleave the amide bond (peptide bond) formed between a carboxyl group in this type of compound and a compound containing a primary amine (—NH) without the need for an additional self-cleaving moiety.

[0061] In one embodiment, the linker fragment is selected from linker fragments containing asparagine (Asn), such as Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Pro-Asn, and Lys-Asn. Asparagine (Asn) is recognized by legumain, which is not only present in the lysosomes of tumor cells but also distributed in large amounts outside tumor cells, allowing the small molecule to be released extracellularly to exert a bystander effect and exhibit good killing effect against target cells with low endocytosis. In one embodiment, the linker fragment is Ala-Ala-Asn (AAN).

[0062] The cytotoxin containing a primary amino group (—NH) may be selected from small molecule compounds with various cytotoxic activities, such as topoisomerase I inhibitors such as camptothecins and their derivatives (e.g., exatecan, 14-aminocamptothecin, 9-aminocamptothecin, etc.), topoisomerase II inhibitors such as anthracyclines and their derivatives (e.g., daunorubicin), DNA alkylating agents such as maytansinoids and their derivatives, calicheamicins and their derivatives, dokamycins and their derivatives (e.g., duocarmycin derivatives), and pyrrolobenzodiazepines and their derivatives (e.g., PBD), microtubule protein inhibitors such as monomethyl auristatins and their derivatives (e.g., demethylated MMAE or demethylated MMAF), PROTAC compounds, and molecular glue compounds (e.g., lenalidomide). In one embodiment, the cytotoxin containing a primary amino group (—NH) is exatecan.

[0063] In certain embodiments, the cytotoxin containing a primary amino group (—NH2) is selected from exatecan, 14-aminocamptothecin (14-AC), 9-aminocamptothecin (9-AC), demethylated MMAE, demethylated MMAF, daunorubicin, and lenalidomide.

[0064] In one embodiment, the linker drug conjugate of the invention is mc-AAQ-Exatecan, which has the structure shown below. [ka]

[0065] Antibody-drug conjugates A ninth aspect of the present invention relates to an antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, wherein the antibody drug conjugate is formed by conjugating a linker drug conjugate according to the eighth aspect of the invention to an antibody.

[0066] In one embodiment, the antibody drug conjugate has the structure shown in Formula II: Ab-(LD)p Formula II During the ceremony, Ab is antibody; L is a linker, the structure of which is M'-X, where M' binds to Ab and X binds to D; M' is a chemical structure formed by binding M, as defined above, to Ab via a maleimide group; X is a linker fragment of 1 to 4 amino acids as defined above; D is a primary amine (-NH2)-based cytotoxin as defined above; p is any number between 2 and 8 (e.g., 2, 2.5, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.21, 6.22, 6.23, 6.24, 6.25, 6.26, 6.27, 6.28, 6.29 ... 3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5 or 7.5-8).

[0067] In Formula II, LD indicates that the linker and cytotoxin are covalently linked to form the LD molecule, and Ab-(LD)p indicates that p LD molecules are covalently conjugated to Ab.

[0068] In the present invention, the drug-antibody ratio (DAR) refers to the number of drug molecules conjugated to an antibody (e.g., p in Formula II). The number of drug molecules contained in the antibody-drug conjugates described herein may be an integer or a decimal, and in either case refers to the average number of drug molecules conjugated per antibody molecule. The phrase "p is any number between 2 and 8" means that p can be any integer between 2 and 8 (including the endpoints 2 and 8) or any decimal between 2 and 8, such as 2.3, 3.9, 4.0, or 4.2. Furthermore, those skilled in the art will understand that even when using the same preparation method, the DAR values ​​of antibody-drug conjugates prepared in different batches are not necessarily identical and may vary, for example, within a range of ±0.5.

[0069] The drug-antibody ratio (DAR) can be verified by conventional analytical means such as mass spectrometry, ELISA assay, HIC, and HPLC. Additionally, the quantitative distribution of the ADC with respect to p may also be measured. In some cases, separation, purification, and verification of a homogeneous ADC where p is a particular value from ADCs with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0070] In the present invention, one method for covalently conjugating a linker-drug conjugate to an antibody (Ab) is to conjugate maleimide with a thiol group in the antibody to form an -S-succinimide structure. For example, when mc-AAN-Exatecan is covalently conjugated to Ab, the resulting ADC has the following structural formula: [ka]

[0071] In the ADCs formed with the above-mentioned maleimide linker, -S- may be an exogenous sulfhydryl group introduced by engineering, or may be a sulfhydryl group derived from the antibody itself, generated as a result of reduction of the antibody Ab and subsequent cleavage of disulfide bonds.

[0072] Another method is to link the chemical structure of cyclooctyne and its derivative fragments (BCN, DIBO, DIBAC, BARAC, etc.) to an antibody, and conjugate the cyclooctyne with the azide group (-N3) introduced into the engineered antibody to form a triazole structure. For example, when BCN-AAN-Exatecan is covalently conjugated to an Ab, the resulting ADC has the following structural formula: [ka]

[0073] There are no particular limitations on the antibodies that can be used in the present invention, and they can be selected from mouse-derived antibodies, rabbit-derived antibodies, phage display-derived antibodies, yeast display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies.

[0074] In certain embodiments, the antibody is a monoclonal antibody, selected from, but not limited to, a HER2 antibody and an EGFR antibody.

[0075] In one embodiment, the antibody is a HER2 antibody. In one embodiment, the antibody consists of a light chain and a heavy chain, and the light chain comprises CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In one embodiment, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0076] In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence SEQ ID NO: 7. In one embodiment, the light chain further comprises a light chain constant region having the amino acid sequence SEQ ID NO: 8. In one embodiment, the amino acid sequence of the light chain is SEQ ID NO: 9.

[0077] In some embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence SEQ ID NO: 10. In some embodiments, the heavy chain further comprises a heavy chain constant region having the amino acid sequence SEQ ID NO: 11. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 12.

[0078] In some embodiments, the antibody is an EGFR antibody. In some embodiments, the antibody consists of a light chain and a heavy chain, and the light chain comprises CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0079] In one embodiment, the light chain comprises a light chain variable region having the amino acid sequence SEQ ID NO: 19. In one embodiment, the light chain further comprises a light chain constant region having the amino acid sequence SEQ ID NO: 20. In one embodiment, the amino acid sequence of the light chain is SEQ ID NO: 21.

[0080] In some embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence SEQ ID NO: 22. In some embodiments, the heavy chain further comprises a heavy chain constant region having the amino acid sequence SEQ ID NO: 23. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 24.

[0081] Information regarding some of the sequences relevant to the present invention is provided in Table 1 above.

[0082] Pharmaceutical compositions and uses for producing drugs A tenth aspect of the present invention provides a pharmaceutical composition comprising the linker drug conjugate described above, or a pharmaceutically acceptable salt, solvate or solvate of said salt, or the antibody drug conjugate described above, or a pharmaceutically acceptable salt, solvate or solvate of said salt.

[0083] In certain embodiments, the pharmaceutical composition further comprises at least one of a chemotherapeutic agent, an immunotherapeutic agent, and an immunosuppressant agent used in the treatment of tumors.

[0084] In certain embodiments, the chemotherapeutic agent is, for example, adriamycin, cyclophosphamide, taxanes (e.g., paclitaxel (Taxol), docetaxel (Taxotere)), capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (letrozole, forvestrol, anastrozole), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, semustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisolone, or the like, or a combination thereof.

[0085] In one embodiment, the immunotherapeutic agent is a PD-1 antibody, a PD-L1 antibody, an EGFR monoclonal antibody, a CD20 monoclonal antibody, a HER2 monoclonal antibody (e.g., trastuzumab, trastuzumab biosimilar, trastuzumab-dkst), or the like, or a combination thereof.

[0086] In certain embodiments, the immunosuppressant is (1) a glucocorticoid, such as cortisone and prednisone; (2) a microbial metabolite, such as cyclosporine and tacrolimus; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal antilymphocyte antibodies, such as antilymphocyte globulin and OKT3; or (5) an alkylating agent, such as cyclophosphamide. In specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, Prograf, Zenapax, Simulect, cyclosporine, tacrolimus, rapamycin, mycophenolic acid, mizoribine, cyclophosphamide, fingolimod, or the like.

[0087] In certain embodiments, the pharmaceutical composition further comprises at least one pharmaceutical adjuvant.

[0088] An eleventh aspect of the present invention relates to the use of a linker drug conjugate as defined above, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof, or an antibody drug conjugate as defined above, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof, in the manufacture of a medicament, wherein said medicament is for use in the prevention and / or treatment of tumors.

[0089] A twelfth aspect of the present invention provides a method of treating and / or preventing tumors, the method comprising administering to a subject in need thereof a therapeutically and / or prophylactically effective amount of a linker drug conjugate as described above, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof, or an antibody drug conjugate as described above, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof.

[0090] In the present invention, the term "tumor" refers to a lesion formed by localized cell hyperproliferation in an organ or tissue, including hematological tumors and solid tumors, and encompassing benign and malignant tumors. In one embodiment, the tumor is selected from tumors that are positive for or highly express the following markers: HER2 and EGFR.

[0091] In certain embodiments, the tumor is selected from breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, intraspinal tumor, mediastinal tumor, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumor, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumor, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal cancer, laryngeal cancer, oral cancer, esophageal cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), kidney cancer, cervical cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, Kaposi's sarcoma, non-melanoma skin cancer (including squamous cell carcinoma and basal cell carcinoma), hemangioma, glioma, and glioblastoma cerebral.

[0092] Preparation method A thirteenth aspect of the present invention provides a process for preparing a linker drug conjugate as described above, or a pharmaceutically acceptable salt, solvate or solvate of said salt thereof, which comprises the steps of: 1. Preparing a linker fragment X consisting of 1 to 4 amino acids; 2. X is coupled to a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to give MX; 3. A cytotoxin D containing a primary amine (-NH2) group is conjugated to MX to give the linker-drug conjugate MXD.

[0093] Next, the above preparation method will be further explained using mc-AAN-Exatecan as an example. A typical preparation process for mc-AAN-Exatecan is as follows: 1. Fmoc-Ala-OH is activated with HOSu and then reacted with L-Ala to obtain intermediate 1. The structural formula of intermediate 1 is as follows: [ka] 2. Intermediate 1 is activated with HOSu and then reacted with L-Asn to obtain intermediate 2. The structural formula of intermediate 2 is as follows: [ka] 3. Intermediate 2 is subjected to a Fmoc removal reaction to obtain intermediate 3. The structural formula of intermediate 3 is as follows: [ka] 4. Intermediate 3 is reacted with 6-(maleimido)hexanoic acid succinimide ester to obtain intermediate 4, the structural formula of which is as follows: [ka] 5. Condensation of intermediate 4 with exatecan gives the target product, whose structural formula is as follows: [ka]

[0094] A fourteenth aspect of the present invention provides a method for preparing the antibody drug conjugate described above, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, comprising the steps of: 1. Dilute a fixed weight of monoclonal antibody with PBS buffer and adjust the pH of the antibody to approximately 7.5. Measure the protein concentration, weigh the net weight of the antibody solution, and calculate the total protein content. Add the TCEP solution to the antibody and allow to react at room temperature with continuous stirring. 2. Add an excess amount of cytotoxic drug solution to the reduced antibody solution, mix evenly, and then react at room temperature while continuing to stir; after the reaction is complete, add an excess amount of N-acetylcysteine ​​solution to the reaction solution, and react at room temperature while continuing to stir; 3. Purify the conjugate product using an ultrafiltration centrifuge tube, exchange it into a storage solution, and then filter it through a sterile filter to obtain the antibody-drug conjugate. [Effects of the Invention]

[0095] In one embodiment, the linker provided by the present invention can release the payload by enzymatic degradation after removing the hydrophobic PAB structure, thereby reducing the hydrophobicity of the linker drug and reducing the content of polymer generated during ADC preparation, while also exhibiting corresponding cytotoxic activity. DETAILED DESCRIPTION OF THE INVENTION

[0096] Definition of Terms Hereinafter, embodiments of the present invention will be described in detail with reference to examples. However, it will be understood by those skilled in the art that the examples described below are merely illustrative examples for the purpose of explaining the present invention and should not be construed as limiting the scope of the present invention. Unless specific conditions are specified in the examples, they are carried out according to general conditions or conditions recommended by the manufacturer. Unless the manufacturer of the reagents or instruments used is specified, they are general products available on the market.

[0097] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.In addition, the terms used herein relating to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and experimental procedures are terms and conventional procedures widely used in the corresponding fields.Meanwhile, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0098] For purposes of the present invention, unless otherwise specified, any numerical range should be understood to include any value or any sub-range contained within that range.

[0099] In the present invention, the term "antibody" generally refers to an immunoglobulin molecule consisting of two identical pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be divided into two categories: kappa and lambda. Heavy chains can be divided into five types: μ, δ, γ, α, or ε. Antibodies can be divided into five types based on their heavy chains: IgM, IgD, IgG, IgA, and IgE. Within the light and heavy chains, the variable and constant regions are connected by a "J" region consisting of about 12 or more amino acids, and heavy chains also contain a "D" region consisting of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the complement system component C1q. The VH and VL regions can be divided into multiple, more variable regions called complementarity-determining regions (CDRs), interspersed with more highly conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy-light chain pair (VH and VL) form the antibody-binding site. The assignment of amino acids to each region or structural domain follows Kabat's Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883.

[0100] In certain embodiments, in the conjugation reaction, fewer drug moieties than the theoretical maximum are bound to the antibody.Generally, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be bound to drug moieties, and in fact, most of the cysteine ​​thiol groups in antibodies exist as disulfide bridges.In certain embodiments, antibodies can be reduced with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) under partial or complete reducing conditions to generate reactive cysteine ​​thiol groups.

[0101] In the present invention, the term "pharmaceutically acceptable salt" refers to (i) a salt of an acidic functional group present in the conjugate provided by the present invention, including, but not limited to, alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; other metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic base salts such as ammonium salt; tert-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucosamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylamine salt, and tris(hydroxymethyl)aminomethane salt. and (ii) salts formed by a basic functional group present in the conjugates provided by the present invention with a suitable inorganic or organic cation (base), including salts of organic bases such as methyl fluoride, methyl hydrochloride, methyl bromide, methyl hydroiodide, etc.; salts of inorganic acids such as nitrate, perchlorate, sulfate, phosphate, etc.; salts of lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, etc.; salts of arylsulfonates such as benzenesulfonate, p-toluenesulfonate, etc.; salts of organic acids such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc.; salts of amino acids such as glycinate, trimethylglycinate, alginate, ornithine, glutamate, aspartate, etc.

[0102] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid to provide a pharmaceutically acceptable anion, or by reacting a sufficient amount of an acidic compound with a suitable base to provide a pharmaceutically acceptable cation.

[0103] In the present invention, the term "solvate" refers to the following form of the antibody-drug conjugate according to the present invention: a solid or liquid complex formed by coordination of the antibody-drug conjugate with solvent molecules. A hydrate is a specific form of a solvate having coordinated water molecules. In the present invention, the preferred solvate is a hydrate.

[0104] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known to those skilled in the art, or will become apparent in light of this disclosure. As described in REMINGTON'S PHARMACEUTICAL SCIENCES (Martin, EW, ed., Mack Publishing Company, 19th ed. (1995)), methods for preparing such pharmaceutical compositions include incorporating suitable pharmaceutical excipients, carriers, diluents, etc. that are not toxic to cells or mammals when exposed at the dosages and concentrations used.

[0105] In the present invention, adjuvants refer to excipients and additives used in the manufacture and formulation of drugs, and refer to substances that, in addition to active ingredients, are incorporated into pharmaceutical preparations after their safety has been reasonably evaluated. Adjuvants have important functions such as shaping, acting as a vehicle, and improving stability, as well as solubilization, dissolution assistance, sustained release, and controlled release, and are important components that may affect the quality, safety, and efficacy of drugs. Depending on their source, they can be divided into natural substances, semi-synthetic substances, and fully synthetic substances. According to their roles and uses, they can be divided into solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, bulking agents, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavorings, preservatives, suspending agents, coating agents, fragrances, anti-caking agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. According to the route of administration, they can be divided into oral administration, injection, mucosal administration, transdermal or topical administration, nasal or oral inhalation administration, intraocular administration, etc. The same adjuvant can be used for different administration routes of pharmaceutical formulations and has different roles and uses.

[0106] In the present invention, the pharmaceutical composition can be prepared in various appropriate dosage forms depending on the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder aerosols, and sprays. Here, the pharmaceutical composition or appropriate dosage form may contain 0.01 mg to 1000 mg of the antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof.

[0107] As used herein, the term "treating" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic, in terms of partial or complete stabilization or cure of the disease and / or disease side effects. As used herein, "treatment" encompasses any treatment for a disease in a patient, including (a) prevention of a disease or condition in a patient who is susceptible to, but has not yet been diagnosed with, the disease or condition, (b) suppression of symptoms of the disease, i.e., prevention of its occurrence, or (c) alleviation of symptoms of the disease, i.e., regression of the disease or condition.

[0108] In the present invention, "subject" refers to a vertebrate. In certain embodiments, a vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cows), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, a mammal refers to a human.

[0109] In the present invention, an "effective amount" refers to an amount effective to achieve a desired therapeutic or preventive effect at the necessary dosage and frequency. The "therapeutically effective amount" of a substance / molecule according to the present invention may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance / molecule to induce a desired response in the individual. A therapeutically effective amount also encompasses an amount in which any toxic or adverse consequences of the substance / molecule are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective to achieve a desired preventive effect at the necessary dosage and frequency. Usually, but not always, a prophylactically effective amount is less than a therapeutically effective amount because a prophylactic dose is administered to a subject prior to the onset of disease or at an early stage of disease. In the case of cancer, a therapeutically effective amount of a drug reduces the number of cancer cells, reduces the size of a tumor, inhibits (i.e., somewhat slows, preferably stops) the infiltration of cancer cells into surrounding organs, inhibits (i.e., somewhat slows, preferably stops) the metastasis of tumors, somewhat inhibits the growth of tumors, and / or somewhat alleviates one or more symptoms associated with cancer.

[0110] For purposes of the present invention, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), incorporated herein by reference.

[0111] In the present invention, when X is Ala-Ala-Gln, X refers to a linker fragment formed after Ala, Ala, and Gln are linked in order to form a peptide bond, and those skilled in the art will understand that this does not limit the specific steric configuration (e.g., L- or D-form) of Ala, Ala, and Gln. Similarly, when X is Ala-Ala-(3-cyano-alanine), X refers to a linker fragment formed after Ala, Ala, and (3-cyano-alanine) are linked in order to form a peptide bond, and those skilled in the art will similarly understand that this does not limit the specific steric configuration (e.g., L- or D-form, or S- or R-form) of Ala, Ala, and (3-cyano-alanine). Other similar limitations on X in the present invention can be understood by reference to the above content.

[0112] In the present invention, when X is selected from Ala-Ala-Gln, Ala-Ala-(3-cyano-alanine) and deuterated versions thereof, this means that X may be Ala-Ala-Gln, Ala-Ala-(3-cyano-alanine), a deuterated version of Ala-Ala-Gln, or a deuterated version of Ala-Ala-(3-cyano-alanine).

[0113] The term "deuteride" refers to a compound formed after one or more (e.g., two, three, four, or more) hydrogen atoms (the positions of the hydrogen atoms are not particularly limited, and include hydrogen atoms on an alkyl group, hydrogen atoms on an amino group, etc.) in a compound are replaced with deuterium atoms.

[0114] The term "chimeric antibody" refers to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another species, such as an antibody whose variable region sequences are derived from a murine antibody and whose constant region sequences are derived from a human antibody.

[0115] "Humanized" antibodies refer to non-human (e.g., murine) forms of antibodies, which are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are derived from a human immunoglobulin (recipient antibody) in which residues from the complementarity-determining regions (CDRs) of the recipient antibody are substituted by CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0116] Furthermore, during humanization, amino acid residues within the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated, which may improve one or more binding characteristics (e.g., affinity) of the antibody. For example, mutations can be introduced by PCR-mediated mutagenesis, and their effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Furthermore, typically, no more than one or two mutations are made within a single CDR. [Example]

[0117] The present invention will be further described below using specific examples, but these examples are not intended to limit the scope of the present invention.

[0118] Example 1. Preparation of linker drug MCC-AAQ-Exatecan 1. Synthesis of Intermediate 1 Fmoc-Gln-Exatecan Fmoc-Gln-OH (N-fluorenylmethyloxycarbonyl-L-glutamine) (76.3 mg 1.1 eq.) and Exatecan (100 mg 1.0 eq.) were added to a reaction flask, followed by DMF (1 mL), DIEA (29 mg 1.5 eq.), and TBTU (72.5 mg 1.1 eq.), and the reaction was allowed to proceed at room temperature for 2 hours. The reaction was monitored by HPLC and continued until the starting material disappeared. After completion of the reaction, purification was carried out by medium-pressure column chromatography (DCM / MeOH). The product was collected and concentrated to give Intermediate 1. The reaction formula is as follows: [ka]

[0119] 2. Synthesis of Intermediate 2 Gln-Exatecan Intermediate 1 Fmoc-Gln-Exatecan (130 mg 1.0 eq.) was added to a reaction flask, followed by DCM (2 mL) and stirred at room temperature. DEA (0.5 mL) was added and the reaction was allowed to proceed at room temperature. After 4 hours, the reaction was monitored by HPLC and continued until the starting material disappeared. MTBE (10 mL) was slowly added, causing a large amount of solid to precipitate. After stirring for 30 minutes, it was filtered and washed with MTBE to obtain an off-white solid, which was dried to give 95 mg of Intermediate 2.

[0120] The reaction formula is as follows: [ka]

[0121] 3. Synthesis of Intermediate 3 Fmoc-Ala-Ala-Gln-Exatecan Intermediate 2: Gln-Exatecan (100 mg 1.0 eq.) was added to a reaction flask, and DMF (1 mL), Fmoc-Ala-Ala-OH (67.8 mg 1.0 eq.), TBTU (68.4 mg 1.2 eq.), and DIEA (34.4 mg 1.5 eq.) were added. The reaction was allowed to proceed at room temperature for 3 hours. The reaction was monitored by HPLC until completion. Purification was carried out by medium-pressure column chromatography (DCM / MeOH). The product was recovered to give Intermediate 3.

[0122] The reaction formula is as follows: [ka]

[0123] 4. Synthesis of Intermediate 4 Ala-Ala-Gln-Exatecan Intermediate 3: Fmoc-Ala-Ala-Gln-Exatecan (140 mg 1.0 eq.) was added to a reaction flask, followed by DCM (2 mL) and stirred at room temperature. DEA (0.5 mL) was added and the reaction was allowed to proceed at room temperature. After 4 hours, the reaction was monitored by HPLC until completion. MTBE (10 mL) was added slowly, causing a large amount of solid to precipitate. After stirring for 30 minutes, the mixture was filtered and washed with MTBE to obtain an off-white solid, which was dried to give Intermediate 4. The reaction formula is as follows: [ka]

[0124] 5. Synthesis of the target product MCC-Ala-Ala-Gln-Exatecan Intermediate 4 Ala-Ala-Gln-Exatecan (50 mg, 1.0 eq.) was added to a reaction flask, followed by DMF (1 mL), MCC (18 mg, 1.1 eq.), HATU (80.8 mg, 3.0 eq.), and DIEA (36.6 mg, 4.0 eq.). The reaction was allowed to proceed at room temperature. After 2 hours, the reaction was monitored by HPLC until the disappearance of Intermediate 4, and then the reaction was stopped. Purification was performed by prep-HPLC, and the product was collected, concentrated, and dried to give the desired product (NMR spectrum is shown below).

[0125] Reaction equation [ka]

[0126] 1 H NMR (400 MHz, DMSO-d6) δ 8.17~8.26 (d, J =2.0 Hz, 1H), 7.83~7.91 (d, J =2.0 Hz, 2H), 7.65~7.74(d, J =2.0 Hz, 2H), 7.21~7.28(d, J =8.0 Hz,2H), 6.98~7.04(m,2H), 5.43~5.50(m,1H), 5.36~5.43(m,2H), 5.15~5.24(m,1H), 5. 01~5.11(m,1H), 4.14~4.24(m,1H), 3.97~4.08(m,1H), 3.47~3.57(m,1H), 3.20~3.27(m, 1H), 3.05~3.16(m,2H), 2.30~2.36(m,2H), 2.00~2.15(m,4H), 1.77~1.92(m,3H), 1.65~2 .00(m,5H), 1.45~1.64(m,4H), 1.09~1.24(m,5H), 0.95~1.07(m,4H), 0.79~0.94(m,5H).

[0127] Example 2. Preparation of linker drug MCC-Ala-Ala-Asn-Exatecan 1. Synthesis of intermediate 1 Intermediate 1 was obtained by activating Fmoc-Ala-Ala-OH (N-[fluorenylmethyloxycarbonyl]-L-alanyl-L-alanine) with HOSu (N-hydroxysuccinimide) and then reacting it with L-Asn (L-asparagine).

[0128] The specific procedure is as follows: Fmoc-Ala-Ala-OH (1.0 eq), HOSu (1.3 eq), and THF (10 v / w) were added to a reaction flask at room temperature, and DCC (1.3 eq) was slowly added while stirring and maintaining room temperature. The reaction proceeded at room temperature and was monitored by HPLC until the remaining amount of Fmoc-Ala-Ala-OH (area normalized method) reached a low level. The reaction solution was then filtered and the filter cake was washed with THF (2.5 v / w). Purified water (11.5 v / w) was added to the filtrate, followed by the addition of L-Asn (1.1 eq) and solid sodium bicarbonate (1.0 eq). The reaction was continued with stirring at room temperature. The remaining amount of Fmoc-Ala-Ala-Osu was monitored by HPLC until it reached a low level. Citric acid monohydrate (1.0 eq) was then added and the mixture was stirred. The reaction solution was concentrated to remove most of the solvent. DMF (4 v / w) was added to dissolve the product. After filtration, reverse-phase separation was performed using preparative liquid chromatography. The separated solution was concentrated until no significant droplets were observed, and then extracted four times with ethyl acetate. The ethyl acetate layer was concentrated and dried to obtain Intermediate 1. The reaction scheme is as follows: [ka]

[0129] 2. Synthesis of intermediate 2 DEA (diethylamine) is added to intermediate 1 to remove Fmoc, thereby obtaining intermediate 2.

[0130] The specific procedure is as follows: Add intermediate 1 (1.0 eq) to a reaction flask at room temperature, add DMF (15 v / w), control the temperature at room temperature, add DEA (3 v / w) dropwise, and allow the reaction to proceed at room temperature. Detect by HPLC until no intermediate 1 remains, concentrate to remove DMF, add DCM (40 v / w) and pure water (40 v / w), stir, and then separate. The aqueous layer was concentrated and dried to obtain intermediate 2. The reaction scheme is as follows: [ka]

[0131] 3. Synthesis of intermediate 3 Intermediate 2 was reacted with N-succinimidyl-4-(N-maleimidomethylcyclohexanecarboxylate) to give Intermediate 3.

[0132] The specific procedure is as follows: At room temperature, intermediate 2 (1.0 eq) was added to a reaction flask, followed by N-succinimidyl-4-(N-maleimidomethyl)cyclohexanecarboxylate (1.1 eq), DMF (22 v / w), water (9.5 v / w), and DIPEA (1.0 eq). The reaction was then allowed to proceed at room temperature until the amount of intermediate 2 remaining was low (detected by QDA). After the reaction was completed, most of the solvent was concentrated to remove, and water (50 v / w) and EA (50 v / w) were added. 1.0 eq of DIEA was added, and the mixture was stirred to dissolve and clarify, and then allowed to stand for separation. The aqueous layer was extracted twice with EA (50 v / w*2), and the aqueous layer was concentrated and dried to obtain intermediate 3. The reaction scheme is as follows: [ka]

[0133] 4. Synthesis of the target product Intermediate 3 was subjected to a condensation reaction with exatecan mesylate (CAS No.: 169869-90-3) using HATU to obtain intermediate 4.

[0134] The specific procedure is as follows: At room temperature, intermediate 3 (1.2 eq) was added to a reaction flask, followed by DMF (15 v / w), exatecan mesylate (1.0 eq), TBTU (1.4 eq), and DIPEA (4.5 eq) in that order. The reaction was then allowed to proceed at room temperature and monitored by HPLC. Upon completion, the product was purified by prep-HPLC. The fraction was concentrated, extracted with DCM, and then concentrated to obtain the target product (NMR spectrum is shown below). The reaction scheme is as follows: [ka]

[0135] 1 H NMR (400 MHz, DMSO-d6) δ 8.15~8.25 (d, J =2.0 Hz, 1H), 7.85~8.0 (d, J =7.5Hz, 2H), 7.7~7.85(d, J =2.0 Hz, 1H), 7.33~7.4 (d, J =8.0 Hz,1H), 7.28~7.32 (m,1H), 6.98~7.05(d, J =8.0 Hz,2H), 6.85~6.95(m,1H), 6.45~6.57(m,1H), 5.45~5.55(m,1H), 5.40~5.45(m,1H), 5.20~5 .27(m,1H), 4.40~4.50(m,1H), 3.95~4.05(m,1H), 3.20~3.27(m,2H), 3.10~3.18(m,2H), 2.86 ~2.97(m,2H), 2.36~2.43(m,3H), 2.15~2.25(m,1H), 1.95~2.10(m,3H), 1.80~1.93(m,3H), 1 .73~1.78(m,1H), 1.55~1.70(m,3H), 1.45~1.55(m,3H), 1.0~1.3(m,10H), 0.80~0.95(m,5H).

[0136] Example 3. Preparation of linker drug MCC-Ala-Ala-(3-cyano-L-alanine)-Exatecan 1. Synthesis of Intermediate 1 (3-cyano-L-alanine)-Exatecan Fmoc-Asn-OH (40 mg, 1.2 eq) was added to a reaction flask, followed by DMF (1.5 mL), TBTU (45 mg, 1.5 eq), and DIEA (60 mg, 2.5 eq). The mixture was stirred until complete dissolution. After stirring for 30 minutes, exatecan mesylate (50 mg, 1.0 eq) was added and stirred for 4 hours. The reaction was monitored by HPLC to confirm completion. Further TBTU (45 mg, 1.5 eq) and DIEA (60 mg, 2.5 eq) were added, and the reaction was continued at room temperature. The reaction was monitored by HPLC until no starting material, exatecan, remained and approximately 70% of the product was Fmoc-(3-cyano-L-alanine)-Exatecan. The reaction was then stopped and purified by medium-pressure chromatography. The product was collected and concentrated to dryness under reduced pressure at 30 °C to obtain the desired product.

[0137] The target product was added to a reaction flask, and DCM (4 mL) was added and stirred. DEA (1 mL) was added and stirred at room temperature until completely dissolved. The reaction was monitored by HPLC and continued until no starting material remained. MTBE (10 mL) was added and stirred at room temperature. A large amount of solid precipitated. Filtration gave an off-white solid. The filter cake was washed with MTBE and dried to give 35 mg of a solid (Intermediate 1).

[0138] The reaction formula is as follows: [ka]

[0139] 2. Synthesis of Intermediate 2 MCC-Ala-Ala-OH SMCC (460 mg, 1.1 eq) was added to the reaction flask, followed by NH2-Ala-Ala-OH (200 mg, 1.0 eq) and DMF (4 mL, 20 V). The mixture was stirred and then DIPEA (161.4 mg, 1.0 eq) was added. The mixture was heated to 60 °C and allowed to react. As the reaction proceeded, the reaction mixture gradually dissolved and became clear. The reaction was continued for at least 8 h. A sample was taken for LC-MS detection. After the reaction was completed, the mixture was cooled to room temperature. The DMF was removed by concentration under reduced pressure at 35 °C, followed by the addition of purified water (4 mL, 20 V) and DIPEA (161.4 mg, 1.0 eq). EA (4 mL, 20 V) was added, stirred, and washed. The organic phase was discarded. The mixture was then extracted twice with EA (2 mL, 10 V). The organic phase was discarded. The aqueous phase was collected and the acidity was adjusted to pH 1-2 with 2M hydrochloric acid. A large amount of white solid precipitated. The mixture was stirred for 1 hour and filtered to obtain a white solid. The solid was washed with pure water and dried to obtain 320 mg of a white solid (Intermediate 2).

[0140] The reaction formula is as follows: [ka]

[0141] 3. Synthesis of the target product: MCC-Ala-Ala-(3-cyano-L-alanine)-Exatecan Intermediate 1 (3-cyano-L-alanine)-Exatecan (35 mg, 1.0 eq) was added to a reaction flask, followed by DMF (1 mL), Intermediate 2 MCC-Ala-Ala-OH (28 mg, 1.1 eq), TBTU (25.3 mg, 1.2 eq), and DIPEA (12.8 mg, 1.5 eq). The reaction was allowed to proceed at room temperature for 2 hours, after which it was monitored by HPLC until no more Intermediate 1 remained. The product was purified by prep-HPLC and recovered. The product was concentrated and dried to give the desired product as a yellow glassy solid (NMR spectrum is shown below).

[0142] The reaction formula is as follows: [ka]

[0143] 1 H NMR (400 MHz, DMSO-d6) δ 8.45~8.55 (d, J =2.0 Hz, 1H), 8.23~8.29 (d, J =2Hz, 2H), 7.95~8.03 (d, J =2.0 Hz, 1H), 7.84~7.92 (d, J =8.0 Hz,1H), 7.76~7.84 (m,1H), 7.27~7.33(d, J =8.0 Hz,1H), 6.98~7.02(m,1H), 5.47~5.56(m,1H), 5.40~5.45(m,1H), 5.26~5.36(m,1H), 5.1~5.2(m ,1H), 4.44~4.55(m,1H), 4.07~4.19(m,1H), 3.88~4.0(m,2H), 3.19~3.26(m,2H), 3.13~3.18(m,1 H), 2.90~3.0(m,2H), 2.76~2.88(m,2H), 2.36~2.43(m,3H), 2.00~2.12(m,2H), 1.80~1.93(m,3H) , 1.80~1.92(m,2H), 1.55~1.70(m,3H), 1.13~1.26(m,5H), 1.05~1.12(m,3H), 0.80~0.95(m,5H).

[0144] Example 4. Synthesis route of リンカーMCC-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan 1. Synthesis of Intermediate 1 ((S)-2-amino-4-cyanobutanoic acid)-Exatecan (S)-2-((((9H-Fluoren-9-yl) methoxy) carbonyl) amino)-cyano-butanoate (79.8 mg 1.1 eq) and exatecan mesylate (110 mg 1.0 eq) were added to a reaction flask, followed by DMF (1.5 mL), TBTU (79.73 mg, 1.5 eq), and DIEA (80 mg 2.5 eq), and the reaction was allowed to proceed at room temperature for 3 hours. The reaction was monitored by HPLC. After completion of the reaction, 9 mL of ACN was added and stirred at room temperature. 1.0 mL of DEA was then added, and the reaction was allowed to proceed at room temperature for 1 hour. Samples were taken to monitor the reaction progress, and the reaction was continued until no starting material remained. 10 mL of MTBE was added. Stirring did not result in any significant precipitation. After stirring for 1 hour, no obvious change was observed.

[0145] The solvent was concentrated under reduced pressure at 30°C. A large amount of solid precipitated during the concentration process. At this point, only approximately 3 mL of solvent had been removed, so concentration was stopped and stirring was continued at room temperature for 1 hour. After filtration, the solid was washed with MTBE and dried to give an off-white solid (Intermediate 1).

[0146] The reaction formula is as follows: [ka]

[0147] 2. Synthesis of Intermediate 2 MCC-Ala-Ala-OH SMCC (460 mg, 1.1 eq) was added to the reaction flask, followed by NH2-Ala-Ala-OH (200 mg, 1.0 eq) and DMF (4 mL, 20 V). The mixture was stirred, and then DIPEA (161.4 mg, 1.0 eq) was added. The mixture was heated to 60 °C and reacted for 8 h. A sample was collected for LC-MS analysis. After the reaction was completed, the mixture was cooled to room temperature. The DMF was removed by concentration under reduced pressure at 35 °C, followed by the addition of purified water (4 mL, 20 V) and DIPEA (161.4 mg, 1.0 eq). EA (4 mL, 20 V) was added, stirred, and washed. The organic phase was discarded. The mixture was then washed twice with EA (2 mL, 10 V). The organic phase was discarded. The aqueous phase was recovered, and the acidity was adjusted to pH 1-2 with 2 M hydrochloric acid. A large amount of white solid precipitated. The mixture was stirred for 1 h and filtered to obtain the white solid. The solid was washed with pure water and dried to obtain 320 mg of a white solid (Intermediate 2).

[0148] The reaction formula is as follows: [ka]

[0149] 3. Synthesis of the target product MCC-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan Intermediate 1 ((S)-2-amino-4-cyanobutanoic acid)-Exatecan (25 mg, 1.0 eq) was added to a reaction flask, followed by DMF (1 mL), Intermediate 2 MCC-Ala-Ala-OH (19 mg, 1.1 eq), TBTU (17.7 mg, 1.2 eq), and DIPEA (8.88 mg, 1.5 eq). The reaction was allowed to proceed at room temperature for 2 hours and then monitored by HPLC until completion. The product was purified by prep-HPLC and recovered. The acetonitrile was mostly removed by concentration, followed by lyophilization to yield the desired product as a yellow solid (NMR spectrum is shown below).

[0150] The reaction formula is as follows: [ka]

[0151] 1 H NMR (400 MHz, DMSO-d6) δ 8.30~8.37 (d, J =2.0 Hz, 1H), 7.87~7.96 (d, J =2.0 Hz, 2H), 7.75~7.86(d, J =2.0 Hz, 2H), 7.27~7.33(d, J =8.0 Hz,1H), 6.98~7.02(m,2H), 5.45~5.54(m,1H), 5.40~5.45(m,2H), 5.23~5.32(m,1H), 5. 04~5.13(m,1H), 4.25~4.37(m,1H), 3.97~4.09(m,1H), 3.56~3.66(m,4H), 3.10~3.26(m, 4H), 2.35~2.46(m,4H), 2.00~2.13(m,3H), 1.77~1.92(m,3H), 1.66~1.75(m,2H), 1.56~1 .64(m,3H), 1.45~1.53(m,1H), 1.12~1.26(m,4H), 1.01~1.13(m,4H), 0.80~0.94(m,4H).

[0152] Example 5. Synthesis route of リンカーMCC-Ala-Ala-Ala-Ala-Gln-Exatecan 1. Synthesis of intermediate 1 MCC-Ala-Ala-Ala-Ala-Gln-OH H-Gln(Trt)-OH 2-CTC resin (1.0 g, 0.5 mmol, 0.5 mmol / g) was added to a solid-phase reactor, and DCM (20 mL) was added. The resin was shaken on a shaker for 20 min to allow complete swelling. The resin was filtered and washed three times with DMF. 20 mL of a DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol) was added and thoroughly shaken. DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) was added, and the reaction was continued on a shaker for 1 h. The resin showed a negative color with Kaiser's reagent. The resin was filtered and washed six times with DMF. 20 mL of 20% piperidine / DMF solution was added and the mixture was allowed to react for 15 minutes on a shaker. After filtration, 20 mL of 20% piperidine / DMF solution was added again and the mixture was allowed to react for 15 minutes on a shaker. The resin showed a positive color when stained with Kaiser's reagent. The resin was washed six times with DMF to obtain H-Ala-Gln(Trt)-2-CTC-resin.

[0153] To the reaction tube containing H-Ala-Gln(Trt)-2-CTC-resin, 20 mL of a DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol) was added. After thorough shaking, DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) was added and the mixture was incubated in a shaker for 1 hour. The resin showed a negative Kaiser's reagent color. The resin was filtered and washed six times with DMF. 20 mL of 20% piperidine / DMF solution was added and the mixture was incubated in a shaker for 15 minutes. After filtration, another 20 mL of 20% piperidine / DMF solution was added and the mixture was incubated in a shaker for 15 minutes. The resin showed a positive Kaiser's reagent color. The resin was washed six times with DMF to obtain H-Ala-Ala-Gln(Trt)-2-CTC-resin.

[0154] To the reaction tube containing H-Ala-Ala-Gln(Trt)-2-CTC-resin, 20 mL of a DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol) was added. After thorough shaking, DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) was added and the mixture was incubated in a shaker for 1 hour. The resin showed a negative Kaiser's reagent color. The resin was filtered and washed six times with DMF. 20 mL of 20% piperidine / DMF solution was added and the mixture was incubated in a shaker for 15 minutes. After filtration, 20 mL of 20% piperidine / DMF solution was added again and the mixture was incubated in a shaker for 15 minutes. The resin showed a positive Kaiser's reagent color. The resin was washed six times with DMF to obtain H-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin.

[0155] To the reaction tube containing H-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin, 20 mL of a DMF solution containing Fmoc-Ala-OH (3.0 eq, 467 mg, 1.5 mmol), HBTU (3.0 eq, 569 mg, 1.5 mmol), and HOBT (3.0 eq, 202 mg, 1.5 mmol) was added. After thorough shaking, DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) was added and the mixture was incubated in a shaker for 1 hour. The resin showed a negative Kaiser's reagent color. The resin was filtered and washed six times with DMF. 20 mL of 20% piperidine / DMF solution was added and the mixture was incubated in a shaker for 15 minutes. After filtration, 20 mL of 20% piperidine / DMF solution was added again and the mixture was incubated in a shaker for 15 minutes. The resin showed a positive Kaiser's reagent color. The resin was washed six times with DMF to give H-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin.

[0156] A 20 mL DMF solution containing SMCC (3.0 eq, 501 mg, 1.5 mmol) was added to the reaction tube containing H-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin. After thorough shaking, DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) was added and the mixture was incubated in a shaker for 1 h. The resin showed a negative color response with Kaiser's reagent. The resin was filtered and washed sequentially with DMF six times, MeOH three times, and MTBE three times. The resin was dried in a vacuum oven at 30 °C for 1 h to obtain MCC-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin (1.42 g, 0.5 mmol).

[0157] The resulting resin was transferred to a 50 mL centrifuge tube. A cold 95% TFA / Tis solution (15 mL) was added, and the mixture was allowed to react in a shaker for 1 hour. After filtration, the filtrate was slowly added to a cold MTBE solution (150 mL). After standing for 1 hour, a white precipitate formed. The precipitate was centrifuged and washed three times with cold MTBE solution. The washed precipitate was collected and dried in a vacuum drying oven at 30°C for 4 hours to obtain the intermediate 1MCC-Ala-Ala-Ala-Ala-Gln-OH as a white solid.

[0158] The reaction formula is as follows: [ka]

[0159] 2. Synthesis of the target product, MCC-Ala-Ala-Ala-Ala-Gln-Exatecan Intermediate 1: MCC-Ala-Ala-Ala-Ala-Gln-OH (244 mg, 2.0 eq) was added to a reaction flask, followed by DMF (2 mL), exatecan mesylate (100 mg, 1.0 eq), TBTU (132 mg, 2.2 eq), and DIPEA (73 mg, 3.0 eq). The reaction was stirred at room temperature for 1 hour. The reaction was monitored by HPLC and continued until 10% of exatecan remained.

[0160] The target product was recovered by prep-HPLC purification, and the collected solution was concentrated and dried to give a yellow solid (NMR spectrum is as follows).

[0161] The reaction formula is as follows: [ka]

[0162] 1 H NMR (400 MHz, DMSO-d6) δ 8.25~8.33(d, J =2.0 Hz, 1H), 7.87~7.95 (d, J =2.0 Hz, 2H), 7.76~7.86(d, J =2.0 Hz, 2H), 7.28~7.34(d, J =8.0 Hz,1H), 7.20~7.26(m,1H), 6.98~7.02(m,1H), 6.73~6.76(m,1H), 5.47~5.55(m,1H), 5.40~5.41(m,1H), 5.26~5.36(m,2H), 5.05~5.15 (m,2H), 4.12~4.20(m,2H), 4.03~4.09(m,1H), 3.87~3.94(m,1H), 3.20~3 .27(m,2H), 3.12~3.18(m,1H), 2.65~2.68(m,1H), 2.36~2.43(m,2H), 2.31 ~2.35(m,1H), 2.02~2.13(m,2H), 1.78~1.93(m,3H), 1.6~1.76(m,2H), 1. 56~1.64(m,2H), 1.21~1.27(m,1H), 1.06~1.20(m,6H), 0.85~0.94(m,3H).

[0163] Example 6: Synthetic Route of Linker Drug MCC-Ala-Ala-Gln-Belotecan MCC-Ala-Ala-Gln-Belotecan synthesis pathway [ka] 1. Intermediate 3 Fmoc-Gln-Belotecan synthesis [ka]

[0164] Intermediate 1 (212 mg, 0.575 mmol, 5.0 eq), Belotecan (50 mg, 0.115 mmol, 1.0 eq), and HATU (219 mg, 0.575 mmol, 5 eq) were added to a reaction flask, and DMF (3 mL) was added and the mixture was shaken to dissolve. DIEA (190 μL, 1.15 mmol, 10 eq) was added at 10°C, and the mixture was stirred at 10°C for 1 hour. The reaction was monitored by HPLC until Belotecan was completely reacted. The product was then purified and isolated. The product was concentrated and lyophilized to obtain product 3 Fmoc-Gln-Belotecan as a yellow solid (51 mg, purity: 98.71%, yield: 55.84%). MS (ESI): m / z 784.3 [M+H] + 6.

[0165] 2. Synthesis of Intermediate 4 Gln-Belotecan [ka]

[0166] Intermediate 3 Fmoc-Gln-Belotecan (51 mg, 0.065 mmol, 1.0 eq) was added to a reaction flask, followed by the addition of DMF (4 mL) and diethylamine (1 mL), and the mixture was stirred at 20°C for 2 hours. The reaction was monitored by HPLC and continued until no starting material remained. The reaction mixture was concentrated in vacuo, and DMF (2 mL) was added for preparative purification. The preparative mixture was concentrated and then lyophilized to give 4 Gln-Belotecan as a yellow solid (24 mg, purity: 91.96%, yield: 60.40%). MS (ESI): m / z 562.34 [M+H] + .

[0167] 3. Synthesis of Intermediate 5 MCC-Ala-Ala-OH [ka] The starting materials, 5-1 MCC-OSu (9.5 g, 59.31 mmol, 1.0 eq) and 5-2 Ala-Ala-OH (21.8 g, 65.24 mmol, 1.1 eq), were added to a reaction flask. At 25 °C, DMF (190 mL) was added, followed by DIEA (9.8 mL, 59.31 mmol, 1.0 eq). The reaction was stirred at 60 °C for 8 h. The reaction was monitored by HPLC to confirm completion. The reaction mixture was cooled to 25 °C, concentrated in vacuo, and then HO (190 mL), DIEA (9.8 mL), and EA (190 mL) were added sequentially. The mixture was stirred at 25 °C for 1 h, then transferred to a separatory funnel and allowed to stand for separation. The aqueous layer was separated and extracted twice with EA (95 mL). The EA layer was removed, and 2M hydrochloric acid solution (71.3 mL) was slowly added dropwise to the aqueous layer. The mixture was stirred at 25°C for 1 hour and then filtered. The resulting solid was dried in vacuo to give a white solid, 5MCC-Ala-Ala-OH (16.55 g, purity: 95%, yield: 73.54%). MS (ESI): m / z 380.2 [M+H] + .

[0168] 4.MCC-Ala-Ala-Gln-Belotecan synthesis [ka]

[0169] Intermediate 4 (15 mg, 0.027 mmol, 1.0 eq), 5MCC-Ala-Ala-OH (15.2 mg, 0.04 mmol, 1.5 eq), and HOAT (7.3 mg, 0.054 mmol, 2.0 eq) were added to a reaction flask, and DMF (2 mL) was added and the mixture was shaken to dissolve. DIC (8.0 μL, 0.054 mmol, 2.0 eq) was added at 10 °C, and the reaction was stirred at 20 °C for 3 hours. The reaction was monitored by HPLC until intermediate 4 was completely reacted. The product was purified by preparative liquid chromatography and recovered. The product was concentrated and lyophilized to obtain the desired product, MCC-Ala-Ala-Gln-Belotecan, as a yellow solid (11 mg, purity: 96.42%, yield: 43.03%). MS (ESI): m / z 462.5 [M+2H] / 2+ .

[0170] 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J= 10.4 Hz, 1H), 8.19 (d, J= 8.0 Hz, 1H), 8.15 (d, J= 8.0 Hz, 1H), 7.92~7.87 (m, 4H), 7.78 (t, J= 8.0 Hz, 1H), 7.35 (s, 1H), 7.21 (brs, 1H), 6.99 (s, 2H), 6.77 (brs, 1H), 5.46~5.43 (m, 4H), 4.79~4.76 (m, 1H), 4.35~4.28 (m, 1H), 4.26~4.21 (m, 2H), 3.42~3.38 (m, 2H), 3.23 (d, J= 6.8 Hz, 2H), 3.22~3.18 (m, 1H), 2.22~2.07 (m, 3H), 1.93~1.84 (m, 3H), 1.75~1.66 (m, 3H), 1.62~1.59 (m, 2H), 1.27~1.26 (m, 4H), 1.23~1.21 (m, 5H), 1.18~1.16 (m, 6H), 0.90~0.86 (m, 6H).

[0171] Furthermore, those skilled in the art can obtain other linker-drug conjugates of the present invention, such as MCC-AAQ-Rapamycin, by referring to the preparation methods of Examples 1 to 6 above. [ka] MCC-AAQ-Rapamycin

[0172] Example 7. Preparation of EGFR antibody drug conjugates 1. Preparation of antibody conjugate EGFR antibody-MCC-AAQ-Exatecan 1. The pH of the EGFR antibody was adjusted to approximately 7.5 with Tris-EDTA solution. The protein concentration was measured using a Nanodrop™ system, and the net weight of the antibody solution was weighed to calculate the total protein content. The antibody was then added to the TCEP solution, placed on a 3D shaker, and incubated at room temperature for over 120 minutes while shaking to completely reduce the interchain disulfide bonds of the antibody.

[0173] 2. An excess amount of MCC-AAQ-Exatecan solution (dissolved in DMSO) was added to the reduced antibody solution, mixed uniformly, and then placed on a 3D shaker and allowed to react for at least 30 minutes while shaking at room temperature. After the reaction was complete, an excess amount of N-acetylcysteine ​​solution was added to the reaction solution. The mixture was placed on a 3D shaker and allowed to react for at least 30 minutes while shaking at room temperature.

[0174] 3. The conjugated target product was purified using a 30KD ultrafiltration centrifuge tube and exchanged into a storage solution (10 mM histidine, pH 5.5) at an exchange ratio of 1000 or more. It was then filtered through a 0.22 μm sterile filter to obtain the antibody-drug conjugate EGFR antibody-MCC-AAQ-Exatecan, which was stored at 4°C.

[0175] 4. The protein concentration of the antibody-drug conjugate was measured by UV / BCA method, DAR by HIC, and purity by SEC.

[0176] II. Preparation of other EGFR antibody-drug conjugates For a method for preparing an antibody-drug conjugate using another linker-drug conjugate of the present invention (the linker-drug conjugate shown in Formula I) and an EGFR antibody, reference may be made to the method for preparing the antibody conjugate EGFR antibody-MCC-AAQ-Exatecan described above.

[0177] Example 8. Preparation of HER2 antibody drug conjugates For a method for preparing an antibody-drug conjugate (e.g., antibody-drug conjugate HER2 antibody-MCC-AAN-Exatecan) using the linker-drug conjugate of the present invention (the linker-drug conjugate shown in Formula I) and a HER2 antibody, reference can be made to the method for preparing the antibody conjugate EGFR antibody-MCC-AAQ-Exatecan in Example 7 above.

[0178] Example 9: In vitro activity test of various antibody-drug conjugates For various antibody drug conjugates of the present invention, in vitro activity tests were carried out to detect cell killing activity.

[0179] The test method is as follows: Target cells were seeded in a fixed number into a 96-well black-bottom clear cell culture plate. After 24 hours of incubation, serially diluted samples were added. After 72 or 144 hours of incubation, the cells were developed with a colorimetric reagent for 60 minutes and the plate was read using a microplate reader. The higher the number of viable cells, the higher the signal value. Based on the signal values ​​obtained from different concentration gradient points, a four-parameter fitting curve was constructed on the microplate reader, and the C value (i.e., IC50 value) was calculated.

[0180] Some of the data is as follows:

[0181] [Table 7]

[0182] Furthermore, the present invention also provides the following examples.

[0183] Example 10. Preparation of linker-drug conjugate mc-AAN-Exatecan 1. Synthesis of intermediate 1 Fmoc-Ala-OH (N-fluorenylmethyloxycarbonyl-L-alanine, CAS No.: 35661-39-3) was activated with HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6) and then reacted with L-Ala (L-alanine, CAS No.: 56-41-7) to obtain intermediate 1. Specifically, Fmoc-Ala-OH (3 g, 1.0 eq) and HOSu (1.45 g, 1.3 eq) were added to a reaction flask, followed by the addition of 21 mL of THF. The temperature was controlled at room temperature, and DCC (2.59 g, 1.3 eq) was slowly added with stirring. The reaction was then allowed to proceed at room temperature and monitored by HPLC until completion. The reaction mixture was then filtered, and the filter cake was washed with THF (6 mL). Purified water (15 mL) was added to the filtrate, followed by the addition of L-Ala (1.12 g, 1.3 eq) and solid sodium bicarbonate (0.81 g, 1.0 eq), and the mixture was stirred at room temperature. The reaction was monitored by HPLC until completion. Citric acid (2.02 g, 1.0 eq) was then added and the mixture was stirred. The reaction mixture was extracted with ethyl acetate, and the organic layer was concentrated. DMF (12 mL) was added to dissolve the target product. After filtration, the product was separated by pre-HPLC. The separated solution was concentrated until no significant droplets were observed, followed by extraction three times with ethyl acetate. The ethyl acetate layer was concentrated and dried to obtain 1.75 g of intermediate 1. The yield of this step was 57%, and the reaction scheme is as follows: [ka]

[0184] 2. Synthesis of intermediate 2 Intermediate 1 (N-[fluorenylmethyloxycarbonyl]-L-alanyl-L-alanine, CAS No.: 87512-31-0) was activated with HOSu (N-hydroxysuccinimide, CAS No.: 6066-82-6) and then reacted with L-Asn (L-asparagine, CAS No.: 70-47-3) to obtain intermediate 2. Specifically, intermediate 1 (0.87 g, 1.0 eq), HOSu (0.34 g, 1.3 eq), and THF (9 mL) were added to a reaction flask, and DCC (0.61 g, 1.3 eq) was slowly added with stirring at room temperature. The reaction proceeded at room temperature and was monitored by HPLC until completion. The reaction mixture was then filtered, and the filter cake was washed with THF (2 mL). Purified water (10 mL) was added to the filtrate, followed by the addition of L-Asn (0.34 g, 1.1 eq) and solid sodium bicarbonate (0.19 g, 1.0 eq), and the mixture was stirred at room temperature. The reaction was monitored by HPLC until completion. Citric acid monohydrate (0.48 g, 1.0 eq) was then added and the mixture was stirred. The reaction mixture was concentrated to remove most of the solvent, and the residue was purified by fractionation. The fraction was concentrated until no significant droplets were observed, and then extracted with ethyl acetate. The organic layer was concentrated and dried to obtain 826 mg of intermediate 2. The yield of this step was 73%, and the reaction scheme is as follows: [ka]

[0185] 3. Synthesis of intermediate 3 DEA (diethylamine, CAS No.: 109-89-7) was added to intermediate 2, and the Fmoc was removed to obtain intermediate 3. Specifically, intermediate 2 (100 mg) and DMF (1.5 mL) were added to a reaction flask, and the temperature was controlled at room temperature. DEA (300 μL) was added dropwise, and the reaction proceeded at room temperature. The reaction was monitored by HPLC until the disappearance of intermediate 2, and then the reaction was stopped. The mixture was concentrated to remove DMF, and DCM (4 mL) and pure water (4 mL) were added. After stirring, the mixture was separated. The aqueous layer was concentrated and dried to obtain 92 mg of intermediate 3. The yield of this step was 166% (including the solvent), and the reaction scheme is as follows: [ka]

[0186] 4. Synthesis of intermediate 4 Intermediate 3 was reacted with 6-(maleimido)hexanoic acid succinimide ester (CAS No.: 55750-63-5) to obtain intermediate 4. Specifically, intermediate 3 (92 mg, 1.0 eq) was added to a reaction flask, and 6-(maleimido)hexanoic acid succinimide ester (135 mg, 1.3 eq), DMF (1.5 mL), and DIPEA (0.059 mL, 1.0 eq) were added. The reaction was monitored by HPLC. After completion of the reaction, the product was purified and concentrated to obtain 56 mg of intermediate 4. The yield of this step was 35.7%, and the reaction scheme is as follows: [ka]

[0187] 5. Synthesis of the target product Intermediate 4 was obtained by condensation of intermediate 4 with exatecan mesylate (CAS No.: 169869-90-3). Specifically, intermediate 4 (26 mg, 1.0 eq) was added to a reaction flask at room temperature, followed by DMF (1.5 mL), exatecan mesylate (29.6 mg, 1.0 eq), EEDQ (20.7 mg, 1.5 eq), HATU (31.8 mg, 1.5 eq), DMAP (0.7 mg, 0.1 eq), and DIPEA (29.2 μL, 3.0 eq) were added sequentially. The reaction was allowed to proceed at room temperature and monitored by HPLC. Upon completion of the reaction, the product was purified and concentrated to obtain 16.5 mg of the desired product (NMR spectrum is shown below). The yield of this step was 33.5%, and the reaction scheme is as follows: [ka]

[0188] 1H NMR (400 MHz, DMSO-d6) δ 8.22~8.29 (d, J =2.0 Hz, 1H), 7.94~8.05 (d, J =2.0Hz, 3H), 7.83~7.88(d, J =2.0 Hz,1H), 7.75~7.82(d, J =2.0 Hz, 1H), 7.34~7.40 (m,1H), 7.28~7.33 (m,1H), 6.96~7.02(d, J =8.0 Hz,2H), 6.87~6.94(m,1H), 5.42~5.55(m,1H), 5.41~5.46(m,2H), 5.21~5.26(m,1H) ), 4.41~4.49(m,1H), 4.00~4.12(m,2H), 3.32~3.40(m,2H), 3.12~3.17(m,2H), 2.86 ~2.96(m,2H), 2.71~2.76(m,2H), 2.36~2.43(m,2H), 2.14~2.24(m,1H), 1.97~2.07( m,3H), 1.81~1.93(m,2H), 1.36~1.52(m,4H), 1.05~1.25(m,8H), 0.83~0.93(m,3H).

[0189] Example 11. Preparation of linker-drug conjugate mc-Ala-Ala-Gln-Exatecan Intermediate 1 Synthesis of Fmoc-Gln-Exatecan Fmoc-Gln-OH (N-fluorenylmethyloxycarbonyl-L-glutamine) (76.3 mg 1.1 eq.) and Exatecan (100 mg 1.0 eq.) were added to a reaction flask, followed by DMF (1 mL), DIEA (29 mg 1.5 eq.), and TBTU (72.5 mg 1.1 eq.). The reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC and continued until the starting material disappeared. After completion of the reaction, the mixture was concentrated onto silica gel and purified by column chromatography. The product was recovered, concentrated, and dried to obtain 140 mg of the desired product. The purity was 95% (approximately 4% of Hobt, a by-product of TBTU, was not removed), and the yield was 94%.

[0190] The reaction formula is as follows: [ka]

[0191] 1. Synthesis of Intermediate 2 Gln-Exatecan Intermediate 1: Fmoc-Gln-Exatecan (130 mg 1.0 eq.) was added to a reaction flask, followed by DCM (2 mL). The mixture was stirred at room temperature, but did not dissolve or clear. DEA (0.5 mL) was added, and the reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC and continued until the starting material disappeared. MTBE (10 mL) was slowly added, causing a large amount of solid to precipitate. After stirring for 30 minutes, the mixture was filtered to obtain an off-white solid, which was then dried to give 95 mg of the desired product. The purity was 96% and the yield was 94%.

[0192] The reaction formula is as follows: [ka]

[0193] 2. Synthesis of Intermediate 3 Fmoc-Ala-Ala-Gln-Exatecan Intermediate 2: Gln-Exatecan (100 mg 1.0 eq.) was added to a reaction flask, followed by DMF (1 mL), Fmoc-Ala-Ala-OH (67.8 mg 1.0 eq.), TBTU (68.4 mg 1.2 eq.), and DIEA (34.4 mg 1.5 eq.), and the reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC until no intermediate 2 remained, at which point the reaction was stopped. The product was concentrated onto silica gel and purified by column chromatography. 140 mg of the desired product was recovered. The purity was 95% and the yield was 85%.

[0194] The reaction formula is as follows: [ka]

[0195] 3. Synthesis of Intermediate 4 Ala-Ala-Gln-Exatecan Intermediate 3: Fmoc-Ala-Ala-Gln-Exatecan (140 mg, 1.0 eq.) was added to a reaction flask, followed by DCM (2 mL) and DEA (0.5 mL), and the reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC and continued until the starting material disappeared. MTBE (10 mL) was slowly added, causing a large amount of solid to precipitate. After stirring for 30 minutes, the off-white solid was filtered and dried to give 70 mg of the desired product. Purity: 96%. Yield: 66%.

[0196] The reaction formula is as follows: [ka]

[0197] 4. Synthesis of the target product mc-Ala-Ala-Gln-Exatecan Intermediate 4 Ala-Ala-Gln-Exatecan (50 mg 1.0 eq.) was added to a reaction flask, and DMF (1 mL), 6-(maleimido)hexanoic acid succinimide ester (21.8 mg 1.1 eq.), and DIEA (12.1 mg 1.5 eq.) were added. The reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC until the disappearance of Intermediate 4, and then the reaction was stopped. The target product was recovered and concentrated to dryness, yielding 35 mg of the target product (NMR spectrum is shown below). The purity was 98% and the yield was 55%.

[0198] The reaction formula is as follows: [ka]

[0199] 1H NMR (400 MHz, DMSO-d6) δ 8.28~8.36 (d, J =2.0 Hz, 1H), 7.90~7.96(d, J =2.0Hz, 2H), 7.75~7.87(d, J =2.0 Hz,2H), 7.27~7.33(d, J =2.0 Hz, 1H), 7.20~7.25 (m,1H), 6.97~7.03(d, J =8.0 Hz,2H), 6.69~6.75(m,1H), 6.49~6.53(m,1H), 5.46~5.55(m,1H), 5.40~5.45(m,2H), 5.24~5. 34(m,1H), 5.04~5.14(m,1H), 4.31~4.38(m,1H), 4.02~4.23(m,2H), 3.86~3.96(m,1H), 3.40~ 3.49(m,1H), 3.33~3.40(m,2H), 3.12~3.20(m,1H), 2.36~2.42(m,2H), 2.15~2.25(m,1H), 2.0 0~2.12(m,4H), 1.71~1.93(m,3H), 1.36~1.52(m,4H), 1.01~1.20(m,10H), 0.83~0.92(m,3H).

[0200] Example 12. Preparation of linker-drug conjugate mc-Pro-Exatecan 1. Synthesis of Intermediate 1 Fmoc-Pro-Exatecan Fmoc-Pro-OH (N-fluorenylmethyloxycarbonyl-L-proline) (41 mg, 1.3 eq.) and Exatecan (50 mg, 1.0 eq.) were added to a reaction flask, and DMF (1 mL) and DIEA (49 mg, 4 eq.) were added to dissolve the product. HATU (46 mg, 1.3 eq.) and HOBt (12.7 mg, 1.0 eq.) were added, and the reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC and continued until the starting material disappeared. After completion of the reaction, the mixture was concentrated onto silica gel and purified by column chromatography. The target product was recovered, concentrated, and dried to obtain 50 mg of the target product. The purity was 95% and the yield was 73%.

[0201] The reaction formula is as follows: [ka]

[0202] 2. Synthesis of Intermediate 2 Pro-Exatecan Intermediate 1: Fmoc-Pro-Exatecan (50 mg, 1.0 eq.) was added to a reaction flask, followed by DCM (1 mL) and DEA (0.2 mL), and the reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC and continued until the starting material disappeared. MTBE (10 mL) was slowly added, causing a large amount of solid to precipitate. After stirring for 30 minutes, the off-white solid was filtered and dried to give 30 mg of the desired product. The purity was 96% and the yield was 86%.

[0203] The reaction formula is as follows: [ka]

[0204] 3. Synthesis of the target compound mc-Pro-Exatecan Intermediate 2: Pro-Exatecan (30 mg 1.0 eq.) was added to a reaction flask, followed by DMF (1 mL), 6-(maleimido)hexanoic acid succinimide ester (21.2 mg 1.1 eq.), and DIEA (12.1 mg 1.5 eq.). The reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC until the disappearance of Intermediate 2, at which point the reaction was stopped. The mixture was concentrated onto silica gel and purified by column chromatography. 25 mg of the desired product was recovered (NMR spectrum is shown below). The purity was 96% and the yield was 56%.

[0205] The reaction formula is as follows: [ka] 1H NMR (400 MHz, DMSO-d6) δ 8.17~8.26 (d, J =2.0 Hz, 1H), 7.80~7.90 (d, J =2.0 Hz, 2H), 7.21~7.28(d, J =8.0 Hz,1H), 6.98~7.04(m,1H), 5.01~5.11(m,1H), 4.14~4.24(m,1H), 3.97~4 .08(m,1H), 3.47~3.57(m,1H), 3.20~3.27(m,1H), 3.05~3.16(m,2H), 2.30 ~2.36(m,2H), 2.00~2.15(m,4H), 1.77~1.92(m,3H), 1.65~2.00(m,6H), 1. 45~1.64(m,4H), 1.09~1.24(m,4H), 0.95~1.07(m,2H), 0.79~0.94(m,3H).

[0206] Example 13. Preparation of antibody conjugate HER2 antibody-mc-AAN-Exatecan (DAR8) 1.10 mg of HER2 antibody was diluted with PBS buffer to a concentration of 5 mg / mL. The pH of the antibody was adjusted to approximately 7.5 with Tris-EDTA solution. The protein concentration was measured using a Nanodrop, and the net weight of the antibody solution was weighed to calculate the total protein content. An 8-fold molar amount of TCEP solution was added to the antibody, placed on a 3D shaker, and allowed to react for at least 120 minutes with continuous stirring at room temperature. 2. An excess amount of mc-AAN-Exatecan solution (dissolved in DMSO) was added to the reduced antibody solution, mixed evenly, and then placed on a 3D shaker and allowed to react for at least 30 minutes while shaking at room temperature. After the reaction was complete, an excess amount of N-acetylcysteine ​​solution was added to the reaction solution. The solution was placed on a 3D shaker and allowed to react for at least 30 minutes while shaking at room temperature. 3. The conjugate product was purified in a 15 mL 30 KD ultrafiltration centrifuge tube and exchanged into a storage solution (10 mM histidine, pH 5.5). The exchange ratio was 1000 times or more. It was then filtered through a 0.22 μm sterile filter to obtain the antibody-drug conjugate HER2 antibody-mc-AAN-Exatecan, which was stored at 4°C. 4. The protein concentration of the antibody-drug conjugate was measured by UV / BCA method, DAR by HIC, and purity by SEC.

[0207] Example 14: In vitro activity test of various antibody-drug conjugates Referring to the steps in Example 13, the linker-drug conjugate mc-AAN-Exatecan was conjugated to a HER2 antibody, an EGFR antibody, and a non-specific binding antibody (Anti-HEL), respectively, to prepare the corresponding ADCs. The linker-drug conjugate mc-Pro-Exa was conjugated to a HER2 antibody to prepare an ADC. The cell-killing activity was detected.

[0208] The test method is as follows: Target cells were seeded in a fixed number into a 96-well black-bottom clear cell culture plate. After 24 hours of incubation, serially diluted samples were added. After 72 or 144 hours of incubation, the cells were developed with a colorimetric reagent for 60 minutes and the plate was read using a microplate reader. The higher the number of viable cells, the higher the signal value. Based on the signal values ​​obtained from different concentration gradient points, a four-parameter fitting curve was constructed on the microplate reader, and the C value (i.e., IC50 value) was calculated.

[0209] The detailed data is as follows:

[0210] [Table 8]

[0211] [Table 9]

[0212] [Table 10]

[0213] Example 15. Preparation, detection, and analysis of ADCs with different linker structures or cytotoxic agents. The following ADC stock solutions were prepared with reference to the preparation processes for the Linker-Payload in Example 10 and the ADC stock solution in Example 13, and the basic quality characteristics of the obtained ADC stock solutions were detected and analyzed.

[0214] After conjugation to the same HER2 antibody, the DAR values ​​were all around 8. Based on the detection data analysis of these ADCs, the following conclusions were reached: 1. The polymer content (HMW%) of ADCs prepared from linker payloads without PAB was found to be significantly lower than that of ADCs prepared from linker payloads containing PAB fragments. This indicates that PAB fragments are a disadvantage in preparing ADCs from this type of linker payload. Specific data are shown in Table 5. 2. Payloads with a primary amine (-NH) structure were releasable by linkers that did not contain PAB fragments, but payloads with a non-primary amine group (-NH-(i-Pr) structure) were not releasable by linkers that did not contain PAB fragments. Specific data are shown in Table 6.

[0215] The inventors investigated the polymer content in ADCs obtained by conjugating mc-Pro-PAB-Exatecan to the antibody TF (TF-mAb-H39 described in CN201610705557.4) and mc-Pro-Exatecan to a HER2 antibody, and confirmed that the PAB structure has a significant effect. Removal of the PAB from the linker reduced the polymer content from approximately 70-80% to 1%.

[0216] [Table 11]

[0217] We investigated the in vitro cell-killing activity of ADCs obtained by conjugating mc-Pro-Belotecan and mc-Pro-Exatecan to HER2 antibodies. The mc-Pro-Belotecan-conjugated ADC did not exhibit tumor cell-killing activity, indicating that the structure was stable and that the payload was not released by enzymatic degradation. On the other hand, the mc-Pro-Exatecan-conjugated ADC exhibited good cell-killing activity, suggesting that the payload could be released from this structure. Furthermore, the mc-Pro-Exatecan-conjugated ADC to a non-binding antibody (without targeted endocytosis) did not exhibit specific cell-killing activity, suggesting that the structure was stable outside the cell and that the payload was released only after the structure was endocytosed into the cell. Furthermore, a comparison of the chemical structures of mc-Pro-Belotecan and mc-Pro-Exatecan revealed that the main difference between the two is that Belotecan is linked to proline via an -NH-(i-Pr) structure, whereas Exatecan is linked to proline via an -NH2 structure. Therefore, if the amino acid fragment in the linker is linked to the payload containing a primary amine (-NH2) structure, it is possible that the payload can be released by enzymatic degradation by removing the hydrophobic PAB structure (in most ADCs currently under investigation, the self-cleaving PAB structure is essential for effective payload release). This reduces the hydrophobicity of the linker-drug, thereby reducing the amount of polymer produced during ADC preparation and demonstrating the corresponding cell killing activity.

[0218] [Table 12]

[0219] Although specific embodiments of the present invention have been described in detail, it is understood by those skilled in the art that, according to all the teachings disclosed, various modifications and substitutions to these details are possible, and all such modifications fall within the scope of protection of the present invention. The full scope of the present invention is defined by the appended claims and any equivalents thereof.

Claims

1. A linker drug conjugate or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein said linker drug conjugate has the structure shown in Formula I: MXD Formula I During the ceremony, M is a chemical structure containing a maleimide (m) fragment or a chemical structure containing a cyclooctyne and its derivative fragments; X is a linker fragment consisting of 1 to 5 amino acids or derivatives thereof, and is preferably Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), o-alanine), 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys- Pro, Ala-Ala-Ala-Ala-Gln, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Ala-Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-(2-amino-4-cyanobutanoic acid), 2-amino-4-cyanobutanoic acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln and deuterated versions thereof; A linker drug conjugate or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein D is a cytotoxin.

2. 2. The linker-drug conjugate of claim 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, wherein the chemical structure comprising the maleimide (m) fragment is selected from 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl (MCC), 6-maleimidohexanoyl (MC), M-(PEG)n, MC-(PEG)n, MCC-(PEG)n, and the structures thereof are as follows: Table 1 Here, n represents the degree of polymerization of PEG, and is preferably an integer of 2 to 12.

3. 2. The linker-drug conjugate of claim 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, wherein the chemical structure comprising the cyclooctyne fragment is selected from BCN, DIBO, DIBAC, BARAC, etc., and the structure thereof is as follows: Table 2

4. The linker fragment consisting of 1 to 5 amino acids or derivatives thereof may be Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-Ala-(3-cyano-alanine), Ala-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), anine), 3-cyano-alanine, Ala-Ala-Ala-Pro, Ala-Ala-Pro, Ala-Pro, Pro, Pro-Asn, Asn-Pro, Lys, Lys-Asn, Lys-Pr o, Ala-Ala-Ala-Ala-Gln, Ala-Ala-Ala-Gln, Ala-Ala-Gln, Ala-Gln, Ala-Ala-Ala-Ala-(2-amino-4-cyanobutanoic 4. The linker-drug conjugate of claim 1, wherein the linker-drug conjugate is selected from Ala-Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-Ala-(2-amino-4-cyanobutanoic acid), Ala-(2-amino-4-cyanobutanoic acid), 2-amino-4-cyanobutanoic acid, Gly-Gly-Gly-Gln, Gly-Gly-Gln, Gly-Gln, Gln and deuterated versions thereof, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

5. D is a cytotoxin containing a hydroxy group (-OH), a primary amino group (-NH2), or a secondary amino group (-NHR), Preferably, the cytotoxin containing a hydroxy group (-OH), a primary amino group (-NH2) or a secondary amino group (-NHR) is selected from the group consisting of camptothecin-based topoisomerase I inhibitors, daunorubicin-based topoisomerase II inhibitors, monomethyl auristatin derivatives, molecular glue compounds and immunosuppressants; More preferably, the cytotoxin containing a hydroxy group (-OH), a primary amino group (-NH2) or a secondary amino group (-NHR) is selected from exatecan, 14-aminocamptothecin (14-AC), 9-aminocamptothecin (9-AC), belotecan, MMAE and its derivatives, MMAF and its derivatives, lenalidomide, rapamycin and its derivatives; 5. The linker drug conjugate of any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the cytotoxin containing a hydroxy group (-OH), a primary amino group (-NH2), or a secondary amino group (-NHR) is selected from exatecan, belotecan, and rapamycin.

6. 6. The linker drug conjugate of any one of claims 1 to 5, wherein the linker drug conjugate is MCC-AAQ-Exatecan and has the structure shown below: or a pharmaceutically acceptable salt, solvate, or solvate of said salt. 【Chemistry 1】

7. An antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, formed by conjugating the linker drug conjugate of any one of claims 1 to 6 to an antibody. Preferably, the antibody drug conjugate has the structure shown in Formula II: Ab-(LD)p Formula II During the ceremony, Ab is antibody; L is a linker, the structure of which is M'-X, where M' binds to Ab and X binds to D; M' is a chemical structure formed by binding M defined in any one of claims 1 to 6 to Ab via a maleimide group; X is a linker fragment consisting of 1 to 5 amino acids or derivatives thereof as defined in any one of claims 1 to 6; D is a cytotoxin as defined in any one of claims 1 to 6; p is any number between 2 and 8.

8. the antibody is selected from a mouse-derived antibody, a rabbit-derived antibody, a phage display-derived antibody, a yeast display-derived antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody, and a multispecific antibody; The antibody-drug conjugate of claim 7, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, wherein preferably the antibody is a monoclonal antibody selected from a HER2 antibody and an EGFR antibody.

9. A drug composition comprising the linker drug conjugate of any one of claims 1 to 6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the antibody drug conjugate of claim 6 or 7, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, Preferably, the pharmaceutical composition further comprises at least one of a chemotherapeutic agent, an immunotherapeutic agent, and an immunosuppressant agent used in the treatment of tumors; Preferably, the pharmaceutical composition further comprises at least one pharmaceutical adjuvant.

10. 10. Use of the linker-drug conjugate according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the antibody-drug conjugate according to claim 7 or 8, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, in the manufacture of a medicament for use in the prevention and / or treatment of tumors.

11. 10. A method for preparing the linker drug conjugate of any one of claims 1 to 6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, comprising the steps of:

1. Preparing a linker fragment X consisting of 1 to 5 amino acids or derivatives thereof; 2. X is coupled to a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to give MX; 3. Conjugating the cytotoxin D to MX to give the linker-drug conjugate MXD.

12. 12. The method of claim 11, wherein the linker-drug conjugate is MCC-AAQ-Exatecan, comprising the steps of:

1. Fmoc-Gln is reacted with Exatecan to obtain intermediate 1, the structural formula of which is as follows: 【Chemistry 2】 2. Intermediate 1 was deprotected with DEA ​​to remove the Fmoc group to obtain intermediate 2, the structural formula of which is as follows: 【Transformation 3】 3. Intermediate 2 is reacted with Fmoc-Ala-Ala to obtain intermediate 3, the structural formula of which is as follows: 【Chemistry 4】 4. The Fmoc protecting group of intermediate 3 is deprotected to obtain intermediate 4, the structural formula of which is as follows: 【Transformation 5】 5. Intermediate 4 is condensed with MCC to obtain the target product, the structural formula of which is as follows: 【Transformation 6】

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