Preparation methods and applications of a class of linker drugs and their antibody-drug conjugates

Abstract

This invention relates to the pharmaceutical field, specifically to linker drug conjugates, antibody drug conjugates, their preparation methods, and applications. Specifically, this invention relates to a linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, wherein the linker drug conjugate has the structure shown in Formula I, wherein M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne fragment, X is a linker fragment consisting of 1-5 amino acids or their derivatives, and D is a cytotoxic agent. The linker drug conjugate of this invention can release the payload through enzymatic hydrolysis after removing the hydrophobic PAB structure, thereby reducing the hydrophobicity of the linker-drug and decreasing the polymer content generated during ADC preparation, while also exhibiting corresponding cytotoxic activity, M-X-D Formula I.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on January 29, 2024, with Chinese application number 202480009220.2 and invention title "Preparation method and application of a class of linker drugs and their antibody-drug conjugates".

[0002] Cross-reference to related applications

[0003] This application is based on and claims priority to CN application No. 202310093383.0 filed on January 31, 2023, and CN application No. 202310947390.2 filed on July 28, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] This invention relates to the field of pharmaceuticals, and more specifically, to linker drug conjugates, antibody drug conjugates, their preparation methods and applications. Background Technology

[0005] Research on antibody-drug conjugates (ADCs) dates back to the 1980s, but it wasn't until 2000 that the first ADC (trade name Mylotarg, developed by Pfizer) was approved by the FDA for the treatment of acute myeloid leukemia. However, due to limitations in conjugation technology, targeting, and efficacy, the intact ADC was unstable in the blood, leading to fatal toxicity, and was withdrawn from the market in 2010. This cast a further shadow over the already uncertain research on ADC drugs.

[0006] However, Seagen improved upon its existing technology and developed brentuximab vedotin (SGN-35, trade name Adcetris) using its novel antibody-drug conjugate (ADC) technology. This new ADC was approved by the FDA in 2011 for the treatment of Hodgkin's lymphoma and systemic anaplastic large cell lymphoma. In 2013, ADCs achieved another breakthrough when Ado-trastuzumab emtansine (T-DM1, trade name Kadcyla), jointly developed by Genentech and ImmunoGen, was approved by the FDA for HER2-positive breast cancer, marking the first ADC targeting a solid tumor. With the successful development of these two drugs, ADCs once again entered the research field with great interest.

[0007] In recent years, ADCs have developed rapidly, with 15 ADC drugs currently approved for marketing and more than 200 ADCs entering clinical trials. ADCs are playing an increasingly important role in the field of targeted cancer therapy.

[0008] Antibody-drug conjugates (ADCs) consist of three parts: the antibody (Ab), the linker, and the small molecule cytotoxic drug (payload / warhead). The antibody portion of an ADC should possess tumor specificity, good endocytosis efficiency, good antigen affinity, and be non-immunogenic or low-immunogenic; IgG1 is the most commonly used antibody portion. The warhead portion is generally a cytotoxic drug that targets microtubules, DNA, or RNA, such as camptothecin, maytansine, or olistatin. It needs to have sufficient toxicity, sufficient water solubility, and its target site must be located intracellularly. The linker connecting the antibody and the small molecule cytotoxic drug must be stable in the bloodstream, rapidly disintegrate within the cell, and efficiently release the toxin. Summary of the Invention

[0009] The inventors of this application have creatively designed a new connector substructure, thereby providing the present invention.

[0010] Linker drug conjugates

[0011] A first aspect of the present invention relates to a linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said linker drug conjugate having the structure shown in Formula I.

[0012] MXD

[0013] Formula I

[0014] in,

[0015] M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.);

[0016] X is a linker fragment consisting of 1 to 5 amino acids or their derivatives, preferably selected from 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-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), Ala-(3-cyano-alanine). ine), 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 their deuterated compounds;

[0017] D stands for cytotoxin.

[0018] In the linker drug conjugate of the present invention, the linker is composed of a chemical structure containing a maleimide (m) fragment or a cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.) and a linker fragment consisting of 1 to 5 amino acids or their derivatives. It has the following characteristics: it is stable in the blood circulation, can be rapidly disintegrated in cells or tumor microenvironment, and can efficiently release toxins.

[0019] The chemical structure containing the maleimide (m) fragment can be selected from 4-(N-maleiminomethyl)-cyclohexane-1-formyl (MCC), 6-maleiminohexanoyl (MC), and M-(PEG). n MC-(PEG) n MCC-(PEG) n The structures are as follows:

[0020]

[0021] Where n represents the degree of polymerization of PEG, preferably an integer between 2 and 12.

[0022] In some embodiments, the chemical structure containing the maleimide (m) fragment is 6-maleiminohexanoyl (MC). In some embodiments, the chemical structure containing the maleimide (m) fragment is MC-(PEG). n， Wherein, n is selected from 4, 8, and 12. The inventors have found that a lower degree of polymerization of PEG is beneficial to reducing the polymer content (HMW%) in ADCs prepared from linker drug conjugates to some extent; therefore, the degree of polymerization n of PEG is preferably 4.

[0023] Chemical structures containing cyclooctyne fragments can be selected from BCN, DIBO, DIBAC, BARAC, etc., as shown below:

[0024] .

[0025] For most currently under development ADCs, the PAB structure (p-aminobenzyl-carbamoyl) serves as a self-releasing segment in the linker, essential for efficient payload release. However, the hydrophobicity of the PAB structure tends to generate excessive polymers during ADC fabrication. Therefore, further research and development of novel linker structures are needed to overcome these issues.

[0026] Therefore, through experimental research, the inventors of this application discovered that if D is a cytotoxin containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) groups, then the amino acid fragment contained in the linker drug conjugate of this application is linked to a payload containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) structures. This allows the payload to be released through enzymatic hydrolysis after the hydrophobic structure PAB is removed. This not only reduces the hydrophobicity of the linker-drug and the amount of polymer generated during ADC preparation, but also exhibits corresponding cell-killing activity.

[0027] Therefore, in some embodiments, D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group.

[0028] In some embodiments, the cytotoxic agents containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) groups may be selected from topoisomerase I inhibitors such as camptothecin compounds and their derivatives (e.g., eczema, 14-aminocamptothecin, 9-aminocamptothecin, belotecone, etc.), topoisomerase II inhibitors such as doxorubicin compounds and their derivatives (e.g., daunorubicin), DNA alkylating agents such as maytansine compounds and their derivatives, calichimycin compounds and their derivatives, docarmicin compounds and their derivatives (e.g., duocarmycin derivatives), and pyrrolobenzodiazepine compounds and their derivatives (e.g., PBD), microtubule inhibitors such as calendulatin compounds and their derivatives (e.g., MMAE and its derivatives or MMAF and its derivatives), Protac compounds and molecular gel compounds (e.g., lenalidomide), immunosuppressants such as rapamycin and its derivatives, and other small molecule compounds with cytotoxic activity. In some embodiments, the cytotoxic agent containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group is selected from Exatecan, beloteccan, and rapamycin. In some embodiments, the cytotoxic agent containing a hydroxyl (-OH) group is rapamycin. In some embodiments, the cytotoxic agent containing a secondary amine (-NHR) group is beloteccan. In some embodiments, the cytotoxic agent containing a primary amine (-NH2) group is Exatecan.

[0029] In some embodiments, the linker fragment consisting of the 1 to 5 amino acids or their derivatives in Formula I is selected from 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-cyano-alanine), Ala-(3-cyano-alanine) -alanine), 3-cyano-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-(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 their deuterated compounds.

[0030] The amino acid monomers or their derivatives in Formula I, or fragments composed of multiple amino acids or their derivatives, can be recognized by enzymes in the lysosomes of tumor cells and dissociate the carboxyl group in these compounds from the ester or amide bonds (peptide bonds) formed with compounds containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR), without requiring additional self-immolative moiety.

[0031] In some embodiments, the linker fragment consisting of the 1 to 5 amino acids or their derivatives in Formula I is selected from Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Ala-Ala-Ala-Ala-(3-cyano-L-alanine), A ...L-alanine), Ala-(3-cyano-L-L-alanine), Ala-(3-cyano-L-L-alanine), Ala-(3-cyano-L-L-alanine), Ala-(3-cyano-L-L-alanine), Ala-(3-cyano-L-L-alanine), Ala-(3-cyano-L-L-alanine), Ala-(3-cyano-L-L-alanine), Ala-(3-cyano-L-L-alanine), Ala-(3-cyano-L-L-L-alanine), Ala-(3-cyano-L-L-L-alanine), Ala-( L-alanine), 3-cyano-L-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-((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.

[0032] In some embodiments, the linker is selected from linkers containing asparagine (Asn), such as Ala-Ala-Ala-Ala-Asn, Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Pro-Asn, Lys-Asn. In some embodiments, the linker is Ala-Ala-Asn (AAN).

[0033] In some embodiments, the linker drug conjugate of the present invention is MCC-AAQ-Exatecan, which has the following structure:

[0034] .

[0035] Antibody-drug conjugates

[0036] The second aspect of the 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 the linker drug conjugate described in the first aspect of the invention with an antibody.

[0037] In some embodiments, the antibody-drug conjugate has the structure shown in Formula II.

[0038] Ab-(LD) p

[0039] Formula II

[0040] in,

[0041] Ab represents antibodies;

[0042] L is a connector with a structure of M'-X, where M' is connected to Ab and X is connected to D;

[0043] M' is the chemical structure formed by M as defined above being linked to Ab through a maleimide group;

[0044] X is a linker consisting of 1 to 5 amino acids or their derivatives as defined above;

[0045] D stands for cytotoxin as defined above;

[0046] p is any value 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, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or such as 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).

[0047] In Formula II, LD represents the linker covalently linked to the cytotoxin, forming an LD molecule; Ab-(LD) p This indicates that p LD molecules are covalently coupled to Ab.

[0048] In this invention, the drug-to-antibody ratio (DAR) refers to the number of drug molecules conjugated to the antibody (e.g., p in Formula II). The number of drug molecules contained in the antibody-drug conjugates described herein can be an integer or a decimal. Whether integer or decimal, it refers to the average number of drug molecules conjugated to each antibody. "p is any value between 2 and 8" means that p can be any integer selected from 2 to 8 (inclusive of the endpoints 2 and 8), or any decimal selected from 2 to 8, such as 2.3, 3.9, 4.0, and 4.2. Furthermore, those skilled in the art will understand that even using the same preparation method, the DAR values ​​of antibody-drug conjugates prepared in different batches may not be exactly the same; for example, they may fluctuate within a range not exceeding 0.5.

[0049] Drug-antibody ratio (DAR) can be verified using conventional analytical methods such as mass spectrometry, ELISA, HIC, and HPLC. The quantitative distribution of ADCs with respect to p-value can also be determined. In some cases, the separation, purification, and verification of homogeneous ADCs with a specific p-value from ADCs carrying other drug loads can be achieved using methods such as reversed-phase HPLC or electrophoresis.

[0050] In this invention, when the linker drug conjugate is covalently coupled to the antibody (Ab), one method is to form a -S-succinimide structure by coupling maleimide with the thiol group in the antibody. For example, when MCC-AAQ-Exatecan is covalently coupled to the Ab, the resulting ADC has the following structural formula:

[0051] .

[0052] In the maleimide-linked ADCs formed above, -S- can be either an external thiol group introduced through engineering modification, or a thiol group contained in the antibody itself after the antibody Ab is reduced and the disulfide bond is opened.

[0053] Another approach is to couple cyclooctyne with an azide group (-N3) introduced into the modified antibody to form a triazole structure. For example, when BCN-AAQ-Exatecan is covalently coupled to the antibody, the resulting ADC structure is shown below:

[0054]

[0055] There are no particular limitations on the antibodies that can be used in this invention. They can be selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies.

[0056] In some embodiments, the antibody is a monoclonal antibody, and is not limited to being selected from: HER2 antibody, EGFR antibody.

[0057] In some embodiments, the antibody is a HER2 antibody. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the light chain comprises CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0058] In some embodiments, the light chain comprises a light chain variable region with the amino acid sequence SEQ ID NO: 7. In some embodiments, the light chain further comprises a light chain constant region with the amino acid sequence SEQ ID NO: 8. In some embodiments, the amino acid sequence of the light chain is SEQ ID NO: 9.

[0059] In some embodiments, the heavy chain comprises a heavy chain variable region with the amino acid sequence SEQ ID NO: 10. In some embodiments, the heavy chain further comprises a heavy chain constant region with the amino acid sequence SEQ ID NO: 11. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 12.

[0060] In some embodiments, the antibody is an EGFR antibody. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the light chain comprises CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown 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, the amino acid sequences of which are shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0061] In some embodiments, the light chain comprises a light chain variable region with the amino acid sequence SEQ ID NO: 19. In some embodiments, the light chain further comprises a light chain constant region with the amino acid sequence SEQ ID NO: 20. In some embodiments, the amino acid sequence of the light chain is SEQ ID NO: 21.

[0062] In some embodiments, the heavy chain comprises a heavy chain variable region with the amino acid sequence SEQ ID NO: 22. In some embodiments, the heavy chain further comprises a heavy chain constant region with the amino acid sequence SEQ ID NO: 23. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 24.

[0063] Information on some of the sequences involved in this invention is provided in Table 1.

[0064] Table 1: Sequence Description

[0065]

[0066]

[0067] Pharmaceutical Compositions and Pharmaceutical Uses

[0068] A third aspect of the invention provides a pharmaceutical composition comprising the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0069] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapy drug, an immunotherapy drug, and an immunosuppressant for treating tumors.

[0070] In some embodiments, the chemotherapeutic agents are, for example, adriamycin, cyclophosphamide, taxanes (such as taxol, docetaxel), capecitabine, gemcitabine, vinorelbine, tamoxifen, aromatase inhibitors (renin, fenofibrate, arnoxin), 5-FU plus leucovorin, irinotecan, oxaliplatin, cisplatin, carboplatin, estradiol, Novantrone, prednisone, vincristine, doxorubicin, prednisone, etc., or combinations thereof.

[0071] In some embodiments, the immunotherapeutic agent is, for example, 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 a combination thereof.

[0072] In some embodiments, the immunosuppressant is selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tebufenozide; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; and (5) alkylating agents, such as cyclophosphamide. In some specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, prazosin, selenophora, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, imidazolidin, cyclophosphamide, fingolimod, etc.

[0073] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

[0074] A fourth aspect of the invention provides the use of the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, in the preparation of a medicament for the prevention and / or treatment of tumors.

[0075] A fifth aspect of the invention provides a method for treating and / or preventing tumors, comprising: administering to a subject in need a therapeutic and / or preventative effective amount of the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0076] In this invention, "tumor" refers to a lesion formed by excessive proliferation of cells in a localized area of ​​an organ or tissue, including hematologic malignancies and solid tumors, including benign and malignant tumors. In some embodiments, the tumor is selected from tumors that are positive for or highly express the following markers: HER2, EGFR.

[0077] In some embodiments, the tumor is selected from: breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, spinal canal 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 carcinoma, 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 brain glioma.

[0078] Preparation method

[0079] A sixth aspect of the present invention provides a method for preparing the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, the method comprising the following steps:

[0080] 1. Prepare a linker fragment X consisting of 1-5 amino acids or their derivatives;

[0081] 2. X is linked to a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to obtain MX;

[0082] 3. Cytotoxin D is linked to MX to obtain the linker drug conjugate MXD.

[0083] The preparation method described above is further illustrated below using MCC-AAQ-Exatecan as an example. An exemplary preparation process for MCC-AAQ-Exatecan is as follows:

[0084] 1. Fmoc-Gln was reacted with Exatecan to obtain intermediate one, the structural formula of which is as follows:

[0085]

[0086] 2. Intermediate 1 is deprotected from the Fmoc protecting group via DEA to obtain intermediate 2, wherein the structural formula of intermediate 2 is as follows:

[0087]

[0088] 3. Intermediate 2 is reacted with Fmoc-Ala-Ala to obtain intermediate 3, the structural formula of which is as follows:

[0089]

[0090] 4. The intermediate ter was deprotected from the Fmoc protecting group to obtain intermediate 4, the structural formula of which is as follows:

[0091]

[0092] 5. The intermediate four is condensed with MCC to obtain the product, the product structure of which is as follows:

[0093]

[0094] A seventh aspect of the present invention provides a method for preparing the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, the method comprising the following steps:

[0095] 1. Take a certain weight of monoclonal antibody, dilute it with PBS buffer and adjust the antibody pH to about 7.5; detect the protein concentration, weigh the net weight of the antibody solution, and calculate the total protein; add TCEP solution to the antibody, react at room temperature, and shake continuously.

[0096] 2. Add an excess of cytotoxic drug solution to the reduced antibody solution, mix well, and react at room temperature, mixing continuously. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction solution, react at room temperature, and mix continuously.

[0097] 3. The conjugate product was purified using an ultrafiltration centrifuge tube and transferred to a storage solution. Then, it was filtered using a sterile filter to obtain the antibody-drug conjugate.

[0098] In addition, the present invention also provides the following technical solutions:

[0099] Linker drug conjugates

[0100] The eighth aspect of the present invention relates to a linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said linker drug conjugate having the structure shown in Formula I.

[0101] MXD

[0102] Formula I

[0103] in,

[0104] M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.);

[0105] 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, Gln;

[0106] D is a cytotoxin containing a primary amine (-NH2) group.

[0107] In the linker drug conjugate of the present invention, the linker is composed of a linker fragment consisting of a chemical structure containing a maleimide (m) fragment or a cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.) and 1 to 4 amino acids. It has the following characteristics: it is stable in the blood circulation, can be rapidly disintegrated in cells or tumor microenvironment, and can efficiently release toxins.

[0108] The chemical structure containing the maleimide (m) fragment can be selected from 4-(N-maleiminomethyl)-cyclohexane-1-formyl (mcc), 6-maleiminohexanoyl (mc), and m-(PEG). n mc-(PEG) n mcc-(PEG) n The structures are as follows:

[0109]

[0110] Where n represents the degree of polymerization of PEG, preferably an integer between 2 and 12.

[0111] In some embodiments, the chemical structure containing the maleimide (m) fragment is 6-maleiminohexanoyl (mc). In some embodiments, the chemical structure containing the maleimide (m) fragment is mc-(PEG). n， Wherein, n is selected from 4, 8, and 12. The inventors have found that a lower degree of polymerization of PEG is beneficial to reducing the polymer content (HMW%) in ADCs prepared from linker drug conjugates to some extent; therefore, the degree of polymerization n of PEG is preferably 4.

[0112] Chemical structures containing cyclooctyne fragments can be selected from BCN, DIBO, DIBAC, BARAC, etc., as shown below:

[0113] .

[0114] The linker fragments consisting of 1 to 4 amino acids in Formula I are 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. The above-mentioned amino acid monomers or fragments composed of multiple amino acids can be recognized by specific enzymes within the lysosomes of tumor cells, and the amide bond (peptide bond) formed between the carboxyl group and the compound containing a primary amine in this type of compound can be dissociated, without requiring additional self-immolative moiety.

[0115] In some embodiments, the linker fragment is selected from those containing asparagine (Asn), such as Ala-Ala-Ala-Asn, Ala-Ala-Asn, Ala-Asn, Asn, Pro-Asn, and Lys-Asn. Asparagine (Asn) can be recognized by legumains, which are present not only within the lysosomes of tumor cells but also extensively distributed extracellularly, thus enabling the extracellular release of small molecules to exert a by-stander effect and exhibit good killing activity against cells with low endocytosis target sites. In some embodiments, the linker fragment is Ala-Ala-Asn (AAN).

[0116] The cytotoxin containing a primary amine (-NH2) group can be selected from various small molecule compounds with cytotoxic activity, such as topoisomerase I inhibitors (e.g., eczemab, 14-aminocamptothecin, 9-aminocamptothecin, etc.), topoisomerase II inhibitors (e.g., doxorubicin, maytansine, calichiomycin, docarmicin, and pyrrolobenzodiazepines and their derivatives (e.g., PBD), microtubule inhibitors (e.g., demethyl MMAE or demethyl MMAF, etc.), Protac compounds, and molecular gel compounds (e.g., lenalidomide, etc.). In some embodiments, the cytotoxin containing a primary amine (-NH2) group is eczemab.

[0117] In some embodiments, the cytotoxic substance containing a primary amine (-NH2) group is selected from Exatecan, 14-aminocamptothecin (14-AC), 9-aminocamptothecin (9-AC), demethyl MMAE, demethyl MMAF, Daunorubicin, and lenalidomide.

[0118] In some embodiments, the linker drug conjugate of the present invention is mc-AAN-Exatecan, which has the following structure:

[0119] .

[0120] Antibody-drug conjugates

[0121] The ninth aspect of the present invention relates to an antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt thereof, said antibody-drug conjugate being formed by conjugating an antibody with a linker drug conjugate as described in the eighth aspect of the present invention.

[0122] In some embodiments, the antibody-drug conjugate has the structure shown in Formula II.

[0123] Ab-(LD) p

[0124] Formula II

[0125] in,

[0126] Ab represents antibodies;

[0127] L is a connector with a structure of M'-X, where M' is connected to Ab and X is connected to D;

[0128] M' is the chemical structure formed by M as defined above being linked to Ab through a maleimide group;

[0129] X is a linker consisting of 1 to 4 amino acids as defined above;

[0130] D represents a cytotoxic substance containing a primary amine (-NH2) group as defined above;

[0131] p is any value 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, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, or such as 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).

[0132] In Formula II, LD represents the linker covalently linked to the cytotoxin, forming an LD molecule; Ab-(LD) p This indicates that p LD molecules are covalently coupled to Ab.

[0133] In this invention, the drug-to-antibody ratio (DAR) refers to the number of drug molecules conjugated to the antibody (e.g., p in Formula I). ​​The number of drug molecules contained in the antibody-drug conjugates described herein can be an integer or a decimal. Whether integer or decimal, it refers to the average number of drug molecules conjugated to each antibody. "p is any value between 2 and 8" means that p can be any integer selected from 2 to 8 (inclusive of the endpoints 2 and 8), or any decimal selected from 2 to 8, such as 2.3, 3.9, 4.0, and 4.2. Furthermore, those skilled in the art will understand that even using the same preparation method, the DAR values ​​of antibody-drug conjugates prepared in different batches may not be exactly the same; for example, they may fluctuate within a range not exceeding 0.5.

[0134] Drug-antibody ratio (DAR) can be verified using conventional analytical methods such as mass spectrometry, ELISA, HIC, and HPLC. The quantitative distribution of ADCs with respect to p-value can also be determined. In some cases, the separation, purification, and verification of homogeneous ADCs with a specific p-value from ADCs carrying other drug loads can be achieved using methods such as reversed-phase HPLC or electrophoresis.

[0135] In this invention, when the linker drug conjugate is covalently coupled to the antibody (Ab), one method is to form a -S-succinimide structure by coupling maleimide with the thiol group in the antibody. For example, when mc-AAN-Exatecan is covalently coupled to the Ab, the resulting ADC has the following structural formula:

[0136] .

[0137] In the maleimide-linked ADCs formed above, -S- can be either an external thiol group introduced through engineering modification, or a thiol group contained in the antibody itself after the antibody Ab is reduced and the disulfide bond is opened.

[0138] Another approach involves linking antibodies with the chemical structures of cyclooctyne and its derivative fragments (such as BCN, DIBO, DIBAC, BARAC, etc.), where the cyclooctyne couples with the azide group (-N3) introduced into the modified antibody to form a triazole structure. For example, when BCN-AAN-Exatecan is covalently coupled to the antibody, the resulting ADC structure is shown below:

[0139] .

[0140] There are no particular limitations on the antibodies that can be used in this invention. They can be selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies.

[0141] In some embodiments, the antibody is a monoclonal antibody, and is not limited to being selected from: HER2 antibody, EGFR antibody.

[0142] In some embodiments, the antibody is a HER2 antibody. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the light chain comprises CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. In some embodiments, the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, the amino acid sequences of which are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0143] In some embodiments, the light chain comprises a light chain variable region with the amino acid sequence SEQ ID NO: 7. In some embodiments, the light chain further comprises a light chain constant region with the amino acid sequence SEQ ID NO: 8. In some embodiments, the amino acid sequence of the light chain is SEQ ID NO: 9.

[0144] In some embodiments, the heavy chain comprises a heavy chain variable region with the amino acid sequence SEQ ID NO: 10. In some embodiments, the heavy chain further comprises a heavy chain constant region with the amino acid sequence SEQ ID NO: 11. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 12.

[0145] In some embodiments, the antibody is an EGFR antibody. In some embodiments, the antibody comprises a light chain and a heavy chain, wherein the light chain comprises CDR-L1, CDR-L2, and CDR-L3, the amino acid sequences of which are shown 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, the amino acid sequences of which are shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0146] In some embodiments, the light chain comprises a light chain variable region with the amino acid sequence SEQ ID NO: 19. In some embodiments, the light chain further comprises a light chain constant region with the amino acid sequence SEQ ID NO: 20. In some embodiments, the amino acid sequence of the light chain is SEQ ID NO: 21.

[0147] In some embodiments, the heavy chain comprises a heavy chain variable region with the amino acid sequence SEQ ID NO: 22. In some embodiments, the heavy chain further comprises a heavy chain constant region with the amino acid sequence SEQ ID NO: 23. In some embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 24.

[0148] Information on some sequences involved in this invention is provided in Table 1 above.

[0149] Pharmaceutical Compositions and Pharmaceutical Uses

[0150] The tenth aspect of the present invention provides a pharmaceutical composition comprising the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0151] In some embodiments, the pharmaceutical composition further comprises at least one of a chemotherapy drug, an immunotherapy drug, and an immunosuppressant for treating tumors.

[0152] In some embodiments, the chemotherapeutic agents are, for example, adriamycin, cyclophosphamide, taxanes (such as taxol, docetaxel), capecitabine, gemcitabine, vinorelbine, tamoxifen, aromatase inhibitors (renin, fenofibrate, arnoxin), 5-FU plus leucovorin, irinotecan, oxaliplatin, cisplatin, carboplatin, estradiol, Novantrone, prednisone, vincristine, doxorubicin, prednisone, etc., or combinations thereof.

[0153] In some embodiments, the immunotherapeutic agent is, for example, 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 a combination thereof.

[0154] In some embodiments, the immunosuppressant is selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tebufenozide; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; and (5) alkylating agents, such as cyclophosphamide. In some specific embodiments, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, prazosin, selenophora, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, imidazolidin, cyclophosphamide, fingolimod, etc.

[0155] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

[0156] The eleventh aspect of the present invention provides the use of the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, in the preparation of a medicament for the prevention and / or treatment of tumors.

[0157] The twelfth aspect of the present invention provides a method for treating and / or preventing tumors, comprising: administering to a subject in need a therapeutic and / or preventative effective amount of the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the aforementioned antibody drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0158] In this invention, "tumor" refers to a lesion formed by excessive proliferation of cells in a localized area of ​​an organ or tissue, including hematologic malignancies and solid tumors, including benign and malignant tumors. In some embodiments, the tumor is selected from tumors that are positive for or highly express the following markers: HER2, EGFR.

[0159] In some embodiments, the tumor is selected from: breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, primary liver cancer, spinal canal 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 carcinoma, 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 brain glioma.

[0160] Preparation method

[0161] The thirteenth aspect of the present invention provides a method for preparing the aforementioned linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, the method comprising the following steps:

[0162] 1. Prepare linker fragment X consisting of 1-4 amino acids;

[0163] 2. X is linked to a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to obtain MX;

[0164] 3. Cytotoxin D containing a primary amine (-NH2) group is linked to MX to obtain the linker drug conjugate MXD.

[0165] The preparation method described above is further illustrated below using mc-AAN-Exatecan as an example. An exemplary preparation process for mc-AAN-Exatecan is as follows:

[0166] 1. Fmoc-Ala-OH was activated with HOSu and then reacted with L-Ala to obtain intermediate one, the structural formula of which is as follows:

[0167]

[0168] 2. Intermediate 1 is activated with HOSu and then reacted with L-Asn to obtain intermediate 2, the structural formula of which is as follows:

[0169]

[0170] 3. Intermediate 2 is subjected to a deFmoc reaction to obtain intermediate 3, the structural formula of which is as follows:

[0171]

[0172] 4. Intermediate III is reacted with succinimide 6-(maleimino)hexanoate to obtain intermediate IV, wherein the structural formula of intermediate IV is as follows:

[0173]

[0174] 5. The intermediate tetrahydropalmatine (THP) is condensed with eczemab to obtain the product, the structural formula of which is as follows:

[0175] .

[0176] The fourteenth aspect of the present invention provides a method for preparing the aforementioned antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, the method comprising the following steps:

[0177] 1. Take a certain weight of monoclonal antibody, dilute it with PBS buffer and adjust the antibody pH to about 7.5; detect the protein concentration, weigh the net weight of the antibody solution, and calculate the total protein; add TCEP solution to the antibody, react at room temperature, and shake continuously.

[0178] 2. Add an excess of cytotoxic drug solution to the reduced antibody solution, mix well, and react at room temperature, mixing continuously. After the reaction is complete, add an excess of N-acetylcysteine ​​solution to the reaction solution, react at room temperature, and mix continuously.

[0179] 3. The conjugate product was purified using an ultrafiltration centrifuge tube and transferred to a storage solution. Then, it was filtered using a sterile filter to obtain the antibody-drug conjugate.

[0180] Beneficial effects

[0181] In some embodiments, the linker provided by the present invention can release the payload through an enzymatic reaction after removing the hydrophobic PAB structure. This can reduce the hydrophobicity of the linker-drug, reduce the polymer content generated during ADC preparation, and also exhibit corresponding cell-killing activity. Detailed Implementation

[0182] Terminology Definition

[0183] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0184] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0185] In this invention, unless otherwise stated, any numerical range should be understood to include any value within the range or any subrange.

[0186] In this invention, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of identical polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). The light chains of antibodies can be classified into two types: κ and λ. The heavy chains can be classified into five types: μ, δ, γ, α, or ε. Based on the different heavy chains, antibodies can be classified into five classes: IgM, IgD, IgG, IgA, and IgE. Within both the light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids. The heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C...). H 1. C H 2 and C H 3) Composition. Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and C1q, a component of the complement system. V H and V L The region can be further subdivided into highly variable regions (called complementarity-determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V LIt consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L Each amino acid forms an antibody binding site. The allocation of amino acids to each region or domain follows the definitions in Kabat 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.

[0187] In some embodiments, the drug module is coupled to the antibody in a coupling reaction with less than the theoretical maximum. Generally, antibodies do not contain many free and reactive cysteine ​​thiols that can link to the drug module; in fact, most cysteine ​​thiols in antibodies exist as disulfide bridges. In some embodiments, the antibody can be reduced under partially or completely reducing conditions with reducing agents such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiols.

[0188] In this invention, the term "pharmaceutically acceptable salt" refers to (i) the salt formed by the acidic functional group present in the couplings provided by this invention and a suitable inorganic or organic cation (base), including but not limited to, alkali metal salts such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts such as calcium salts, magnesium salts, etc.; other metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts such as ammonium salts; organic base salts such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenylethylamine salts, piperazine salts, tetramethylamine salts, and tris(hydroxymethyl)aminomethane salts. (ii) The salts formed by the basic functional groups present in the couplings provided by the present invention and suitable inorganic or organic anions (acids), including but not limited to, hydrohalides, such as hydrofluoric acid, hydrochloride, hydrobromide, hydroiodide, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkyl sulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates, such as benzenesulfonates, p-benzenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamate salts, aspartate salts, etc.

[0189] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting an adequate amount of a basic substance with a suitable acid providing a pharmaceutically acceptable anion, or by reacting an adequate amount of an acid with a suitable base providing a pharmaceutically acceptable cation.

[0190] In this invention, solvates refer to these forms of the antibody-drug conjugates of the invention: solid or liquid complexes formed by coordination with solvent molecules. Hydrates are a specific form of solvate having coordinated water molecules. Hydrates are preferred solvates in this invention.

[0191] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known, or will be obvious to those skilled in the art according to the disclosure of the present invention. As described in REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, EW, ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to cells or mammals exposed thereto at the doses and concentrations used.

[0192] In this invention, pharmaceutical excipients refer to the excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical formulation. Besides acting as a formulator, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified into natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0193] In this invention, the pharmaceutical composition can be formulated into various suitable dosage forms depending on the route of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder inhalers, and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the antibody-drug conjugate of this invention, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0194] As used herein, the term “treatment” generally refers to achieving the desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of the disease or its symptoms; and / or therapeutic based on the partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a patient’s disease, including: (a) prevention of the disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppression of the symptoms of the disease, i.e., prevention of its progression; or (c) relief of the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0195] In this invention, "subject" refers to a vertebrate. In some embodiments, vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, mammal refers to a human.

[0196] In this invention, "effective amount" refers to the amount that effectively achieves the desired therapeutic or preventative effect at the necessary dose and time. The "therapeutic effective amount" of the substance / molecule of this invention may vary depending on factors such as an individual's disease state, age, sex, weight, and the substance / molecule's ability to elicit the desired response in the individual. Therapeutic effective amount also encompasses the amount in which the beneficial therapeutic effect of the substance / molecule outweighs any toxic or harmful consequences. "Preventative effective amount" refers to the amount that effectively achieves the desired preventative effect at the necessary dose and time. Typically, but not necessarily, the preventative effective amount will be lower than the therapeutic effective amount because the preventative dose is administered to the subject before the onset of the disease or in the early stages of the disease. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; shrink the tumor volume; inhibit (i.e., slow down, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent.

[0197] In this invention, the 20 common amino acids and their abbreviations follow their usual usage. See Immunology-ASynthesis (2nd edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0198] In this invention, if X is Ala-Ala-Gln, it means that X is a linker fragment composed of Ala, Ala, and Gln sequentially linked to form peptide bonds. Those skilled in the art will understand that it does not limit the specific stereochemical configuration of Ala, Ala, and Gln, such as L-type or D-type. Similarly, if X is Ala-Ala-(3-cyano-alanine), it means that X is a linker fragment composed of Ala, Ala, and (3-cyano-alanine) sequentially linked to form peptide bonds. Those skilled in the art will also understand that it does not limit the specific stereochemical configuration of Ala, Ala, and (3-cyano-alanine), such as L-type, D-type, S-type, or R-type. Other similar limitations on X in this invention should be understood with reference to the foregoing.

[0199] In this invention, if X is selected from Ala-Ala-Gln, Ala-Ala-(3-cyano-alanine) and their deuterated derivatives, it means that X can be Ala-Ala-Gln, Ala-Ala-(3-cyano-alanine), a deuterated derivative of Ala-Ala-Gln, or a deuterated derivative of Ala-Ala-(3-cyano-alanine).

[0200] The term "deuterated compound" refers to a compound formed by replacing one or more (e.g., 2, 3, 4 or more) hydrogen atoms (the positions of the hydrogen atoms are not specifically limited; they can be hydrogen atoms on alkyl groups, hydrogen atoms on amino groups, etc.) with deuterium atoms.

[0201] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0202] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0203] Furthermore, during humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties of the antibody (e.g., affinity). Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs are usually limited to one or two.

[0204] The present invention will be further explained and described below with reference to specific embodiments, but these embodiments are not intended to limit the scope of the present invention.

[0205] Example 1: Preparation of the linker drug MCC-AAQ-Exatecan

[0206] 1. Synthesis of Intermediate 1: Fmoc-Gln-Exatecan

[0207] Fmoc-Gln-OH (N-fluorenylmethoxycarbonyl-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 carried out at room temperature for 2 hours, monitored by HPLC, and no starting material remained. After the reaction was completed, the product was purified by medium-pressure column chromatography (DCM / MeoH). The product was collected, concentrated, and intermediate one was obtained.

[0208] The reaction formula is as follows:

[0209]

[0210] 2. Synthesis of intermediate two Gln-Exatecan

[0211] 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 then added, and the reaction was continued at room temperature. After 4 hours, HPLC monitoring showed no starting material remaining. The reaction was stopped, and MTBE (10 ml) was slowly added, resulting in the precipitation of a large amount of solid. The mixture was stirred for 30 min. After filtration and washing with MTBE, a grayish-white solid was obtained, which was dried to give 95 mg of intermediate 2.

[0212] The reaction formula is as follows:

[0213]

[0214] 3. Synthesis of intermediate three: Fmoc-Ala-Ala-Gln-Exatecan

[0215] Intermediate II, Gln-Exatecan (100 mg 1.0 eq.), was added to a reaction flask along with 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.). The reaction was carried out at room temperature for 3 hours under HPLC monitoring until completion. The product was then purified by medium-pressure column chromatography (DCM / MeOH) to obtain intermediate III.

[0216] The reaction formula is as follows:

[0217]

[0218] 4. Synthesis of intermediate four: Ala-Ala-Gln-Exatecan

[0219] Intermediate triFmoc-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 continued at room temperature. After 4 hours, the reaction was monitored by HPLC until completion. MTBE (10 ml) was slowly added, resulting in the precipitation of a large amount of solid. The mixture was stirred for 30 min. After filtration and washing with MTBE, a grayish-white solid was obtained, which was then dried to give intermediate tetraFmoc-Ala-Ala-Gln-Exatecan.

[0220] The reaction formula is as follows:

[0221]

[0222] 5. Product Synthesis: MCC-Ala-Ala-Gln-Exatecan

[0223] Intermediate tetra(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 mixture was reacted at room temperature for 2 hours, monitored by HPLC, until no intermediate tetra(Ala-Gln-Exatecan) remained, at which point the reaction was stopped. The product was purified by Prep-HPLC, collected, concentrated to dryness, and the final product was obtained (NMR data are shown below).

[0224] Reaction

[0225]

[0226] 1H 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).

[0227] Example 2: Preparation of the linker drug MCC-Ala-Ala-Asn-Exatecan

[0228] 1. Synthesis of Intermediate 1

[0229] Fmoc-Ala-Ala-OH (N-[fluorenylmethoxycarbonyl]-L-alanyl-L-alanine) was activated with HOSu (N-hydroxysuccinimide) and then reacted with L-Asn (L-asparagine) to obtain intermediate one.

[0230] The specific steps are as follows: Add Fmoc-Ala-Ala-OH (1.0 eq), HOSu (1.3 eq), and THF (10 v / w) to the reaction flask at room temperature. Control the temperature at room temperature and slowly add DCC (1.3 eq) under stirring. The reaction is carried out at room temperature and detected by HPLC. When the residual Fmoc-Ala-Ala-OH is small (area normalization method), filter the reaction solution and wash the filter cake with THF (2.5 v / w). Purified water (11.5 v / w) was added to the filtrate, along with L-Asn (1.1 eq) and sodium bicarbonate solid (1.0 eq). The mixture was stirred at room temperature. HPLC analysis showed minimal Fmoc-Ala-Ala-OSu residue. Citric acid monohydrate (1.0 eq) was then added and stirred. The reaction mixture was concentrated to remove most of the solvent. DMF (4 v / w) was added to dissolve the product. After filtration, the product was prepared by reverse-phase chromatography. The prepared solution was concentrated until no significant droplets flowed out, and then extracted four times with ethyl acetate. The ethyl acetate phase was concentrated to dryness to obtain intermediate one. The reaction formula is as follows:

[0231]

[0232] 2. Synthesis of Intermediate Two

[0233] After adding DEA (diethylamine) to intermediate one to remove Fmoc, intermediate two is obtained.

[0234] The specific steps are as follows: Add intermediate one (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 proceed with the reaction at room temperature. Detect the reaction using HPLC until no intermediate one remains. Concentrate to remove DMF, add DCM (40 v / w) and purified water (40 v / w), stir, and separate the liquid phase. Concentrate the aqueous phase to dryness to obtain intermediate two. The reaction formula is as follows:

[0235]

[0236] 3. Synthesis of intermediate three

[0237] Intermediate II was reacted with 4-(N-maleimidemethyl)cyclohexanecarboxylic acid-N-succinimide ester to give intermediate III.

[0238] The specific steps are as follows: At room temperature, add intermediate two (1.0 eq), 4-(N-maleimidemethyl)cyclohexanecarboxylic acid-N-succinimide ester (1.1 eq), DMF (22 v / w), water (9.5 v / w), and DIPEA (1.0 eq) to a reaction flask. Then, proceed with the reaction at room temperature until minimal residue of intermediate two remains (QDA detection). After the reaction is complete, concentrate to remove most of the solvent, add water (50 v / w), EA (50 v / w), and 1.0 eq of DIEA. Stir until dissolved, allow to stand, and separate the layers. Extract the aqueous phase twice with EA (50 v / w * 2). Concentrate the aqueous phase to dryness to obtain intermediate three, as shown in the following reaction formula:

[0239]

[0240] 4. Product synthesis

[0241] Intermediate III was reacted with eczema mesylate (CAS No.: 169869-90-3) via a HATU condensation reaction to obtain intermediate IV.

[0242] The specific steps are as follows: At room temperature, intermediate trioxide (1.2 eq) was added to the reaction flask, followed by DMF (15 v / w), then eczema mesylate (1.0 eq), TBTU (1.4 eq), and DIPEA (4.5 eq). The reaction was then carried out at room temperature, with HPLC monitoring. After the reaction was completed, the solution was purified by prep-HPLC, concentrated, extracted with DCM, and concentrated again to obtain the product (NMR data shown below). The reaction formula is as follows:

[0243]

[0244] 1H 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).

[0245] Example 3: Preparation of the linker drug MCC-Ala-Ala-(3-cyano-L-alanine)-Exatecan

[0246] 1. Synthesis of Intermediate 1 (3-cyano-L-alanine) - Exatecan

[0247] Add Fmoc-Asn-OH (40 mg 1.2 eq) to a reaction flask, then add DMF (1.5 ml), TBTU (45 mg 1.5 eq), and DIEA (60 mg 2.5 eq). Stir until completely dissolved. After stirring for 30 min, add ixotecan mesylate (50 mg 1.0 eq) and stir for 4 hs. HPLC analysis showed no reaction. Add TBTU (45 mg 1.5 eq) and DIEA (60 mg 2.5 eq) and continue the reaction at room temperature. HPLC monitoring showed no remaining Exatecan, and Fmoc-(3-cyano-L-alanine)-Exatecan comprised approximately 70%. Stop the reaction and purify under medium pressure. Collect the product. 30 o C-pressure concentration and drying yield the product.

[0248] Add the above product to a reaction flask, then add DCM (4 ml) and stir. Add DEA (1 ml), stir at room temperature until completely dissolved. HPLC monitoring showed no raw material remaining. Add 10 ml MTBE, stir at room temperature, and a large amount of solid precipitates. Filter to obtain a grayish-white solid. Wash the filter cake with MTBE and dry to obtain 35 mg of solid, i.e., intermediate one.

[0249] The reaction formula is as follows:

[0250]

[0251] 2. Synthesis of Intermediate Two: MCC-Ala-Ala-OH

[0252] Add SMCC (460 mg, 1.1 eq) to the reaction flask, then add NH2-Ala-Ala-OH (200 mg, 1.0 eq), followed by DMF (4 ml, 20V). Stir, then add DIPEA (161.4 mg, 1.0 eq). Heat to 60°C. o C reaction. The reaction solution gradually dissolves as the reaction proceeds. The reaction time is at least 8 hours. Sampling is performed for LC-MS analysis. Once the reaction is complete, the solution is cooled to room temperature. 35 o DMF was removed by vacuum concentration at 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, and the mixture was stirred and washed. The organic phase was discarded. EA (2 ml, 10 V) was added again for extraction twice. The organic phase was discarded. The aqueous phase was retained, and the acidity was adjusted to pH 1-2 with 2M hydrochloric acid, precipitating a large amount of white solid. The mixture was stirred for one hour. The solid was filtered to obtain a white solid. The solid was washed with purified water and dried. 320 mg of white solid, intermediate two, was obtained.

[0253] The reaction formula is as follows:

[0254]

[0255] 3. Product Synthesis: MCC-Ala-Ala-(3-cyano-L-alanine)-Exatecan

[0256] Intermediate 1 (3-cyano-L-alanine)-Exatecan (35 mg 1.0 eq) was added to a reaction flask, followed by DMF (1 mL), then 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 carried out at room temperature for 2 hours. HPLC monitoring showed no residue of intermediate 1, at which point the reaction was stopped. The product was purified by Prep-HPLC and collected. It was then concentrated to dryness. The resulting yellow glassy solid was the product (NMR data are shown below).

[0257] The reaction formula is as follows:

[0258]

[0259] 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).

[0260] Example 4: Synthetic route of the linker drug MCC-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan

[0261] 1. Synthesis of Intermediate 1 ((S)-2-amino-4-cyanobutanoic acid) - Exatecan

[0262] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-cyanobutyric acid (79.8 mg 1.1 eq) and iccinotecan 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). The reaction was carried out at room temperature for 3 hours under HPLC monitoring. After the reaction was complete, 9 ml of ACN was added and the mixture was stirred at room temperature. Then, 1.0 ml of DEA was added, and the reaction was continued at room temperature for 1 hour. A sample was taken for monitoring; no starting material remained. 10 ml of MTBE was added. The mixture was stirred, and no obvious solid precipitation was observed. After stirring for 1 hour, no significant change was observed.

[0263] 30 o C. Reduced pressure concentration removes the solvent. A large amount of solid precipitates during concentration, while only a small amount of solvent, approximately 3 ml, is removed. Concentration is stopped, and stirring continues at room temperature for 1 hour. The solid is then filtered and washed with MTBE. After drying, a grayish-white solid, intermediate one, is obtained.

[0264] The reaction formula is as follows:

[0265]

[0266] 2. Synthesis of Intermediate Two: MCC-Ala-Ala-OH

[0267] Add SMCC (460 mg, 1.1 eq) to the reaction flask, then add NH2-Ala-Ala-OH (200 mg, 1.0 eq) and DMF (4 ml, 20V), stir, and then add DIPEA (161.4 mg, 1.0 eq). Heat to 60°C. o After 8 hours of reaction at step C, samples were taken for LC-MS analysis. Once the reaction was complete, the mixture was cooled to room temperature. 35 o DMF was removed by vacuum concentration at 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, and the mixture was stirred and washed. The organic phase was discarded. EA (2 ml, 10 V) was added again, and the mixture was washed twice. The organic phase was discarded. The aqueous phase was retained, and the pH was adjusted to 1-2 with 2M hydrochloric acid, resulting in the precipitation of a large amount of white solid. The mixture was stirred for one hour. The solid was filtered to obtain a white solid. The solid was washed with purified water and dried. 320 mg of the white solid, intermediate two, was obtained.

[0268] The reaction formula is as follows:

[0269]

[0270] 3. Product Synthesis: MCC-Ala-Ala-((S)-2-amino-4-cyanobutanoic acid)-Exatecan

[0271] 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), then 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 carried out at room temperature for 2 hours, monitored by HPLC, and the reaction was considered complete. The product was purified by Prep-HPLC and collected. Most of the acetonitrile was removed by concentration. The product was lyophilized to obtain a yellow solid (NMR spectrum shown below).

[0272] The reaction formula is as follows:

[0273]

[0274] 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).

[0275] Example 5: Synthetic route of the linker drug MCC-Ala-Ala-Ala-Ala-Gln-Exatecan

[0276] 1. Synthesis of Intermediate 1: MCC-Ala-Ala-Ala-Ala-Gln-OH

[0277] H-Gln(Trt)-OH 2-CTC resin (1.0 g, 0.5 mmol, 0.5 mmol / g) was added to a solid-phase reactor, followed by 20 mL of DCM. The mixture was shaken for 20 min to fully swell the resin. The resin was then 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 after mixing, DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) was added. The mixture was reacted in a shaker for 1 hr. The resin color was negative according to Kaiser assay. The resin was then filtered and washed six times with DMF. Add 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min, filter, and add 20 mL of 20% piperidine / DMF solution again, react in a shaker for 15 min. The resin color was positive according to Kaiser test. Wash the resin 6 times with DMF to obtain H-Ala-Gln (Trt)-2-CTC-resin.

[0278] Add 20 mL of 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) to a reaction tube containing H-Ala-Gln(Trt)-2-CTC-resin. Shake well, then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) and react in a shaker for 1 hour. The resin color was negative according to Kaiser assay. Filter and wash the resin 6 times with DMF. Add 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min, filter, and add another 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min. The resin color was positive according to Kaiser test. Wash the resin 6 times with DMF to obtain H-Ala-Ala-Gln (Trt)-2-CTC-resin.

[0279] Add 20 mL of 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) to a reaction tube containing H-Ala-Ala-Gln (Trt)-2-CTC-resin. Shake well, then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) and react in a shaker for 1 hour. The resin color was negative according to Kaiser assay. Filter and wash the resin 6 times with DMF. Add 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min, filter, and add another 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min. The resin color was positive according to Kaiser test. Wash the resin 6 times with DMF to obtain H-Ala-Ala-Ala-Gln (Trt)-2-CTC-resin.

[0280] Add 20 mL of 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) to a reaction tube containing H-Ala-Ala-Ala-Gln (Trt)-2-CTC-resin. Shake well, then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol) and react in a shaker for 1 hour. The resin color was negative according to Kaiser assay. Filter and wash the resin 6 times with DMF. Add 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min, filter, and add another 20 mL of 20% piperidine / DMF solution, react in a shaker for 15 min. The resin color was positive according to Kaiser test. Wash the resin 6 times with DMF to obtain H-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin.

[0281] Add 20 mL of DMF solution containing SMCC (3.0 eq, 501 mg, 1.5 mmol) to a reaction tube containing H-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC-resin, shake well, and then add DIPEA (3.0 eq, 0.26 mL, 1.5 mmol). React in a shaker for 1 hour. The resin color was negative according to Kaiser assay. Filter the solution and wash the resin 6 times with DMF, 3 times with MeOH, and 3 times with MTBE. Dry in a vacuum oven at 30°C. oDry at C for 1 hr to obtain MCC-Ala-Ala-Ala-Ala-Gln(Trt)-2-CTC- resin (1.42 g, 0.5 mmol).

[0282] Transfer the resin obtained above to a 50 mL centrifuge tube, add 15 mL of cold 95% TFA / Tis solution, and react in a shaker for 1 hour. Filter, and slowly add the filtrate to 150 mL of cold MTBE solution. Let stand for 1 hour, and a white precipitate will form. Centrifuge and wash the precipitate three times with cold MTBE solution. Collect the washed precipitate and dry it in a vacuum oven at 30°C. o Drying at C for 4 hours yields a white solid, namely the intermediate MCC-Ala-Ala-Ala-Ala-Gln-OH.

[0283] The reaction formula is as follows:

[0284]

[0285] 2. Product Synthesis: MCC-Ala-Ala-Ala-Ala-Gln-Exatecan

[0286] Intermediate MCC-Ala-Ala-Ala-Ala-Gln-OH (244 mg, 2.0 eq) was added to a reaction flask, followed by DMF (2 mL), ixotecan sulfonate (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. HPLC analysis showed that 10% of Exatecan remained.

[0287] Prep-HPLC purification was performed, and the product was collected. The solution was concentrated and dried to obtain a yellow solid (NMR data are shown below).

[0288] The reaction formula is as follows:

[0289]

[0290] 1H 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).

[0291] Example 6: Synthetic route of the linker drug MCC-Ala-Ala-Gln-Belotecan

[0292] MCC-Ala-Ala-Gln-Belotecan Synthetic Route

[0293]

[0294] 1. Synthesis of intermediate 3 Fmoc-Gln-Belotecan

[0295]

[0296] 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 shaken to dissolve. o Add DIEA (190 μL, 1.15 mmol, 10 eq) at C, 10 oThe reaction was stirred at C for 1 hour. HPLC analysis confirmed the Belotecan reaction was complete. The product was purified and collected. It was concentrated and lyophilized to give a yellow solid, 3Fmoc-Gln-Belotecan (51 mg, purity: 98.71%, yield: 55.84%). MS (ESI): m / z 784.3 [M+H] + .

[0297] 2. Synthesis of intermediate 4 Gln-Belotecan

[0298]

[0299] Intermediate 3 Fmoc-Gln-Belotecan (51 mg, 0.065 mmol, 1.0 eq) was added to a reaction flask, followed by DMF (4 mL), diethylamine (1 mL), and 20 mL of 20 mmol / L. o Stirring at C for 2 hours, HPLC analysis showed no residue of the starting material. The reaction solution was concentrated under vacuum, and DMF (2 mL) was added. The solution was purified preparatively, concentrated, and lyophilized to obtain a yellow solid, 4 Gln-Belotecan (24 mg, purity: 91.96%, yield: 60.40%). MS (ESI): m / z 562.34 [M+H] + .

[0300] 3. Synthesis of intermediate 5 MCC-Ala-Ala-OH

[0301]

[0302] Add 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) to a reaction flask, and heat to 25 °C. o Add DMF (190 mL) to C, then add DIEA (9.8 mL, 59.31 mmol, 1.0 eq), 60 o The reaction mixture was stirred at room temperature for 8 hours. HPLC analysis confirmed the reaction was complete. The reaction solution was cooled to 25°C. o C, vacuum concentration, then add H2O (190 mL), DIEA (9.8 mL), and EA (190 mL) sequentially, 25 o After stirring for 1 hour, the mixture was added to a separatory funnel and allowed to stand for separation. The aqueous phase was separated and extracted twice with EA (95 mL). The EA layer was separated, and 2M hydrochloric acid solution (71.3 mL) was slowly added dropwise to the aqueous phase at 25°C. oAfter stirring at C for 1 hour, the mixture was filtered. The resulting solid was then vacuum dried 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] + .

[0303] 4 MCC-Ala-Ala-Gln-Belotecan synthesis

[0304]

[0305] Intermediate 4 (15 mg, 0.027 mmol, 1.0 eq), 5 MCC-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, followed by the addition of DMF (2 mL) and shaking to dissolve. o Add DIC (8.0 μL, 0.054 mmol, 2.0 eq) at C, 20 o The reaction was stirred at C for 3 hours. HPLC analysis confirmed the reaction was complete. The product was prepared, purified, and collected. It was then concentrated and lyophilized to give a yellow solid, MCC-Ala-Ala-Gln-Belotecan (11 mg, purity: 96.42%, yield: 43.03%). MS (ESI): m / z 462.5 [M+2H] / 2 + .

[0306] 1H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J= 10.4 Hz, 1H), 8.19 (d, J= 8.0Hz, 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).

[0307] In addition, those skilled in the art can also obtain other linker drug conjugates of the present invention by referring to the preparation methods of Examples 1 to 6 above, such as MCC-AAQ-Rapamycin.

[0308]

[0309] MCC-AAQ-Rapamycin.

[0310] Example 7: Preparation of EGFR antibody-drug conjugates

[0311] I. Preparation of the antibody-drug conjugate EGFR antibody-MCC-AAQ-Exatecan

[0312] 1. Take EGFR antibody and adjust its pH to approximately 7.5 using Tris-EDTA solution. Detect the protein concentration using Nanodrop and weigh the net antibody solution to calculate the total protein content. Add TCEP solution to the antibody and place it on a 3D shaker at room temperature for at least 120 minutes, continuously mixing to completely reduce the disulfide bonds between the antibody chains.

[0313] 2. Add excess MCC-AAQ-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix well, and place on a 3D shaker. Incubate at room temperature for at least 30 minutes, mixing continuously. After the reaction is complete, add excess N-acetylcysteine ​​solution to the reaction solution, place on a 3D shaker, and incubate at room temperature for at least 30 minutes, mixing continuously.

[0314] 3. The conjugate product was purified using a 30KD ultrafiltration centrifuge tube and displaced into a storage solution (10mM Histidine, pH 5.5) at a displacement factor greater than 1000-fold. Then, it was filtered through a 0.22µm sterile filter to obtain the antibody-drug conjugate EGFR antibody-MCC-AAQ-Exatecan, which was stored at 4°C.

[0315] 4. The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, DAR was detected using HIC, and purity was detected using SEC.

[0316] II. Preparation of other EGFR antibody-drug conjugates

[0317] Other linker drug conjugates of the present invention (such as the linker drug conjugate shown in Formula I) and EGFR antibody can be prepared by referring to the preparation method of the above-mentioned antibody conjugate EGFR antibody-MCC-AAQ-Exatecan.

[0318] Example 8: Preparation of HER2 antibody-drug conjugates

[0319] The method for preparing antibody-drug conjugates (such as antibody-drug conjugate HER2 antibody-MCC-AAN-Exatecan) using the linker drug conjugate of the present invention (as shown in Formula I) and HER2 antibody can refer to the preparation method of antibody-drug conjugate EGFR antibody-MCC-AAQ-Exatecan in Example 7 above.

[0320] Example 9: In vitro activity testing of different antibody-drug conjugates

[0321] In vitro activity tests were performed on different antibody-drug conjugates of the present invention to detect their cell-killing activity.

[0322] The testing method is as follows:

[0323] Target cells were seeded in a specific quantity into 96-well black transparent cell culture plates. After 24 hours of incubation, serially diluted samples were added, followed by incubation for another 72 or 144 hours. A chromogenic reagent was then applied for 60 minutes, and the plates were read using a microplate reader. A higher number of viable cells resulted in a higher signal value. Based on the different signal values ​​obtained at different concentration gradients, a four-parameter fitting curve was generated on the microplate reader to obtain the C-value, i.e., the IC50 value. 50 value.

[0324] The following is a partial list of data.

[0325] Table 1. Activity data of MCC-AAQ-Exatecan ADC in MDA-MB-468 cell line.

[0326]

[0327] In addition, the present invention also provides the following embodiments.

[0328] Example 10: Preparation of the linker drug conjugate mc-AAN-Exatecan

[0329] 1. Synthesis of Intermediate 1

[0330] Fmoc-Ala-OH (N-fluorenylmethoxycarbonyl-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 one. Specifically, Fmoc-Ala-OH (3g, 1.0eq) and HOSu (1.45g, 1.3eq) were added to the reaction flask, followed by 21mL of THF. The temperature was controlled at room temperature, and DCC (2.59g, 1.3eq) was slowly added under stirring. The reaction was then carried out at room temperature and monitored by HPLC. After the reaction was completed, the reaction solution was filtered, and the filter cake was washed with THF (6mL). Purified water (15 mL) was added to the filtrate, followed by L-Ala (1.12 g, 1.3 eq) and sodium bicarbonate solid (0.81 g, 1.0 eq). The reaction was stirred at room temperature and monitored by HPLC. After the reaction was complete, citric acid (2.02 g, 1.0 eq) was added and stirred. The reaction solution was then extracted with ethyl acetate, the organic phase was concentrated, and the product was dissolved in DMF (12 mL). After filtration, the solution was passed through pre-HPLC and concentrated until no obvious droplets flowed out. It was then extracted three times with ethyl acetate, and the ethyl acetate phase was concentrated to dryness to obtain intermediate 1.75 g. The yield of this step was 57%. The reaction formula is as follows:

[0331]

[0332] 2. Synthesis of Intermediate Two

[0333] Intermediate 1 (N-[fluorenylmethoxycarbonyl]-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. The temperature was controlled at room temperature, and DCC (0.61 g, 1.3 eq) was slowly added under stirring. The reaction was carried out at room temperature and monitored by HPLC. After the reaction was completed, the reaction solution was filtered, and the filter cake was washed with THF (2 mL). Purified water (10 mL) was added to the filtrate, followed by L-Asn (0.34 g, 1.1 eq) and sodium bicarbonate solid (0.19 g, 1.0 eq). The mixture was stirred at room temperature and monitored by HPLC. After the reaction was complete, citric acid monohydrate (0.48 g, 1.0 eq) was added and stirred. The reaction solution was concentrated to remove most of the solvent, and the purified residue was prepared. The purified residue was concentrated until no obvious droplets flowed out, and then extracted with ethyl acetate. The organic phase was concentrated to dryness to give intermediate II 826 mg. The yield of this step was 73%. The reaction formula is as follows:

[0334]

[0335] 3. Synthesis of intermediate three

[0336] Intermediate 2 was de-Fmoc-reduced by adding DEA (diethylamine, CAS No.: 109-89-7) 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 was carried out at room temperature under HPLC monitoring until intermediate 2 was completely removed. DMF was removed by concentration, and DCM (4 mL) and purified water (4 mL) were added. After stirring, the mixture was separated, and the aqueous phase was concentrated to dryness to obtain 92 mg of intermediate 3. The yield of this step was 166% (including partial solvent encapsulation). The reaction formula is as follows:

[0337]

[0338] 4. Synthesis of intermediate tetramethylpyrrolidone

[0339] Intermediate 3 reacted with succinimide 6-(maleimino)hexanoate (CAS No.: 55750-63-5) to give intermediate 4. Specifically, intermediate 3 (92 mg, 1.0 eq), succinimide 6-(maleimino)hexanoate (135 mg, 1.3 eq), DMF (1.5 mL), and DIPEA (0.059 mL, 1.0 eq) were added to the reaction flask. The reaction was monitored by HPLC. After the reaction was completed, the intermediate was purified and concentrated to obtain 56 mg of intermediate 4. The yield of this step was 35.7%. The reaction formula is as follows:

[0340]

[0341] 5. Product synthesis

[0342] Intermediate 4 was obtained by condensation reaction with eczemab mesylate (CAS No.: 169869-90-3). Specifically, at room temperature, intermediate 4 (26 mg, 1.0 eq) was added to a reaction flask, followed by DMF (1.5 mL), then eczemab 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). The reaction was carried out at room temperature. HPLC monitoring was performed. After the reaction was completed, the product was purified and concentrated to obtain 16.5 mg of the product (NMR results shown below). The yield of this step was 33.5%, and the reaction formula is as follows:

[0343]

[0344] 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).

[0345] Example 11: Preparation of the linker drug conjugate mc-Ala-Ala-Gln-Exatecan

[0346] Synthesis of intermediate one: Fmoc-Gln-Exatecan

[0347] Fmoc-Gln-OH (N-fluorenylmethoxycarbonyl-L-glutamine) (76.3 mg 1.1 eq.) and Exatecan (100 mg 1.0 eq.) were added to a reaction flask, along with DMF (1 ml), DIEA (29 mg 1.5 eq.), and TBTU (72.5 mg 1.1 eq.). The reaction was carried out at room temperature. HPLC monitoring showed no residual starting material. After the reaction was complete, silica gel was added for concentration and the mixture was purified by column chromatography. The product was collected, concentrated to dryness, yielding 140 mg of product with a purity of 95% (approximately 4% TBTU byproduct Hobt remained), and a yield of 94%.

[0348] The reaction formula is as follows:

[0349]

[0350] 1. Synthesis of intermediate two Gln-Exatecan

[0351] Intermediate 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 until it did not dissolve completely. DEA (0.5 ml) was then added, and the reaction was continued at room temperature. HPLC monitoring showed no starting material remaining. The reaction was stopped, and MTBE (10 ml) was slowly added, resulting in the precipitation of a large amount of solid. The mixture was stirred for 30 min. The solid was filtered to obtain a grayish-white solid, which was dried to yield 95 mg of the solid with a purity of 96% and a yield of 94%.

[0352] The reaction formula is as follows:

[0353]

[0354] 2. Synthesis of intermediate three: Fmoc-Ala-Ala-Gln-Exatecan

[0355] Intermediate II Gln-Exatecan (100 mg 1.0 eq.) was added to a reaction flask along with 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.). The reaction was carried out at room temperature. HPLC monitoring showed no intermediate II remaining, at which point the reaction was stopped. Silica gel was added and the mixture was stirred. The product was purified by column chromatography, yielding 140 mg of product with a purity of 95% and a yield of 85%.

[0356] The reaction formula is as follows:

[0357]

[0358] 3. Synthesis of intermediate four: Ala-Ala-Gln-Exatecan

[0359] The intermediate trifurcation thiocarbamate (140 mg 1.0 eq.) was added to a reaction flask, along with DCM (2 ml) and DEA (0.5 ml). The reaction was carried out at room temperature. HPLC monitoring showed no starting material remaining. The reaction was stopped, and MTBE (10 ml) was slowly added, resulting in the precipitation of a large amount of solid. The mixture was stirred for 30 min. Filtration yielded a grayish-white solid, which was dried to give 70 mg of the product. Purity: 96%; Yield: 66%.

[0360] The reaction formula is as follows:

[0361]

[0362] 4. Product synthesis: mc-Ala-Ala-Gln-Exatecan

[0363] Intermediate tetra(Ala-Ala-Gln-Exatecan) (50 mg 1.0 eq.) was added to a reaction flask, followed by 1 mL of DMF, then 21.8 mg 1.1 eq of 6-(maleimide)hexanoate succinimide ester and DIEA (12.1 mg 1.5 eq.). The reaction was carried out at room temperature. HPLC monitoring was maintained until no intermediate tetra(Ala-Gln-Exatecan) remained, at which point the reaction was stopped. The product was purified, collected, concentrated to dryness, and 35 mg was obtained (NMR results are shown below). Purity: 98%; Yield: 55%.

[0364] The reaction formula is as follows:

[0365]

[0366] 1 H 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).

[0367] Example 12: Preparation of linker drug conjugation mc-Pro-Exatecan

[0368] 1. Synthesis of Intermediate 1: Fmoc-Pro-Exatecan

[0369] Fmoc-Pro-OH (N-fluorenylmethoxycarbonyl-L-proline) (41 mg 1.3 eq.) and Exatecan (50 mg 1.0 eq.) were added to a reaction flask, followed by the addition of DMF (1 ml) and DIEA (49 mg 4 eq.) for dissolution. HATU (46 mg 1.3 eq.) and HOBt (12.7 mg 1.0 eq.) were added, and the reaction was carried out at room temperature under HPLC monitoring. No starting material remained. After the reaction was complete, silica gel was added for concentration and the mixture was stirred and purified by column chromatography. The product was collected, concentrated to dryness, and 50 mg of product was obtained with a purity of 95% and a yield of 73%.

[0370] The reaction formula is as follows:

[0371]

[0372] 2. Synthesis of intermediate two (Pro-Exatecan)

[0373] Intermediate Fmoc-Pro-Exatecan (50 mg 1.0 eq.) was added to a reaction flask, followed by DCM (1 ml) and DEA (0.2 ml). The reaction was carried out at room temperature under HPLC monitoring, and no starting material remained. The reaction was stopped, and MTBE (10 ml) was slowly added, resulting in the precipitation of a large amount of solid. The mixture was stirred for 30 min. After filtration, a grayish-white solid was obtained, which was dried to yield 30 mg of 96% purity (86% yield).

[0374] The reaction formula is as follows:

[0375]

[0376] 3. Product synthesis: mc-Pro-Exatecan

[0377] Intermediate II Pro-Exatecan (30 mg 1.0 eq.) was added to a reaction flask, followed by DMF (1 mL), then 6-(maleimide)hexanoic acid succinimide ester (21.2 mg 1.1 eq.) and DIEA (12.1 mg 1.5 eq.). The reaction was carried out at room temperature under HPLC monitoring until no intermediate II remained, at which point the reaction was stopped. Silica gel was added and the mixture was stirred, then purified by column chromatography. 25 mg of the product was collected (NMR results shown below). Purity: 96%; Yield: 56%.

[0378] The reaction formula is as follows:

[0379]

[0380] 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).

[0381] Example 13: Preparation of the antibody-drug conjugate HER2 antibody-mc-AAN-Exatecan (DAR8)

[0382] 1. Take 10 mg of HER2 antibody, dilute it to a concentration of 5 mg / mL with PBS buffer, and adjust the antibody pH to approximately 7.5 with Tris-EDTA solution; detect the protein concentration using Nanodrop, weigh the net antibody solution, and calculate the total protein content. Add 8 molar amounts of TCEP solution to the antibody, place it on a 3D shaker, and react at room temperature for at least 120 min, continuously mixing.

[0383] 2. Add excess mc-AAN-Exatecan solution (dissolved in DMSO) to the reduced antibody solution, mix well, and place on a 3D shaker. Incubate at room temperature for at least 30 minutes, mixing continuously. After the reaction is complete, add excess N-acetylcysteine ​​solution to the reaction solution, place on a 3D shaker, and incubate at room temperature for at least 30 minutes, mixing continuously.

[0384] 3. The conjugate product was purified using a 15 mL 30 KD ultrafiltration centrifuge tube and displaced into a storage solution (10 mM Mistidine, pH 5.5) at a displacement factor greater than 1000-fold. Then, it was 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.

[0385] 4. The protein concentration of the antibody-drug conjugate was detected using the UV / BCA method, DAR was detected using HIC, and purity was detected using SEC.

[0386] Example 14: In vitro activity testing of different antibody-drug conjugates

[0387] Referring to the process in Example 13, corresponding ADCs were prepared by conjugating the linker drug conjugate mc-AAN-Exatecan with HER2 antibody, EGFR antibody and non-specific binding antibody (Anti-HEL), respectively. ADCs were also prepared by conjugating the linker drug conjugate mc-Pro-Exa with HER2 antibody, and cell killing activity was detected.

[0388] The testing method is as follows:

[0389] Target cells were seeded in a specific quantity into 96-well black transparent cell culture plates. After 24 hours of incubation, serially diluted samples were added, followed by another 72 hours of incubation. A chromogenic reagent was then applied, and the plates were incubated for 60 minutes. The plates were then read using a microplate reader. A higher number of viable cells resulted in a higher signal value. Based on the different signal values ​​obtained at different concentration gradients, a four-parameter fitting curve was generated on the microplate reader to obtain the C-value, i.e., the IC50 value. 50 value.

[0390] Detailed data is as follows.

[0391] Table 2 Activity data of mc-AAN-Exatecan ADC in different cell lines

[0392]

[0393] Table 3 Comparison of Exatecan ADC and Payload Activity Data

[0394]

[0395] Table 4. Activity data of mc-Pro-Exa ADC in breast cancer cell lines.

[0396]

[0397] Example 15: Preparation and Detection of ADCs Using Linkers or Cytotoxins with Different Structures

[0398] Referring to the preparation process of Linker-Payload in Example 10 and ADC stock solution in Example 13, the inventors prepared the following ADC stock solution and tested and analyzed the basic quality characteristics of the obtained ADC stock solution.

[0399] After conjugation with the same HER2 antibody, the DAR values ​​were all close to 8. Based on the analysis of the detection data of these ADCs, the following conclusions can be drawn:

[0400] 1. Compared with ADCs containing PAB fragments, the polymer content (HMW%) of ADCs prepared by Linker-Payloads without PAB fragments is significantly lower than that of ADCs prepared with PAB fragments, indicating that PAB fragments are not conducive to the preparation of ADCs by this type of Linker-Payload. Specific data are shown in Table 5.

[0401] 2. Payloads containing primary amine (-NH2) structures can be released through linkers that do not contain PAB fragments, while payloads containing non-primary amine groups (-NH-(i-Pr) structures) cannot be released through linkers that do not contain PAB fragments. Specific data are shown in Table 6.

[0402] The inventors studied the polymer content in ADCs obtained by coupling mc-Pro-PAB-Exatecan with antibody TF (i.e. TF-mAb-H39 in CN201610705557.4) and mc-Pro-Exatecan with HER2 antibody. They found that the PAB structure has a very significant impact. After removing PAB from the linker, the polymer content can be reduced from about 70-80% to 1%.

[0403] Table 5. Analysis data on the impact of PAB fragments on HMW% residual amount

[0404]

[0405] In studying the in vitro cytotoxic activity of ADCs conjugated with HER2 antibodies, mc-Pro-Belotecan and mc-Pro-Exatecan, respectively, it was found that the ADC conjugated with mc-Pro-Belotecan had no tumor cell killing activity, indicating that its structure is stable and cannot release the payload through enzymatic digestion. In contrast, the ADC conjugated with mc-Pro-Exatecan showed better cytotoxic activity, indicating that this structure can release the payload. At the same time, the ADC generated by conjugating mc-Pro-Exatecan with a non-binding antibody (without targeted endocytosis) had no specific cytotoxic activity, indicating that this structure is stable extracellularly and needs to be internalized into the cell before the payload can be released. In addition, by comparing the chemical structures of mc-Pro-Belotecan and mc-Pro-Exatecan, a key difference can be found: Belotecan is linked to Proline with a -NH-(i-Pr) structure, while Exatecan is linked to Proline with a -NH2 structure. Therefore, it is inferred that if the amino acid fragment in the linker is linked to a payload containing a primary amine (-NH2) structure, the payload can be released through enzymatic hydrolysis after removing the hydrophobic PAB structure (for most currently under development ADCs, the PAB structure is an essential structure for effective payload release as a self-releasing fragment). In this way, the hydrophobicity of the linker-drug can be reduced, the polymer content generated during ADC preparation can be reduced, and the corresponding cell-killing activity can also be exhibited.

[0406] Table 6. Data on conjugation of different linker-payloads with HER2 antibody

[0407]

[0408] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

[0409] The present invention relates to the following embodiments.

[0410] 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.

[0411] MXD

[0412] Formula I

[0413] in,

[0414] M is a chemical structure containing a maleimide (m) segment or a cyclooctyne segment;

[0415] X is a linker fragment consisting of 1 to 5 amino acids or their derivatives, preferably selected from 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-Ala-(3-cyano-alanine), Ala-(3-cyano-alanine), Ala-(3-cyano-alanine). ine), 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 their deuterated compounds;

[0416] D stands for cytotoxin.

[0417] 2. The linker drug conjugate of embodiment 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the chemical structure comprising the maleimide (m) fragment is selected from 4-(N-maleiminomethyl)-cyclohexane-1-formyl (MCC), 6-maleiminohexanoyl (MC), M-(PEG). n MC-(PEG) n MCC-(PEG) n The structures are shown below:

[0418]

[0419] Where n represents the degree of polymerization of PEG, preferably an integer between 2 and 12.

[0420] 3. The linker drug conjugate of embodiment 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the chemical structure containing the cyclooctyne fragment is selected from BCN, DIBO, DIBAC, BARAC, etc., and the structures are shown below:

[0421] .

[0422] 4. The linker drug conjugate of any one of embodiments 1-3, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the linker fragment consisting of 1-5 amino acids or their derivatives is selected from 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-Ala-(3-cyano-alanine). anine), Ala-(3-cyano-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 their deuterated compounds.

[0423] 5. The linker drug conjugate of any one of embodiments 1-4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group;

[0424] Preferably, the cytotoxin containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) groups is selected from camptothecin-type topoisomerase I inhibitors, Daunorubicin-type topoisomerase II inhibitors, calendula derivatives, molecular colloidal compounds, and immunosuppressants.

[0425] More preferably, the cytotoxicant containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group 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;

[0426] Most preferably, the cytotoxicant containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group is selected from Exatecan, beloteccan, and rapamycin.

[0427] 6. The linker drug conjugate of any one of embodiments 1-5, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein said linker drug conjugate is MCC-AAQ-Exatecan and has the following structure:

[0428] .

[0429] 7. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the antibody-drug conjugate is formed by conjugating an antibody with a linker drug conjugate as described in any one of embodiments 1-6;

[0430] Preferably, the antibody-drug conjugate has the structure shown in Formula II.

[0431] Ab-(LD) p

[0432] Formula II

[0433] in,

[0434] Ab represents antibodies;

[0435] L is a connector with a structure of M'-X, where M' is connected to Ab and X is connected to D;

[0436] M' is a chemical structure formed by M being linked to Ab via a maleimide group as defined in any of embodiments 1-6;

[0437] X is a linker consisting of 1 to 5 amino acids or their derivatives as defined in any one of embodiments 1-6;

[0438] D is a cytotoxic substance as defined in any of Implementation Schemes 1-6;

[0439] p is any value between 2 and 8.

[0440] 8. The antibody-drug conjugate of embodiment 7, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the antibody is selected from murine antibodies, rabbit antibodies, phage display-derived antibodies, yeast display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies;

[0441] Preferably, the antibody is a monoclonal antibody, selected from: HER2 antibody and EGFR antibody.

[0442] 9. A pharmaceutical composition comprising the linker drug conjugate of any one of embodiments 1-6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the antibody drug conjugate of embodiment 6 or 7, or a pharmaceutically acceptable salt, solvate, or solvate of said salt.

[0443] Preferably, the pharmaceutical composition further comprises at least one of a chemotherapy drug, an immunotherapy drug, and an immunosuppressant for treating tumors;

[0444] Preferably, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

[0445] 10. Use of the linker drug conjugate of any one of embodiments 1-6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, or the antibody drug conjugate of embodiment 7 or 8, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, in the preparation of a medicament for the prevention and / or treatment of tumors.

[0446] 11. A method for preparing the linker drug conjugate according to any one of embodiments 1-6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said method comprising the following steps:

[0447] 1. Prepare a linker fragment X consisting of 1-5 amino acids or their derivatives;

[0448] 2. X is linked to a chemical structure M containing a maleimide (m) fragment or a cyclooctyne fragment to obtain MX;

[0449] 3. Cytotoxin D is linked to MX to obtain the linker drug conjugate MXD.

[0450] 12. The method of implementation scheme 11, wherein the linker drug conjugate is MCC-AAQ-Exatecan, the method includes the following steps:

[0451] 1. Fmoc-Gln was reacted with Exatecan to obtain intermediate one, the structural formula of which is as follows:

[0452]

[0453] 2. Intermediate 1 is deprotected from the Fmoc protecting group via DEA to obtain intermediate 2, wherein the structural formula of intermediate 2 is as follows:

[0454]

[0455] 3. Intermediate 2 is reacted with Fmoc-Ala-Ala to obtain intermediate 3, the structural formula of which is as follows:

[0456]

[0457] 4. The intermediate ter was deprotected from the Fmoc protecting group to obtain intermediate 4, the structural formula of which is as follows:

[0458]

[0459] 5. The intermediate four is condensed with MCC to obtain the product, the product structure of which is as follows:

[0460] .

Claims

1. A linker drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, said linker drug conjugate having the structure shown in Formula I, MXD Formula I in, M is a chemical structure containing a maleimide (m) fragment or a cyclooctyne fragment; X is selected from Ala-Ala-Asn, Ala-Ala-Gln, and their deuterated derivatives; D is a cytotoxin containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group.

2. The linker drug conjugate of claim 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein, The chemical structure containing the maleimide (m) fragment is selected from 4-(N-maleiminomethyl)-cyclohexane-1-formyl (MCC), 6-maleiminohexanoyl (MC), and M-(PEG). n MC-(PEG) n MCC-(PEG) n The structures are shown below: Where n represents the degree of polymerization of PEG, preferably an integer between 2 and 12.

3. The linker drug conjugate of claim 1, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein, The chemical structures containing the cyclooctyne fragment are selected from BCN, DIBO, DIBAC, BARAC, etc., and their structures are shown below: 。 4. The linker drug conjugate of any one of claims 1-3, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein, D represents cytotoxins containing hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) groups, selected from camptothecin-type topoisomerase I inhibitors, Daunorubicin-type topoisomerase II inhibitors, calendula derivatives, molecular colloidal compounds, and immunosuppressants. More preferably, the cytotoxicant containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group 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; Most preferably, the cytotoxicant containing a hydroxyl (-OH), primary amine (-NH2), or secondary amine (-NHR) group is selected from Exatecan, beloteccan, and rapamycin.

5. The linker drug conjugate of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein, The linker drug conjugate is MCC-AAQ-Exatecan, which has the following structure: 。 6. An antibody-drug conjugate, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the antibody-drug conjugate is formed by conjugating an antibody with a linker drug conjugate according to any one of claims 1-5; Preferably, the antibody-drug conjugate has the structure shown in Formula II. Ab-(LD) p Formula II in, Ab represents antibodies; L is a connector with a structure of M'-X, where M' is connected to Ab and X is connected to D; M' is the chemical structure formed by M as defined in any one of claims 1-5 being linked to Ab via a maleimide group; X is defined as in any one of claims 1-5; D is as defined in any one of claims 1-5; p is any value between 2 and 8.

7. The antibody-drug conjugate of claim 6, or a pharmaceutically acceptable salt, solvate, or solvate of said salt, wherein the antibody is selected from murine antibodies, rabbit antibodies, phage-display-derived antibodies, yeast-display-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies, and multispecific antibodies; Preferably, the antibody is a monoclonal antibody, selected from: HER2 antibody and EGFR antibody.

8. A pharmaceutical composition comprising the linker drug conjugate of any one of claims 1-5, 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 chemotherapy drug, an immunotherapy drug, and an immunosuppressant for treating tumors; Preferably, the pharmaceutical composition further comprises at least one pharmaceutical excipient.

9. Use of the linker drug conjugate of any one of claims 1-5, 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, in the preparation of a medicament for the prevention and / or treatment of tumors.

10. The method for preparing the linker drug conjugate MCC-AAQ-Exatecan according to claim 5 comprises the following steps: (1). Fmoc-Gln was reacted with Exatecan to obtain intermediate one, the structural formula of which is as follows: (2). Intermediate 1 is deprotected from the Fmoc protecting group via DEA to obtain intermediate 2, wherein the structural formula of intermediate 2 is as follows: (3). Intermediate 2 is reacted with Fmoc-Ala-Ala to obtain intermediate 3, the structural formula of which is as follows: (4). The intermediate ter was deprotected from the Fmoc protecting group to obtain intermediate 4, wherein the structural formula of intermediate 4 is as follows: (5). The intermediate four is condensed with MCC to obtain the product, the product structure of which is as follows: 。

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