Biologically active conjugate and preparation method and use thereof

Abstract

This disclosure relates to bioactive drug conjugates, their preparation methods and uses, specifically disclosing ligand-drug conjugates of Formula XV, their preparation methods, and their use in the prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to their use in the prevention and / or treatment of tumor diseases.

Description

Technical field

[0001] The present disclosure belongs to the field of medical technology, and relates to bioactive substance conjugates (ligand drug conjugates), compounds, drug linker conjugates and preparation methods thereof, and their functions in prevention and / or treatment and cell activity Abnormality-related diseases, including but not limited to the use in the prevention and / or treatment of tumor diseases.

[0002] This application claims the priority right of the Chinese patent application 2021101781361 whose application date is 2021 / 2 / 9; the priority right of the Chinese patent application 2021103408065 whose application date is 2021 / 3 / 30; the required application date is 2021 / 7 / 21 The priority of the Chinese patent application 202110825932X; the priority of the Chinese patent application 2021108259226 with the filing date of 2021 / 7 / 21 is requested, and the sequence list txt file is also submitted at the same time as the Chinese patent application 2021108259226 on 2021 / 7 / 21; the filing date is required As the priority of Chinese patent application 2021108259067 on July 21, 2021, the sequence listing txt file was also submitted on July 21, 2021 at the same time as Chinese patent application 2021108259067. This application cites the full text of the above-mentioned Chinese patent application. The sequence listing contained in this application is part of the specification and is hereby incorporated by reference in its entirety.

Prior Technology

[0003] Chemotherapy using cytotoxicity was once the standard therapy for cancer, but cytotoxic molecules with high lethality can kill normal cells and cause serious side effects. Targeted anti-tumor drugs have become a hot spot in the field of cancer research because they have both targeting and anti-tumor activities, but they often have relatively large toxic and side effects due to the target selectivity of targeted drugs, which limits the ability of targeted drugs. the therapeutic effect. Biomacromolecular drugs, such as antibodies or antibody fragments, although highly targeted, have limited or no therapeutic effect on solid tumors. ADC is a conjugate of an antibody and a small molecule drug, which combines the targeting effect of the antibody and the activity of bioactive molecules, and becomes a biological missile with very promising efficacy and safety advantages. The antibody guides the ADC to bind to the target cell, and then it is internalized by the cell, and the small molecule drug is released in the cell through enzymatic hydrolysis under the action of a specific enzyme to treat the disease.

[0004] ADC drugs have developed very rapidly in recent years, and there are 14 kinds of ADCs on the market: (1) Mylotarg (Gemtuzumab Ozogamicin, Gemtuzumab (CD33)-calizimycin), CD33-positive acute myeloid leukemia ( AML), approved by the FDA in 2000, off the market in 2010, and re-launched in 2017; (2) Adcetris (Brentuximab Vedotin, CD30 monoclonal antibody-MMAE), classical Hodgkin lymphoma and anaplastic large cell lymphoma, in 2011 FDA approved marketing; (3) Kadcyla (Trastuzumab Emtansine, trastuzumab (Her2)-maytansine alkaloid TM1), HER2 positive breast cancer, FDA approved marketing in 2013; (4) Besponsa (Inotuzumab ozogamicin, CD22 single Anti-calicheamicin), relapsed or refractory B-cell lymphocytic leukemia in adults, approved for marketing in 2017; (5) Lumoxiti (Moxetumomab pasudotox-tdfk, CD22-Pseudomonas exotoxin), relapsed or refractory Hairy cell leukemia (HCL), launched in 2018; (6) Polivy (Polatuzumab vedotin-piiq, CD79b-MMAE), relapsed or refractory diffuse large B-cell lymphoma (R / R DLBCL), launched in 2019 ; (7) Padcev (Enfortumab vedotin-ejfv, Nectin 4-MMAE), locally advanced or metastatic urothelial carcinoma (UC), launched in 2019; (8) Enhertu (Famtrastuzumab deruxtecan-nxki, Her2-topoisomerase I inhibitor), breast cancer, launched in 2019; (9) Trodelvy (Trop2-topoisomerase I inhibitor ADC), triple negative breast cancer, approved by FDA in April 2020; (10) BCMA ADC Belantamab mafodotin ( (11) CD19 ADC Zynlonta (loncastuximab tesirine); (12) EGFR ADC Akalux; (13) Her2 ADC vedicumab RC48; (14) Tissue Factor ADC Tivadak (tisotumab vedotin).

[0005] ADC drug consists of three parts: antibody, bioactive molecule (drug molecule) and linker (Linker). The biologically active molecule is covalently coupled to the antibody via a linker. In addition to targeting and biological activity, the coupling method of bioactive molecules and antibodies is also one of the cores of ADC drugs, which determines the uniformity and stability of drugs, and greatly affects the pharmacokinetic properties and toxicity of ADCs. The improvement of the stability of the link between the bioactive molecule and the antibody will increase the stability of the ADC systemic circulation and reduce the shedding of the drug in non-targeted tissues, thereby relatively increasing the drug brought into the target cell and around the target cell, increasing the biological activity The release of molecules in and near the target cells can further increase the efficiency and attenuate the toxicity. Therefore, increasing the stability of the connection between bioactive molecules and antibodies is one of the most important technical fields in ADC drug research.

[0006] There are mainly two coupling methods of 10 kinds of ADC antibodies and linkers currently on the market: (1) lysine is the most common linking site in antibodies, and its ε-amine group can be combined with the activated carboxyl group of the linker reaction to form an amide bond. At present, there is a technology that can realize site-specific coupling, that is, activate the carboxyl group of the linker with an activation group, and then form an amide bond with the specific ε-amine group of lysine in the antibody to complete the coupling. On the one hand, the fixed-point quantitative coupling through such amide bonds does not have broad-spectrum practicability, and can only be used in some specific cases; on the other hand, under the action of enzymes in vivo, it is prone to hydrolysis, resulting in biological activity Molecules and antibodies are shed before reaching target cells, increasing toxicity. (2) The sulfhydryl group (SH) of antibody cysteine ​​exists in the form of disulfide bonds. Opening disulfide bonds in antibodies can provide multiple free sulfhydryl groups as conjugation sites. Coupling with the antibody sulfhydryl group, one method is Michael addition reaction between the free sulfhydryl group on the antibody and maleimide, or a specific matrix and the free sulfhydryl group on the antibody through two Michael addition reactions to form a Structurally unique sulfur bridges. However, there are many reports in the literature that 30% or more of the ADC obtained by the mercapto Michael addition method will undergo reverse Michael addition in the systemic circulation, resulting in premature shedding of toxins and toxic reactions.

[0007] Antibody-conjugated drugs can be divided into four generations according to the development process:

[0008] The first generation of antibody-drug conjugates is based on murine antibodies, which have strong toxic and side effects due to their complex immunogenicity;

[0009] The second-generation antibody-drug conjugates use humanized or fully human antibodies on the basis of the first-generation antibody-drug conjugates, and the pharmaceutical properties of the second-generation antibody-drug conjugates have been greatly improved. The first-generation and second-generation antibody-drug conjugates are relatively random in the connection between the antibody and the drug, so the final antibody-drug conjugate product is a multi-component mixture. This type of drug is not only highly toxic, but its quality is also difficult to control;

[0010] On the basis of using humanized or fully humanized antibodies, the third-generation antibody-drug conjugates simultaneously realize the fixed-quantitative coupling of toxin-linker (linker) and antibody, thereby generating a homogeneous single-molecule antibody Conjugated drugs, the toxicity and quality control of this type of antibody-conjugated drugs are greatly improved compared with the second-generation antibody-conjugated drugs;

[0011] The fourth generation of antibody-drug conjugates is based on the third generation, and the toxin-linker (linker) has been innovated. The fourth generation of antibody-drug conjugates has changed the previous strategy of antibody-drug conjugates based on highly toxic toxins, and selected relatively low-toxicity camptothecin molecules as biologically active ingredients, and at the same time adopted a high toxin-antibody ratio (DAR), such as With a DAR value of 8, this type of ADC can also produce a good therapeutic effect on tumors with low expression of tumor surface antigens. Although the fourth-generation antibody-drug conjugates started late, due to their remarkable curative effect, they have sprung up and quickly gained clinical approval. The representative drugs Enhertu and Trodelvy have been approved by the FDA for marketing. However, the current fourth-generation ADC still has the problem of poor stability or low solubility. The existing technology lacks universality, and further development of new toxin-linker is still needed to solve these problems.

[0012] Existing antibody-conjugated drugs are generally combined with antibodies on the surface of tumor cells through ADCs, and then endocytosis into endosomes (endosomes), and then transformed from endosomes to lysosomes, and then in Under the action of hydrolytic enzymes in lysosomes, bioactive molecules (toxins or payloads) are dissociated from ADC, and the dissociated bioactive molecules enter the cytoplasm from lysosomes to kill tumor cells, and escape from tumor cells after killing tumor cells. Bioactive molecules can further kill tumor cells with no or low expression of their surrounding antigens (the so-called by-stander effect). During the whole process, changes in any link can cause ADC to develop drug resistance and lose its therapeutic effect, such as changes in antigen expression during treatment, weakened endocytosis or even loss of endocytosis, and endosome or lysosome function. change and so on. Therefore, looking for an ADC that can be stable in the blood circulation system in the body, and at the same time, this ADC can produce extracellular lysis in the tumor microenvironment without the need for endocytosis, and dissociate toxins to kill tumors. This ADC will be able to Overcoming multiple drug resistance mechanisms in traditional ADCs will be of great significance in clinical treatment.

[0013] Although there is evidence that Trodelvy from Immunomedics and Gilead can be cleaved outside tumor cells without endocytosis, since Trodelvy is very unstable in plasma and aqueous solution, its plasma half-life is less than 24 hours, so this type of ADC can only be used The ADC composed of toxins with low biological activity can avoid the non-target toxic side effects caused by the premature shedding of ADC toxins.

[0014] In addition, the function of the linker of the traditional ADC is to complete intracellular enzyme cleavage or use it to regulate water solubility, etc. How to make the linker meet the requirements of enzyme cleavage while utilizing the linker to realize the enrichment of ADC in tumor tissue It is also of great significance to reduce the toxic and side effects of ADC in normal tissues.

[0015] In addition, B7H3 is a type I transmembrane protein of the B7 family, and has two isotypes 2Ig-B7H3 and 4Ig-B7H3 in humans. B7H3 is widely expressed in normal tissues, only constitutively expressed in non-immune quiescent fibroblasts, endothelial cells, osteoblasts and amniotic fluid stem cells, and inducibly expressed on activated T, NK, DC and macrophages. Positive expression was detected in lymphoid organs (Chapoval, A.I., et al., B7H3: A costimulatory molecule for T cell activation and IFN production. Nature Immunology, 2001. 2(3): p. 269-274). Studies have shown that about 76.5% of patients with early liver cancer have serum B7H3 levels three times higher than normal (60.79 ± 19.45 Vs 20.52 ± 8.46ng / mL), but the correlation with disease progression is unclear (Zhao, L., et al., Early Detection of Hepatocellular Carcinoma in Patients with Hepatocirrhosis by Soluble B7H3. Journal of Gastrointestinal Surgery. 21(5): p. 807-812).

[0016] Currently, there are therapeutic strategies targeting B7H3 in preclinical studies. For example, antibodies against mouse B7H3 will enhance infiltrating CD8-positive T cells in tumors and inhibit tumor growth (Mod Pathol. 2010 Aug;23(8):1104-12.). Furthermore, WO2008 / 066691 showed that antibodies recognizing the B7H3 variant B7H3a have antitumor effects on adenocarcinoma in vivo. In a clinical study, a combination drug of mouse B7H3 antibody and radioactive iodine 131 could significantly inhibit the growth of neuroblastoma in patients [Jneufoocol 97(3):409-18(2010)].

Content of invention

[0017] In order to improve the therapeutic effect of antibody drug conjugates (ADC) or other ligand drug conjugates (PDC), reduce drug side effects, and improve therapeutic window, the present disclosure provides a formula (XV) The ligand drug conjugate or a pharmaceutically acceptable salt or solvate thereof, which contains a biologically active molecule (drug molecule), a linker and a targeting moiety, the targeting moiety is passed through an active group (such as a sulfhydryl group) ) is connected with the linker to form a ligand drug conjugate. The present disclosure also develops antibodies comprising variable light chain domains and / or variable heavy chain (VH) domains that bind B7H3 molecules, said variable light chain domains and / or variable heavy chain (VH) domain antibodies from human antibodies.

[0018] For this reason, in the first aspect of the present disclosure, the present disclosure provides the ligand drug conjugate shown in formula XV, the stereoisomer of formula XV or the ligand drug conjugate, and its prodrug , a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof, wherein: Tb is a ligand or a targeting moiety that binds to a target; q is a drug-ligand coupling ratio; D is a biologically active molecular fragment ; L1 is an extension unit; L2 does not exist or is a linking unit; L3 is selected from amino acid residues or short peptides consisting of 2-10 amino acid residues; L4 does not exist or exists, when L4 exists, L4 is selected from From,,,,,,,,, and, 1 bit is connected to L3, and 2 bits are connected to D.

[0019] In addition, it should also be noted that for "the 1 position of L1 is connected to Tb through the S atom", those with ordinary knowledge in the technical field of the present invention can understand that the 1 position of L1 is related to the opening of the disulfide bond ( For example, the reduction of disulfide bonds by the reducing agent TCEP can open the disulfide bonds and generate sulfhydryl groups (SH) after Tb (such as antibodies) itself contains sulfhydryl groups for connection, that is to say, the -S- between L1 and Tb is not otherwise Then the circumscribed sulfur atom. For example, in, -S- is not an additional external sulfur atom, but -S- formed after the sulfhydryl group contained in Tb itself after opening the disulfide bond is connected to the 1-position of L1, for example.

[0020] The extension unit is a component of a ligand-drug conjugate or a drug-linker conjugate or a linker, and its function is to combine the ligand or targeting moiety that binds to the target with the rest of the ligand-drug conjugate part or the rest of the connectome. The extension unit can link the Tb unit to L2 (if present) or L3, specific examples include but are not limited to (where 1 position is linked to the target-binding ligand or targeting moiety, 2 positions are linked to L2 or L3):,, ,,,,,.

[0021] In some embodiments, L1 is selected from: Each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C6-10 aryl group, a 5-10 membered heteroaryl group, an amido group , sulfonamide, imine and CF2; Rx and Ry are independently selected from H and C1-4 alkyl; each m is independently selected from 0, 1, 2, 3, 4, 5 and 6; y1, y2 , y3 and y4 are independently selected from any integer between 0-20; the 1st position is connected to Tb through the S atom, and the 2nd position is connected to L2 or L3.

[0022] The linking unit is a component of a ligand drug conjugate or a drug linker conjugate or a linker, and its function is to combine the extension unit with an amino acid residue or by 2-10 amino acid residues base composed of short peptides. The presence of the connection unit can connect L1 to L3. Specific examples include, but are not limited to (where 1 position is linked to the Stretcher unit and 2 positions are linked to L3): ,,,, and.

[0023] In some embodiments, L2 does not exist or exists, and when L2 exists, L2 is selected from: y1, y2, y3 and y4 are independently selected from any integer between 0-20, and 1 is connected to L1, 2 bits are connected to L3.

[0024] In some embodiments, L1 is.

[0025] In some embodiments, L1 is selected from ,,,,,, and, each Z is independently selected from direct bond, carbon-carbon triple bond, carbon-carbon double bond, C6-10 aryl, 5-10 yuan hetero Aryl and amido (preferably selected from direct bond, carbon-carbon triple bond, carbon-carbon double bond); Rx, Ry are independently selected from H and C1-4 alkyl; each m is independently selected from 0, 1, 2 , 3, 4, 5 and 6; y1 is selected from any integer between 1-6 (such as 4, 5, 6); each y2 is independently selected from any integer between 0-15 (such as 6-15); each y3 independently selected from 1, 2 and 3; each y4 is independently selected from 0 and 1; the 1 position is connected to Tb through the S atom, and the 2 position is connected to L2 or L3.

[0026] In some embodiments, L1 is selected from ,,,,, and, m is selected from 2, 3, 4, y1 is selected from any integer between 1-6 (such as 4, 5, 6), and each y2 is independently is selected from any integer between 0-10 (such as 6-10), each y3 is independently selected from 1 or 2, the 1st position is connected to Tb through the S atom, and the 2nd position is connected to L2 or L3.

[0027] In some embodiments, L1 is selected from ,,,,,, and, the 1st position is connected to Tb through the S atom, and the 2nd position is connected to L2 or L3.

[0028] In some embodiments, L1 is selected from ,,,,,,,.

[0029] In some embodiments, L1 is selected from , , , , , and, the 1st position is connected to Tb through the S atom, and the 2nd position is connected to L2 or L3.

[0030] In some embodiments, L1 is selected from, and, the 1st position is connected to Tb through the S atom, and the 2nd position is connected to L2 or L3.

[0031] In some embodiments, L1 is selected from and, the 1st position is connected to Tb through the S atom, and the 2nd position is connected to L2 or L3.

[0032] In some embodiments, L2 does not exist or exists, when L2 exists, L2 is selected from ,,,,,,, and, y1 is selected from any integer between 1-6 (such as 4, 5, 6), each y2 is independently selected from any integer between 0-10 (such as 6-10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, 1 bit is connected to L1, and 2 bits are connected to L3 .

[0033] In some embodiments, L2 does not exist or exists, and when L2 exists, L2 is selected from the group consisting of , , , , and, position 1 is connected to L1, and position 2 is connected to L3.

[0034] In some embodiments, L2 does not exist or exists, and when L2 exists, L2 is selected from the group consisting of , , , and, 1 position is connected to L1, and 2 positions are connected to L3.

[0035] In some embodiments, L2 does not exist or exists, and when L2 exists, L2 is selected from the group consisting of , , and , and the 1st position is connected to L1, and the 2nd position is connected to L3.

[0036] In some embodiments, L2 is absent.

[0037] In some embodiments, L2 is selected from.

[0038] In some embodiments, L3 is selected from amino acid residues or short peptides consisting of 2-10 amino acid residues; the amino acid residues are selected from natural amino acid residues, Unnatural amino acid residues, or amino acid residues selected from AA1 or stereoisomers thereof.

[0039] In some embodiments, L3 is selected from amino acid residues Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, AA1 or by 2 - A short peptide consisting of 10 amino acid residues selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Asp, AAl.

[0040] In some embodiments, L3 is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, AAl, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Ala-Ala, Val-AA1, Ala-AA1, Gly-AA1, AA1-Gly, Ala-Ala-Ala, Ala- Ala-Asn, Ala-Ala-Asp, Val-AA1-Gly, Ala-AA1-Gly, Gly-AA1-Gly, Lys-Ala-Ala-Asn, Lys-Ala-Ala-Asp, Gly-Phe-Gly, Gly-Gly-Phe-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Phe, Val-Lys-Gly, Val-Lys-Gly-Gly, Val- Lys and Lys-Ala-Asn.

[0041] In some embodiments, L3 is selected from AA1, AA1-Gly, Val-Cit, Val-AA1-Gly, AA1-Ala-Asn, and Gly-Gly-Phe-Gly.

[0042] In some embodiments, L3 is selected from AA1 and Val-AA1-Gly.

[0043] In some embodiments, L3 is selected from Val-AA1-Gly.

[0044] In some embodiments, L3 is selected from the group consisting of,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, and, X -is selected from halide ion, carboxylate ion, sulfate ion, bisulfate ion and OH-, the 1st position is connected to L1 or L2, and the 2nd position is connected to L4 or D.

[0045] In some embodiments, L3 is selected from ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, and, X-is selected from halide ion, Carboxylate ion, sulfate ion, bisulfate ion and OH-, the 1st position is connected to L1 or L2, and the 2nd position is connected to L4 or D.

[0046] In some embodiments, L3 is selected from ,,,,,,,,,,,, and, X-is selected from halide ion, carboxylate ion, sulfate ion, bisulfate ion and OH-, 1 position Connect to L1 or L2, 2 bits to L4 or D.

[0047] In some embodiments, L3 is selected from ,,,,,,,, and, 1 position is connected to L1 or L2, and 2 positions are connected to L4 or D.

[0048] In some embodiments, L3 is selected from the group consisting of , , , , and , 1 position is connected to L1 or L2, and 2 positions are connected to L4 or D.

[0049] In some embodiments, the structure of the amino acid residue shown in AA1 is as follows, AA1 wherein: Ra, Rb are each independently selected from H, , and, and Ra, Rb are not H at the same time; or, Ra and Rb and the carbon atoms connected to them together form a 4-10 membered heterocyclic ring, and the 4-10 membered heterocyclic ring is optionally substituted by one or more R0; r, r1 are each independently selected from 0 Any integer to 20; Rm1, Rn1 are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl and -COORx1; Rx1 is selected from C1-6 alkyl; Or, Rm1 and Rn1 and together with them The connected nitrogen atoms together form a 4-10 membered heterocycle, which is optionally substituted by one or more R0'; Rz is selected from C1-6 alkyl; R0, R0' are independent Rm2, Rn2 are each independently selected from H and C1-6 alkyl.

[0050] In some embodiments, any one of Ra and Rb is H, and the other is selected from , , and .

[0051] In some embodiments, any one of Ra and Rb is H, and the other is selected from and.

[0052] In some embodiments, Ra and Rb, together with the carbon atom to which they are commonly attached, form a 5-6 membered heterocyclic ring substituted by R0.

[0053] In some embodiments, Ra and Rb, together with the carbon atom to which they are commonly attached, form a piperidine or piperidine ring substituted with R0.

[0054] In some embodiments, Ra and Rb, together with the carbon atom to which they are commonly attached, form a piperidine ring substituted with R0.

[0055] In some embodiments, Ra and Rb, together with the carbon atom to which they are commonly attached, form, or, the carbon atom number 1 is the carbon atom to which Ra and Rb are commonly attached.

[0056] In some embodiments, Ra and Rb, together with the carbon atom to which they are jointly attached, form or, the carbon atom number 1 is the carbon atom to which Ra and Rb are commonly attached.

[0057] In some embodiments, r, r1 are each independently selected from 0, 1, 2, 3, 4, and 5.

[0058] In some embodiments, r, r1 are each independently selected from 0 and 4.

[0059] In some embodiments, one of r, r1 is 0 and the other is 4.

[0060] In some embodiments, Rm1 and Rn1 are each independently selected from H, methyl, ethyl, n-propyl, n-butyl, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, - COOC(CH3)3 and -COOCH2CH2CH2CH3.

[0061] In some embodiments, Rm1 and Rn1 are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl and tertiary butoxycarbonyl.

[0062] In some embodiments, Rm1, Rn1 are each independently selected from H and C1-6 alkyl.

[0063] In some embodiments, Rm1, Rn1 are each independently selected from H, methyl, ethyl, and n-propyl.

[0064] In some embodiments, in r and r1, r is 4, and when r1 is 0, Rm1 and Rn1 are each independently selected from H, C1-6 alkyl (such as H, methyl); r is 0, When r1 is 4, Rm1 and Rn1 are each independently selected from C1-6 alkyl (such as methyl, ethyl, n-propyl), preferably selected from C2-6 alkyl (such as ethyl, n-propyl).

[0065] In some embodiments, Rm1 and Rn1 together with the nitrogen atom to which they are jointly attached form a 5-6 membered heterocyclic ring optionally substituted by R0'.

[0066] In some embodiments, Rm1 and Rn1, together with the nitrogen atom to which they are jointly attached, form a piperidine or piperidine ring optionally substituted by R0'.

[0067] In some embodiments, Rm1 and Rn1 together with the nitrogen atom connected to them together form ,,,, nitrogen atom No. 1 is the nitrogen atom connected to Rm1 and Rn1 together.

[0068] In some embodiments, Rz is methyl.

[0069] In some embodiments, R0, R0' are each independently selected from C1-6 alkyl, -NRm2Rn2, and 5-6 membered heterocyclyl optionally substituted by C1-6 alkyl.

[0070] In some embodiments, R is selected from C1-6 alkyl and a 5-6 membered heterocyclic group substituted by C1-6 alkyl, and the 5-6 membered heterocyclic group is selected from piperidinyl and piperidinyl 𠯤 base.

[0071] In some embodiments, R0 is selected from methyl, ethyl, and a 5-6 membered heterocyclic group substituted by methyl, and the 5-6 membered heterocyclic group is piperidinyl.

[0072] In some embodiments, R0 is selected from methyl and a 5-6 membered heterocyclic group substituted by methyl, and the 5-6 membered heterocyclic group is piperidinyl.

[0073] In some embodiments, R0 is selected from methyl, ethyl and .

[0074] In some embodiments, R0 is selected from methyl and .

[0075] In some embodiments, R0' is selected from C1-6 alkyl and -NRm2Rn2.

[0076] In some embodiments, R0' is selected from methyl and -NRm2Rn2.

[0077] In some embodiments, Rm2, Rn2 are methyl.

[0078] In some embodiments, the amino acid residue shown in AA1 is selected from the group consisting of ,,,,,,,,,,,,,,,,,, and.

[0079] In some embodiments, the amino acid residue shown in AA1 is selected from the group consisting of ,,,,,,,,,,,,,, and.

[0080] In some embodiments, the amino acid residue shown in AA1 is selected from the group consisting of ,,,,,,, and.

[0081] In some embodiments, the amino acid residue shown in AA1 is selected from , , , , , and.

[0082] In some embodiments, L4 is absent or present, and when L4 is present, L4 is selected from the group consisting of , , and , the 1 position is linked to L3, and the 2 position is linked to D.

[0083] In some embodiments, L4 is absent or present, and when L4 is present, L4 is or, the 1 position is connected to L3, and the 2 position is connected to D.

[0084] In some embodiments, L4 is absent.

[0085] In some embodiments, L4 is selected from and, the 1st position is connected to L3, and the 2nd position is connected to D.

[0086] In some embodiments, L4 is selected from the group consisting of linking L3 at position 1 and linking D at position 2.

[0087] In some embodiments, the structure of is selected from the following structural fragments: .

[0088] In some embodiments, Tb is an antibody or antigen-binding fragment thereof.

[0089] In some embodiments, the antibodies or antigen-binding fragments thereof and monoclonal antibodies or antigen-binding fragments thereof include: Fab, Fab', F(ab')2, Fd, Fv (e.g., scFv), dAb , complementarity determining region fragments, non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, monoclonal antibody, bispecific antibody or multispecific antibody.

[0090] In some embodiments, Tb is an antibody or antigen-binding fragment thereof having endocytic or non-endocytic activity.

[0091] In some embodiments, Tb is an antibody or antigen-binding fragment thereof having endocytic activity.

[0092] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has the activity of binding free antigen in tumor tissue and / or tumor cell surface antigen tumor cell surface antigen.

[0093] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has the activity of being endocytized by tumor cells and has the activity of binding free antigens in tumor tissues or surface antigens of tumor cells.

[0094] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has the activity of being endocytosed by tumor cells and has the activity of binding free antigens in tumor tissues and surface antigens of tumor cells.

[0095] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has the activity of being endocytosed by tumor cells and has no free antigen-binding activity in tumor tissue.

[0096] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has the activity of being endocytosed by tumor cells and has no tumor cell surface antigen-binding activity.

[0097] In some embodiments, Tb is an antibody or antigen-binding fragment thereof that has no endocytic activity, or has weak endocytic activity.

[0098] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that does not have the activity of binding free antigen in tumor tissue and / or tumor cell surface antigen tumor cell surface antigen.

[0099] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has no or weak endocytosis activity by tumor cells and has binding activity to free antigens in tumor tissues or tumor cell surface antigens.

[0100] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has no or weak endocytosis activity by tumor cells and has binding activity to free antigens in tumor tissues and tumor cell surface antigens.

[0101] In some embodiments, Tb is an antigen-binding fragment of an antibody that has no endocytosis activity by tumor cells, or has weak endocytosis activity, and has no tumor cell surface antigen-binding activity.

[0102] In some embodiments, Tb is an antigen-binding fragment of an antibody that has no endocytosis activity by tumor cells, or has weak endocytosis activity, and has no free antigen-binding activity in tumor tissue.

[0103] In some embodiments, Tb is incapable of endocytic activity by tumor cells, and there is no antibody or antigen-binding fragment thereof corresponding to the antigen in the human body.

[0104] In some embodiments, Tb is an antibody that does not bind a tumor cell associated antigen.

[0105] In some embodiments, the antibody, or antigen-binding fragment thereof, is selected from an IgG isotype antibody.

[0106] In some embodiments, the antibody, or antigen-binding fragment thereof, is selected from isotype IgGl, isotype IgG2, isotype IgG3 or isotype IgG4.

[0107] In some embodiments, the antibody, or antigen-binding fragment thereof, is an anti-chicken lysozyme human IgG1 isotype antibody.

[0108] In some embodiments, Tb is an antigen-binding fragment of an antibody that has the activity of binding a non-endocytosed (eg, ALCAM / CD166) antigen on the surface of a tumor cell.

[0109] In some embodiments, Tb is an antibody that binds to a tumor cell-associated antigen.

[0110] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has binding activity to a free antigen in tumor tissue or to an antigen on the surface of a tumor cell.

[0111] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has binding activity to free antigens in tumor tissue as well as antigens on the surface of tumor cells.

[0112] In some embodiments, Tb is an antibody or antigen-binding fragment thereof having B7H3-2Ig and / or B7H-4Ig.

[0113] In some embodiments, Tb is an antibody or antigen-binding fragment thereof that has a higher B7H3-4Ig binding activity than B7H3-2Ig binding activity.

[0114] In some embodiments of the present disclosure, Tb is a ligand or targeting moiety that binds to a target.

[0115] In some embodiments, the Tb target is selected from targets expressed by tumor cells higher than normal cells.

[0116] In some embodiments, the Tb target is selected from targets that are highly expressed in tumor cells and low in normal cells.

[0117] In some embodiments, the target of Tb is selected from: B7H3, CD20, CD19, CD30, GPNMB, Her2, Trop-2, EGFR, Her3, GD-2, CD79b and BCMA, etc.

[0118] In some embodiments, the Tb is an antibody or antigen-binding fragment thereof.

[0119] In some embodiments, Tb is a non-human antibody, a humanized antibody, a chimeric antibody, a fully human antibody.

[0120] In some embodiments, Tb is a monoclonal antibody, bispecific antibody or multispecific antibody.

[0121] In some embodiments, Tb is a monoclonal antibody or antigen-binding fragment thereof.

[0122] In some embodiments, Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, an anti-Trop-2 antibody or antigen-binding fragment thereof, an anti-Her2 antibody or antigen-binding fragment thereof, an anti-Her3 antibody or antigen-binding fragment thereof, or an anti-Her3 antibody or antigen-binding fragment thereof EGFR antibodies or antigen-binding fragments thereof.

[0123] In some embodiments, Tb is an anti-B7H3 antibody or antigen-binding fragment thereof, eg, 1D1, 1D1-01, 2E3, 2E3-02 antibody, enoblituzumab, mirzotamab, omburtamab or an antigen-binding fragment thereof.

[0124] The sequence of 1D1 is shown in SEQ ID NO:1. The VH sequence of the 1D1-01 is shown in SEQ ID NO:3, and the VL sequence is shown in SEQ ID NO:13. The heavy chain sequence of 1D1-01 is shown in SEQ ID NO:45, and the light chain sequence is shown in SEQ ID NO:46. The sequence of 2E3 is shown in SEQ ID NO:2. The VH sequence of the 2E3-02 is shown in SEQ ID NO:23, and the VL sequence is shown in SEQ ID NO:33. The heavy chain sequence of the 2E3-02 is shown in SEQ ID NO:47, and the light chain sequence is shown in SEQ ID NO:48.

[0125] In some embodiments, Tb is an anti-B7H3 monoclonal antibody or an antigen-binding fragment thereof.

[0126] In some embodiments, Tb is an anti-Trop-2 antibody or antigen-binding fragment thereof, such as datopotamab, sacituzumab or an antigen-binding fragment thereof.

[0127] In some embodiments, Tb is an anti-Trop-2 monoclonal antibody or antigen-binding fragment thereof.

[0128] In some embodiments, Tb is an anti-Her2 antibody or antigen-binding fragment thereof, such as anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, Trastuzumab, Pertuzumab or an antigen-binding fragment thereof.

[0129] In some embodiments, Tb is an anti-Her2 monoclonal antibody or an antigen-binding fragment thereof, such as trastuzumab, pertuzumab or an antigen-binding fragment thereof.

[0130] In some embodiments, Tb is an anti-Her3 antibody or antigen-binding fragment thereof, such as barecetamab, duligotuzumab, elgemtumab, istiratumab, lumretuzumab, patritumab, seribantumab, zenocutuzumab, the 202-2-1 antibody or an antigen-binding fragment thereof.

[0131] In some embodiments, Tb is an anti-Her3 monoclonal antibody or an antigen-binding fragment thereof.

[0132] In some embodiments, Tb is an anti-EGFR antibody or antigen-binding fragment thereof, such as demupitamab, depatuxizumab, futuximab, imgatuzumab, laprituximab, losatuximab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, zalutum umab , Cetuximab or an antigen-binding fragment thereof.

[0133] In some embodiments, Tb is an anti-EGFR monoclonal antibody or an antigen-binding fragment thereof.

[0134] In some embodiments, the antibody has an antibody that binds the antigen but does not have endocytic activity.

[0135] In some embodiments, the antibody has the ability to bind antigens such as B7H3, CD20, CD19, CD30, GPNMB, Her2, Trop-2, EGFR, Her3, GD-2, CD79b and BCMA but does not have endocytic activity Antibody.

[0136] In some embodiments, the antibody has an antibody that binds the B7H3 antigen but does not have endocytic activity. For example, INV721 and I7-01 of WO2021168379A1. More specifically, the Anti-B7H3 antibody has the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 1 described in WO2021168379A1.

[0137] In some embodiments, the antibody has an antibody that binds the GD2 antigen but does not have endocytic activity. For example, INV721 and GD2-5 of WO2021168379A1. More specifically, the Anti-GD2 antibody has the VH of SEQ ID NO:4 and the VL of SEQ ID NO:3 described in WO2021168379A1.

[0138] In some embodiments, the antibody has an antibody that binds to the HER3 antigen but does not have endocytic activity, such as the ANTI-HER3 21F06 antibody shown in SEQ ID NO: 22 of US10808032B2.

[0139] In some embodiments, the antibody has an antibody that binds the CD20 antigen but does not have endocytic activity. Although so-called "type II" CD20-specific antibodies have been shown to be poorly internalized by CD20-positive target cells, other so-called "type I" CD20-specific antibodies have been found to be internalized to some extent and degraded, depending on Initiates and inhibits FcγR expression levels on target cells it interacts with. In some embodiments, the antibody that binds the CD20 antigen but does not have endocytic activity is a type II "CD20-specific antibody, such as obinutuzumab.

[0140] In some embodiments, the antibody has antibody binding activity to a non-endocytic (eg, ALCAM / CD166) antigen. In some embodiments, the antibody comprises VH of SEQ ID No: 73 and VL of SEQ ID No: 74, VH of SEQ ID No: 75 and VL of SEQ ID No: 76 in EP3911682A1, SEQ ID No: 77 The VH of SEQ ID No:78 and the VL of SEQ ID No:78, or the antibody shown by the VH of SEQ ID No:79 and the VL of SEQ ID No:88.

[0141] In some embodiments, the Tb is a targeting moiety, such as a ligand, protein, polypeptide, non-proteinaceous agent (eg, sugar, RNA or DNA), antibody analog, and the like.

[0142] In some embodiments, q is selected from any value between 0.1-16.0; in preferred embodiments, q is selected from any integer between 0.1-16.0.

[0143] In some embodiments, q is selected from any value between 0.1-8.0, and in preferred embodiments, q is selected from any integer between 0.1-8.0.

[0144] In some embodiments, q is selected from any value between 2-8.

[0145] In some embodiments, q is selected from any value between 3-8.

[0146] In some embodiments, q is selected from any value between 4-8.

[0147] In some embodiments, q is selected from any value between 6-8.

[0148] In some embodiments, q is selected from any integer between 2-8.

[0149] In some embodiments, q is selected from any integer between 3-8.

[0150] In some embodiments, q is selected from any integer between 4-8.

[0151] In some embodiments, q is selected from any integer between 6-8.

[0152] In some embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0153] In some embodiments, q is selected from 2, 4, 6 and 8.

[0154] In the present disclosure, the biologically active molecular fragments refer to those known in the art, in antibody-drug conjugates (or antibody-drug conjugate (ADC)), in tumor tissue Occasionally or after linker cleavage / degradation / enzyme cleavage in tumor cells, it can form biologically active drugs (such as small molecule cytotoxic drugs, which include groups after losing one atom or atomic group) or their derivatives (such as Part (fragment or group) of its precursor). In order to avoid ambiguity, "drugs" do not only refer to "drugs" that have been approved by the medical regulatory authorities, but also include any molecule with potential therapeutic biological activity in clinical practice, or in research and development and academic research.

[0155] In some embodiments, D is a molecular fragment having anti-tumor biological activity.

[0156] In some embodiments, D is a molecular fragment with anti-tumor bioactivity, wherein said bioactive molecule is selected from cytotoxic agents or derivatives thereof, such as DNA topoisomerase inhibitors (such as camptothecins) Biologically active molecules such as camptothecin, DXD, substituent-modified camptothecin or substituent-modified DXD) or tubulin inhibitors (eg, MMAF-like tubulin inhibitors, MMAE-like tubulin inhibitors ).

[0157] In some embodiments, the ligand-drug conjugate has a structure of formula I: wherein, R1, R2 are each independently selected from H, halogen, -OH, optionally substituted C1-6 alkyl and any Optionally substituted C1-6 alkoxy, or, R1 and R2 together with the carbon atoms attached to them form a 5-7-membered carbocyclic ring or a 5-7-membered heterocyclic ring, and the heterocyclic ring contains 1 or more O, S , N, carbonyl, pyrenylene or pyrenyl or any combination thereof; R3 is selected from H, halogen, -OH, -NH2, optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, Alternatively, R3 and X together with the carbon atom connected to it form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring, and the heterocyclic ring contains 1 or more O, S, N, carbonyl, arylene or argon or any combination thereof, or, R3 and R2 form a 5-7-membered carbon ring or a 5-7-membered heterocyclic ring together with the carbon atoms connected to it, and the heterocyclic ring contains 1 or more O, S, N, carbonyl , arylene group or argenyl group or any combination thereof; W does not exist or exists, when W exists, W is selected from -O-, -S-, -NR4-,,,,,,,,, and, 1 position and X Linked, the 2-position is linked to L4 or L3; X is selected from direct bonds, optionally substituted -O-(CH2)n3-, -N(R4)-(CH2)n3-, -S-(CH2)n3-, Carbonyl-(CH2)n3, -SO2-(CH2)n3-,,,,-(CH2)n1-, C3-6 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl and 4-10 Membered heterocyclic group, the 1-position is connected to the parent ring, and the 2-position is connected to W or L4; the substituent is selected from one or more C1-4 alkyl, C3-6 cycloalkyl, or multiple C1-4 alkane The group and the carbon atom connected to them at the same time together form a C3-6 cycloalkyl group; each M is independently selected from a direct bond and -CR5aR5b-; R4, R5, R5a, R5b, R6, R7 are each independently selected from H, any Optionally substituted C1-4 alkyl, optionally substituted C1-4 alkoxy and optionally substituted C3-6 cycloalkyl; n, n', n1, n2, n3 are each independently selected from 0 to 6 Any integer between; L4 does not exist or exists, when L4 exists, L4 is selected from ,,,,,,,,,,, 1 position is connected to L3, 2 positions are connected to W or X.

[0158] Tb, L1, L2, L3 and q have the meanings provided above and in any embodiment specifically recited herein.

[0159] In some embodiments, R1, R2 are each independently selected from H, halogen, and C1-4 alkyl.

[0160] In some embodiments, R1 and R2, together with the carbon atoms attached thereto, form a 5-6 membered heterocycle containing 1, 2 or 3 O, S or N or any combination thereof.

[0161] In some embodiments, R1 is selected from H and halogen, and R2 is selected from H and C1-4 alkyl.

[0162] In some embodiments, R1 and R2 and the carbon atom to which they are attached form, or, the dotted line indicates the position where the heterocyclic ring is fused to the benzene ring.

[0163] In some embodiments, R1 is H or F and R2 is H or methyl.

[0164] In some embodiments, R1 is F, R2 is methyl or R1 and R2 are formed together with the carbon atom to which they are attached.

[0165] In some embodiments, R1 is F and R2 is methyl.

[0166] In some embodiments, R1 and R2 are formed together with the carbon atom to which they are attached.

[0167] In some embodiments, R3 is selected from H and C1-4 alkyl.

[0168] In some embodiments, R3 and X, together with the carbon atom to which they are attached, form a 5-6 membered carbocyclic ring.

[0169] In some embodiments, R3 is H or R3 and X are formed together with the carbon atom to which it is attached, and the dashed lines indicate where the carbocycle is fused to the benzene and pyridine rings.

[0170] In some embodiments, R3 is H.

[0171] In some embodiments, W does not exist or exists, when W exists, W is selected from -O-, -S-, -NR4-, ,,,,,, and, 1 position is connected to X, 2 positions Connect to L4 or L3.

[0172] In some embodiments, W does not exist or exists, when W exists, W is selected from -O-, -S-, -NR4-, ,, and, 1 position is connected to X, 2 positions are connected to L4 or L3 connected.

[0173] In some embodiments, W is selected from -O-, -NR4-, and X is linked at position 1 and L4 or L3 at position 2.

[0174] In some embodiments, W is selected from -O- and -NR4-, 1 position is linked to X, and 2 positions are linked to L4 or L3.

[0175] In some embodiments, X is selected from optionally substituted -(CH2)n1-,,,, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl, 1 position It is connected to the parent ring, and the 2-position is connected to W or L4; the substituent is selected from 1 or 2 C1-4 alkyl groups, or 2 C1-4 alkyl groups and the carbon atoms connected to them at the same time form a C3- 6 cycloalkyl.

[0176] In some embodiments, X is selected from optionally substituted,,,,,,,,,,,,,,,,,,, and, the 1 position is connected to the parent ring, and the 2 position is connected to W or L4; The substituent is selected from 1 or 2 C1-4 alkyl groups (such as methyl), or 2 C1-4 alkyl groups (such as methyl) and the carbon atoms connected to them at the same time to form a C3-6 cycloalkane groups (such as cyclopropyl).

[0177] In some embodiments, X is selected from the group consisting of ,,,,,,,,,,,,,,,,,,,, and, the 1st position is connected to the parent ring, and the 2nd position is connected to W or L4.

[0178] In some embodiments, X is selected from the group consisting of , , , and , and the 1st position is connected to the parent ring, and the 2nd position is connected to W.

[0179] In some embodiments, when W does not exist, X is selected from , , and, 1 position is connected to the parent ring, and 2 position is connected to L4; when W exists, X is selected from , , , , , , , ,,,,,, and, 1 bit is connected to the mother ring, and 2 bits are connected to W.

[0180] In some embodiments, W is selected from -O-, -NR4- and, 1 position X is connected, 2 positions are connected to L4 or L3; X is selected from , , and, 1 position is connected to the parent ring, 2 positions Connect to W.

[0181] In some embodiments, R4, R5 are each independently selected from H, C1-4 alkyl and C3-6 cycloalkyl.

[0182] In some embodiments, each R4 is independently selected from H, C1-4 alkyl, and C3-6 cycloalkyl, and R5 is H.

[0183] In some embodiments, each R4 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, tert-butyl, and cyclopropyl, and R5 is H.

[0184] In some embodiments, R5a, R5b are each independently selected from H and C1-4 alkyl.

[0185] In some embodiments, R5a, R5b are each independently selected from H and methyl.

[0186] In some embodiments, each R7 is independently selected from H and C1-4 alkyl.

[0187] In some embodiments, R7 is H.

[0188] In some embodiments, n is selected from 1, 2, and 3.

[0189] In some embodiments, n is 1.

[0190] In some embodiments, n1 is selected from 1, 2, 3 and 4.

[0191] In some embodiments, n2 is 1.

[0192] In some embodiments, n3 is zero.

[0193] In some embodiments, L3 is selected from the group consisting of,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, and, X -is selected from halide ion, carboxylate ion, sulfate ion, bisulfate ion and OH-, the 1st position is connected to L1 or L2, and the 2nd position is connected to L4 or W.

[0194] In some embodiments, L3 is selected from ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, and, X-is selected from halide ion, Carboxylate ion, sulfate ion, bisulfate ion and OH-, the 1st position is connected to L1 or L2, and the 2nd position is connected to L4 or W.

[0195] In some embodiments, L3 is selected from ,,,,,,,,,,, and, X-is selected from halide ion, carboxylate ion, sulfate ion and bisulfate ion, OH-, 1 position Connect to L1 or L2, 2 bits to L4 or W.

[0196] In some embodiments, L3 is selected from the group consisting of , , , , , , , and , and position 1 is connected to L1 or L2, and position 2 is connected to L4 or W.

[0197] In some embodiments, L3 is selected from , , , , , , , , , , , , , , , , , , , 1 position is connected to L1 or L2, and 2 positions are connected to L4 or W.

[0198] In some embodiments, L4 is absent or present, and when L4 is present, L4 is selected from the group consisting of , , and , the 1st position is linked to L3, and the 2nd position is linked to W or X.

[0199] In some embodiments, L4 is absent or present, and when L4 is present, L4 is or, position 1 is linked to L3, position 2 is linked to W or X.

[0200] In some embodiments, L4 is absent.

[0201] In some embodiments, L4 is selected from and, position 1 is linked to L3, and position 2 is linked to W or X.

[0202] In some embodiments, L4 is selected from the group consisting of linking L3 at position 1 and W or X at position 2.

[0203] It should be noted that, as mentioned above, W does not exist or exists, thus, when W does not exist, 1 bit of L4 is connected to L3, and 2 bits are connected to X; when W exists, 1 bit of L4 is connected to L3 is connected, and 2 bits are connected to W. The following connection relationship about L4 can be understood with reference to the foregoing content.

[0204] In some embodiments, the structure of is selected from the following structural fragments: Among them, 1 bit is connected to Tb, and 2 bits are connected to W.

[0205] In some embodiments, D is a structural fragment as shown; position 1 is connected to L3 or L4; eg, , .

[0206] In some embodiments, the structure of is selected from the following structural fragments: wherein position 1 is linked to L4; when L4 is absent, position 1 is linked to L3.

[0207] In some embodiments, W is absent or present, and when W is present, W is selected from -O-, -S-, -NR4-, and; for example absent, -O-, -NR4- or; R4 and R5 are each independently selected from H and C1-4 alkyl; n is independently selected from 0, 1, 2, 3 and 4; X is selected from ,,,,,,,,,,,,,,,,,, ,,,,,,,,and;

[0208] R1 is selected from H, halogen, R2 is selected from H, C1-4 alkyl, or, R1 and R2 form with the carbon atom connected to it, or, the dotted line represents the position where the heterocyclic ring is fused with the benzene ring ; R3 is selected from H and C1-4 alkyl, or, R3 and X form a 5-6 membered carbon ring together with the carbon atom connected to it; preferably, W does not exist or exists, when W exists, W is selected from -O -, -NR4- (eg -NH-, -N(CH3)-, -N(C2H5)-), and; R4 independently selected from H, methyl, ethyl, isopropyl, n-propyl, Tributyl and cyclopropyl; When W does not exist, X is selected from ,, and, 1 position is connected to the parent ring; When W exists, X is selected from ,,,,,,,,,,,,,,,, and In AA1, r is selected from 0, 1, 2, 3, 4 and 5; in Ra, Rb, any one is H, and the other is selected from; or, Ra and Rb and the carbon atoms connected to them together form A 5-6-membered heterocyclic ring substituted by R0; R1 is selected from H, halogen, R2 is selected from H, C1-4 alkyl, or, R1 and R2 are formed with carbon atoms connected to it; R3 is selected from H and C1-4 Alkyl, or, R3 and X and the carbon atom connected to it form together R3 and X and the carbon atom connected to it form together; more preferably, W is selected from -O- and -NR4-, 1 position X is connected, Linked to L4 or L3; X is selected from ,,,, and, the 1st position is connected to the mother ring, and the 2nd position is connected to W; for example, ,,,,,,,,; R1 is F, R2 is methyl or R1 and R2 together with the carbon atom to which it is attached; R3 is H.

[0209] In some embodiments, the ligand drug conjugate has the structure of formula I-1: wherein, Tb, L1, L2, L3, L4, X, R1, R2, R3, R4 and q have the above meaning provided herein and in any embodiment specifically recited herein.

[0210] In some embodiments, the ligand drug conjugate has the structure of formula I-1A or I-1B: wherein, Tb, L2, L3, L4, X, R1, R2, R3, R4 and q have The meaning given above and in any embodiment specifically recited herein.

[0211] In some embodiments, the ligand drug conjugate has the structure of formula I-2: wherein, Tb, L1, L2, L3, L4, X, R1, R2, R3 and q have the above and herein meaning provided in any embodiment specifically recited.

[0212] In some embodiments, the ligand drug conjugate has the structure of formula I-2A or I-2B: wherein, Tb, L2, L3, L4, X, R1, R2, R3 and q have the above meaning provided herein and in any embodiment specifically recited herein.

[0213] In some embodiments, the ligand drug conjugate has the structure of formula 1-3: wherein, Tb, L1, L2, L3, L4, X, R1, R2, R3, R4, R5, n and q has the meaning provided above and in any embodiment specifically recited herein.

[0214] In some embodiments, the ligand drug conjugate has the structure of formula I-3A or I-3B: , wherein, Tb, L2, L3, L4, X, R1, R2, R3, R4 and q has the meaning provided above and in any embodiment specifically recited herein.

[0215] In some embodiments, the Ligand Drug Conjugate has the structure of Formula I-A: wherein Tb, X, R1, R2, R3, Ra, Rb, and q have any of the embodiments described above and specifically described herein The meaning provided in the scheme.

[0216] In some embodiments, the Ligand Drug Conjugate has the structure of Formula I-B: wherein Tb, X, R1, R2, R3, Ra, Rb, and q have any of the embodiments described above and specifically herein The meaning provided in the scheme.

[0217] In some embodiments, the Ligand Drug Conjugate is selected from the following: .

[0218] The Tb antibody or antigen-binding fragment thereof can be prepared by various methods known in the art, for example, by genetic engineering and recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the disclosed antibodies are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions and express the antibodies of the present disclosure.

[0219] In some embodiments of the present disclosure, the targeting moiety is an anti-B7H3 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-B7H3 antibody includes all anti-B7H3 antibodies in the prior art, such as enoblituzumab, mirzotamab, omburtamab, and see CN112521512, WO2021027674, WO2021021543, WO2021006619, CN111662384, CN111454357, WO20 20151384, WO2020140094, WO2020103100, WO2020102779 , WO2020063673, WO2020047257, WO2020041626, CN110684790, CN110642948, WO2019225787, WO2019226017, US20190338030, CN110305213, WO2018209346, WO 2018177393, US9150656, WO2016106004, WO2016044383, WO2016033225, WO2015181267, US20120294796, WO2011109400, CN101104639, WO2004093894, WO200201 0187, Anti-B7H3 antibody in WO2001018021. In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof is selected from the group consisting of antibody numbered M30-H1-L4 in CN103687945B and antibody numbered mAb-C-DUBA in CN109069633A.

[0220] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs), wherein the CDRs are defined by the IMGT numbering system: (a) the sequence is SEQ HCDR1 of ID NO: 10, the sequence is HCDR2 of SEQ ID NO: 11, the sequence is HCDR3 of SEQ ID NO: 12; and / or, the sequence is LCDR1 of SEQ ID NO: 20, the sequence is LCDR2 of GTF, the sequence is SEQ ID NO: 20 LCDR3 of ID NO:22; (b) the sequence is HCDR1 of SEQ ID NO:10, the sequence is HCDR2 of SEQ ID NO:11, the sequence is HCDR3 of SEQ ID NO:12; and / or, the sequence is SEQ ID NO: LCDR1 of 40, the sequence is LCDR2 of GAS, and the sequence is LCDR3 of SEQ ID NO:42; (c) the sequence is HCDR1 of SEQ ID NO:30, the sequence is HCDR2 of SEQ ID NO:31, and the sequence is SEQ ID NO:32 and / or, the sequence is the LCDR1 of SEQ ID NO:40, the sequence is the LCDR2 of GAS, the sequence is the LCDR3 of SEQ ID NO:42; or (d) the sequence is the HCDR1 of SEQ ID NO:30, and the sequence is SEQ HCDR2 of ID NO:31, HCDR3 of SEQ ID NO:32; and / or LCDR1 of SEQ ID NO:20, LCDR2 of GTF, LCDR3 of SEQ ID NO:22.

[0221] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof, as defined by the IMGT numbering system, comprises: VH comprising (a) heavy chain HCDR1, HCDR2, HCDR3 and comprising (a) light chain LCDR1 , LCDR2, LCDR3 VL; VH containing heavy chain HCDR1, HCDR2, HCDR3 in (b) and VL containing light chain LCDR1, LCDR2, LCDR3 in (b); VH containing heavy chain HCDR1, HCDR2, HCDR3 in (c) And VL containing (c) light chain LCDR1, LCDR2, LCDR3; or (d) VH containing heavy chain HCDR1, HCDR2, HCDR3 and (d) VL containing light chain LCDR1, LCDR2, LCDR3.

[0222] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs), wherein the CDRs are defined according to the Chothia numbering system: (a) the sequence is HCDR1 of SEQ ID NO:4, The sequence is HCDR2 of SEQ ID NO:5, the sequence is HCDR3 of SEQ ID NO:6; and / or, the sequence is LCDR1 of SEQ ID NO:14, the sequence is LCDR2 of SEQ ID NO:15, the sequence is SEQ ID NO: 16 LCDR3; (b) the sequence is HCDR1 of SEQ ID NO:24, the sequence is HCDR2 of SEQ ID NO:25, the sequence is HCDR3 of SEQ ID NO:26; and / or, the sequence is LCDR1 of SEQ ID NO:34 , the sequence is LCDR2 of SEQ ID NO:35, the sequence is LCDR3 of SEQ ID NO:36; (c) the sequence is HCDR1 of SEQ ID NO:4, the sequence is HCDR2 of SEQ ID NO:5, and the sequence is SEQ ID NO: 6's HCDR3; and / or, the sequence is LCDR1 of SEQ ID NO:34, the sequence is LCDR2 of SEQ ID NO:35, the sequence is LCDR3 of SEQ ID NO:36; or (d) the sequence is SEQ ID NO:24 HCDR1, the sequence is HCDR2 of SEQ ID NO:25, the sequence is HCDR3 of SEQ ID NO:26; and / or, the sequence is LCDR1 of SEQ ID NO:14, the sequence is LCDR2 of SEQ ID NO:15, the sequence is SEQ ID NO:16 LCDR3.

[0223] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof, as defined by the Chothia numbering system, comprises: VH comprising (a) heavy chain HCDR1, HCDR2, HCDR3 and comprising (a) light chain LCDR1 , LCDR2, LCDR3 VL; VH containing heavy chain HCDR1, HCDR2, HCDR3 in (b) and VL containing light chain LCDR1, LCDR2, LCDR3 in (b); VH containing heavy chain HCDR1, HCDR2, HCDR3 in (c) And VL containing (c) light chain LCDR1, LCDR2, LCDR3; or (d) VH containing heavy chain HCDR1, HCDR2, HCDR3 and (d) VL containing light chain LCDR1, LCDR2, LCDR3.

[0224] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs), wherein the CDRs are defined by the kabat numbering system: (a) the sequence is HCDR1 of SEQ ID NO:7, The sequence is HCDR2 of SEQ ID NO:8, the sequence is HCDR3 of SEQ ID NO:9; and / or, the sequence is LCDR1 of SEQ ID NO:17, the sequence is LCDR2 of SEQ ID NO:18, the sequence is SEQ ID NO: LCDR3 of 19; (b) sequence is HCDR1 of SEQ ID NO:27, sequence is HCDR2 of SEQ ID NO:28, sequence is HCDR3 of SEQ ID NO:29; and / or, sequence is LCDR1 of SEQ ID NO:37 , the sequence is LCDR2 of SEQ ID NO:38, the sequence is LCDR3 of SEQ ID NO:39; (c) the sequence is HCDR1 of SEQ ID NO:7, the sequence is HCDR2 of SEQ ID NO:8, and the sequence is SEQ ID NO: 9 HCDR3; and / or, the sequence is LCDR1 of SEQ ID NO:37, the sequence is LCDR2 of SEQ ID NO:38, the sequence is LCDR3 of SEQ ID NO:39; or (d) the sequence is SEQ ID NO:27 HCDR1, the sequence is HCDR2 of SEQ ID NO:28, the sequence is HCDR3 of SEQ ID NO:29; and / or, the sequence is LCDR1 of SEQ ID NO:17, the sequence is LCDR2 of SEQ ID NO:18, the sequence is SEQ ID NO:19 LCDR3.

[0225] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof, defined by the kabat numbering system, comprises: VH comprising (a) heavy chain HCDR1, HCDR2, HCDR3 and comprising (a) light chain LCDR1 , LCDR2, LCDR3 VL; VH containing heavy chain HCDR1, HCDR2, HCDR3 in (b) and VL containing light chain LCDR1, LCDR2, LCDR3 in (b); VH containing heavy chain HCDR1, HCDR2, HCDR3 in (c) And VL containing (c) light chain LCDR1, LCDR2, LCDR3; or (d) VH containing heavy chain HCDR1, HCDR2, HCDR3 and (d) VL containing light chain LCDR1, LCDR2, LCDR3.

[0226] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NO : the sequence shown in 3 or 23; (ii) compared with the sequence shown in SEQ ID NO: 3 or 23, there is one or several amino acid substitutions, deletions or additions or any combination thereof (such as 1, 2 , 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) with SEQ ID NO :3 or 23 compared to the sequence shown in at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% , a sequence of at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: ( iv) the sequence shown in SEQ ID NO: 13 or 33; (v) compared with the sequence shown in SEQ ID NO: 13 or 33, there is one or several amino acid substitutions, deletions or additions or any combination thereof ( For example, a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi ) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% compared to the sequence shown in SEQ ID NO: 13 or 33 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0227] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises the VH set forth in SEQ ID NO:3, and / or, the VL set forth in SEQ ID NO:13.

[0228] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises the VH set forth in SEQ ID NO:23, and / or, the VL set forth in SEQ ID NO:33.

[0229] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises the VH set forth in SEQ ID NO:3, and / or, the VL set forth in SEQ ID NO:33.

[0230] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises the VH shown in SEQ ID NO:23, and / or, the VL shown in SEQ ID NO:13.

[0231] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises: (a) VH of the sequence shown in SEQ ID NO:3 and VL of the sequence shown in SEQ ID NO:13; (b) SEQ ID NO: The VH of the sequence shown in ID NO:23 and the VL of the sequence shown in SEQ ID NO:33; (c) the VH of the sequence shown in SEQ ID NO:3 and the VL of the sequence shown in SEQ ID NO:13; (d) SEQ ID NO: VH of the sequence shown in ID NO:23 and the VL of the sequence shown in SEQ ID NO:13; (e) heavy chain variable region (VH) and light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) independently have at least 70%, at least 80%, at least 85%, at least 90% compared with the VH and VL described in any group (a) to (f). %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (f) heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) are the same as the VH described in any one of (a) to (d) Compared with VL respectively, there are independently one or several amino acid substitutions, deletions or additions or any combination thereof (such as 1, 2, 3, 4, 5, 6, 7, 8 1, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof). Preferably, said substitution is a conservative substitution. In some embodiments, the heavy chain of the anti-B7H3 antibody comprises a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof having a Conservative substitutions of up to 50 amino acids (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids Conservative substitutions; eg, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids). In certain embodiments, the light chain of the anti-B7H3 antibody comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having at most 50 Conservative substitutions of amino acids (e.g., conservative substitutions of at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids; eg conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).

[0233] In some embodiments, the constant region is altered, e.g., mutated, to modify the properties of the anti-B7H3 antibody molecule (e.g., alter one or more of the following properties: Fc receptor binding, antibody glycosylation, cysteine amino acid residues, effector cell function or complement function). Effector function can be altered by substituting at least one amino acid residue in the constant region of the antibody with a different residue, e.g., altering the affinity of the antibody for an effector ligand such as FcR or complement C1q ( e.g. lower). The Fc region of an antibody mediates several important effector functions such as ADCC, phagocytosis (ADCP), CDC, etc. In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof has a heavy chain constant region (CH) selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. Heavy chain constant region; in particular a heavy chain constant region selected from eg IgGl, IgG2, IgG3 and IgG4, more particularly a heavy chain constant region selected from IgGl (eg human IgGl). In some embodiments, the human IgG1 heavy chain constant region is set forth in SEQ ID NO:43. In some embodiments, an antibody of the disclosure, or an antigen-binding fragment thereof, has a light chain constant region selected from, for example, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (eg, a human kappa light chain constant region). In some embodiments, the light chain constant region has the sequence set forth in SEQ ID NO:44.

[0235] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises CH shown in SEQ ID NO: 43 or a variant thereof having up to 20 CHs as compared to SEQ ID NO: 43 Conservative substitutions of amino acids (e.g. conservative substitutions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g. 1, 2, 3, 4, 5, 6, 7 8, 9 or 10 amino acid conservative substitutions), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% compared to SEQ ID NO:43 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0236] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises a light chain constant region or a variant thereof. In some embodiments, the light chain constant region comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises a light chain constant region (CL) set forth in SEQ ID NO: 44, or a variant thereof having up to 20 amines compared to SEQ ID NO: 44 Conservative substitutions of amino acids (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 amino acid conservative substitutions), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% compared to SEQ ID NO:44 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0237] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown in SEQ ID NO: 43 and a light chain constant region (CL) shown in SEQ ID NO: 44 ).

[0238] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain comprising an amino acid sequence selected from the group consisting of: (i) comprising the amino acid sequence shown in SEQ ID NO:3 The sequence of the VH sequence and the CH sequence shown in SEQ ID NO:43; (ii) compared with the sequence shown in (i), there is one or several amino acid substitutions, deletions or additions or any combination thereof (for example, at most 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions or any of them Combinations; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence of at least 97%, at least 98%, or at least 99% sequence identity; and (b) a light chain comprising an amino acid sequence selected from the group consisting of: (iv) comprising the VL sequence shown in SEQ ID NO:13 and the sequence of the CL sequence shown in SEQ ID NO:44; (v) has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50 amino acids) compared to the sequence shown in (iv) , up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; For example, a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi ) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% compared to the sequence shown in (iv) %, at least 98%, or at least 99% sequence identity.

[0239] In some embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0240] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain comprising an amino acid sequence selected from the group consisting of: (i) comprising the amino acid sequence shown in SEQ ID NO:23 The sequence of the VH sequence and the CH sequence shown in SEQ ID NO:43; (ii) compared with the sequence shown in (i), there is one or several amino acid substitutions, deletions or additions or any combination thereof (for example, at most 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions or any of them Combinations; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence of at least 97%, at least 98%, or at least 99% sequence identity; and (b) a light chain comprising an amino acid sequence selected from the group consisting of: (iv) comprising the VL sequence shown in SEQ ID NO:33 and the sequence of the CL sequence shown in SEQ ID NO:44; (v) has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50 amino acids) compared to the sequence shown in (iv) , up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; For example, a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi ) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% compared to the sequence shown in (iv) %, at least 98%, or at least 99% sequence identity.

[0241] In some embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0242] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain comprising: (i) the sequence shown in SEQ ID NO: 45; (ii) with ( i) substitutions, deletions or additions of one or several amino acids or any combination thereof (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25) compared to the indicated sequence substitutions, deletions or additions of, at most 20, at most 15, at most 10 or at most 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) has at least 80%, at least 85%, at least A sequence of 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and the light chain Comprising: (iv) the sequence shown in SEQ ID NO:46; (v) compared with the sequence shown in (iv), there is one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50 Substitutions, deletions, or additions of, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids, or any combination thereof ; such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or ( vi) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% of the sequence shown in (iv) , sequences with at least 98% or at least 99% sequence identity; preferably, the substitutions described in (ii) or (v) are conservative substitutions.

[0243] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain comprising: (i) the sequence shown in SEQ ID NO: 47; (ii) with ( i) substitutions, deletions or additions of one or several amino acids or any combination thereof (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25) compared to the indicated sequence substitutions, deletions or additions of, at most 20, at most 15, at most 10 or at most 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) has at least 80%, at least 85%, at least A sequence of 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and the light chain Comprising: (iv) the sequence shown in SEQ ID NO:48; (v) compared with the sequence shown in (iv), there is one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50 Substitutions, deletions, or additions of, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids, or any combination thereof ; such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or ( vi) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% of the sequence shown in (iv) , sequences with at least 98% or at least 99% sequence identity; preferably, the substitutions described in (ii) or (v) are conservative substitutions.

[0244] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody or a fully human antibody.

[0245] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof is selected from scFv, Fab, Fab', (Fab')2, Fv fragments, disulfide-linked Fv (dsFv), diabodies ( diabody).

[0246] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof is a scFv. In certain embodiments, the scFv of the present disclosure comprises: (a) VH of the sequence shown in SEQ ID NO:3 and VL of the sequence shown in SEQ ID NO:13; (b) VL of the sequence shown in SEQ ID NO:23 VH and the VL of the sequence shown in SEQ ID NO:33; (c) the VH of the sequence shown in SEQ ID NO:3 and the VL of the sequence shown in SEQ ID NO:33; (d) the sequence shown in SEQ ID NO:23 VH and VL of the sequence shown in SEQ ID NO: 13; (e) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable Region (VL) independently has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% compared with VH and VL described in any group (a) to (d). %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (f) the heavy chain variable region (VH) and the light chain may Variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) are compared with VH and VL described in any one of (a) to (d), respectively, independently Substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amines amino acid substitutions, deletions or additions or any combination thereof). Preferably, said substitution is a conservative substitution.

[0247] In some embodiments, an antibody of the disclosure is a scFv. In certain embodiments, the scFv of the present disclosure comprises: (i) a sequence shown in SEQ ID NO: 1 or 2; (ii) a sequence having one or more amino acids compared to the sequence shown in (i). Substitutions, deletions or additions or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 Substitution, deletion or addition of amino acids or any combination thereof; for example, substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, deletion or addition or any combination thereof); or (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% of the sequence shown in (i) %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; preferably, the substitutions described in (ii) are conservative substitutions.

[0248] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof is a scFv. In certain embodiments, a scFv of the disclosure comprises the sequence set forth in SEQ ID NO: 1 or 2.

[0249] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof is selected from the group consisting of: 1D1 antibody, 1D1-01 antibody, 2E3 antibody, and 2E3-02 antibody.

[0250] In some embodiments of the present disclosure, the targeting moiety is Trastuzumab or Pertuzumab. Trastuzumab is an anti-Her2 monoclonal antibody, its amino acid sequence is known to those with ordinary knowledge in the technical field of the present invention, its schematic sequence can be found in, for example, CN103319599, the last Lys is easily deleted but Does not affect biological activity, see Dick, L. W. et al., Biotechnol. Bioeng., 100: 1132-1143.

[0251] Exemplary heavy and light chain sequences of trastuzumab can be found, eg, in IMGT / mAb-DB ID97. Exemplary heavy and light chain sequences of Pertuzumab can be found in SEQ ID No. 16 and SEQ ID No. 15 of US7560111, and also in IMGT / mAb-DB ID 80.

[0252] In some embodiments of the present disclosure, the targeting moiety is an anti-Her3 antibody or an antigen-binding fragment thereof. In some embodiments, the anti-Her3 antibody includes all prior art anti-Her3 antibodies, for example, see antibodies barecetamab, duligotuzumab, elgemtumab, istiratumab, lumretuzumab, patritumab, seribantumab, zenocutuzumab, 202-2-1 antibody, antibody CN 103189392 B The antibody shown in heavy chain SEQ ID NO: 10 and light chain SEQ ID NO: 14 and the antibody shown in IMGT / mAb-DB NO: 546.

[0253] In some embodiments, the targeting moiety is an anti-Her3 antibody, an antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) as follows, wherein the CDRs are according to the IMGT numbering system Definition: the sequence is HCDR1 of SEQ ID NO:56, the sequence is HCDR2 of SEQ ID NO:57, the sequence is HCDR3 of SEQ ID NO:58; and / or, the sequence is LCDR1 of SEQ ID NO:41, the sequence is AAS LCDR2, the sequence is LCDR3 of SEQ ID NO:21.

[0254] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof, as defined by the IMGT numbering system, comprises: the above-mentioned VH containing the above-mentioned heavy chain HCDR1, HCDR2, HCDR3 and the above-mentioned light chain LCDR1, LCDR2, LCDR3 The VL.

[0255] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof comprises an antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR), wherein the CDR is defined by the kabat numbering system: The sequence is SEQ ID NO: HCDR1 of 53, the sequence is HCDR2 of SEQ ID NO:54, the sequence is HCDR3 of SEQ ID NO:55; and / or, the sequence is LCDR1 of SEQ ID NO:59, the sequence is LCDR2 of SEQ ID NO:60, the sequence is LCDR3 of SEQ ID NO:21.

[0256] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof, defined according to the kabat numbering system, comprises: the above-mentioned VH containing the above-mentioned heavy chain HCDR1, HCDR2, and HCDR3 and the above-mentioned VH containing the above-mentioned light chain LCDR1, LCDR2, LCDR3 The VL.

[0257] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NO : the sequence shown in 49; (ii) compared with the sequence shown in SEQ ID NO: 49, there is one or several amino acid substitutions, deletions or additions or any combination thereof (such as 1, 2, 3 , 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) the sequence shown in SEQ ID NO:49 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least A sequence of 97%, at least 98%, or at least 99% sequence identity; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: (iv) SEQ ID NO: The sequence shown in 50; (v) has one or several amino acid substitutions, deletions or additions or any combination thereof (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi) the sequence shown in SEQ ID NO:50 The sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% %, at least 98%, or at least 99% sequence identity.

[0258] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof comprises: (a) VH of the sequence shown in SEQ ID NO:49 and VL of the sequence shown in SEQ ID NO:50; (b) heavy chain variable region (VH) and light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) are independently related to the VH and VL compared with at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; or (c) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) independently has one or several amino acid substitutions, deletions or additions or any combination thereof (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof). Preferably, said substitution is a conservative substitution.

[0259] In some embodiments, the heavy chain of the anti-Her3 antibody comprises a human immunoglobulin heavy chain constant region (CH) or a variant thereof having a Conservative substitutions of up to 50 amino acids (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids Conservative substitutions; eg, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids). In certain embodiments, the light chain of the anti-B7H3 antibody comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having at most 50 Conservative substitutions of amino acids (e.g., conservative substitutions of at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids; eg conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).

[0260] In some embodiments, the constant region is altered, e.g., mutated, to modify the properties of the anti-Her3 antibody molecule (e.g., alter one or more of the following properties: Fc receptor binding, antibody glycosylation, cysteine amino acid residues, effector cell function or complement function). Effector function can be altered by substituting at least one amino acid residue in the constant region of the antibody with a different residue, e.g., altering the affinity of the antibody for an effector ligand such as FcR or complement C1q ( e.g. lower). The Fc region of an antibody mediates several important effector functions such as ADCC, phagocytosis (ADCP), CDC, etc.

[0261] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof has a heavy chain constant region (CH) selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. Heavy chain constant region; in particular a heavy chain constant region selected from eg IgGl, IgG2, IgG3 and IgG4, more particularly a heavy chain constant region selected from IgGl (eg human IgGl). In some embodiments, an antibody of the disclosure, or an antigen-binding fragment thereof, has a light chain constant region selected from, for example, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (eg, a human kappa light chain constant region).

[0262] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown in SEQ ID NO: 43 and a light chain constant region (CL) shown in SEQ ID NO: 44 ).

[0263] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain comprising: (i) the sequence shown in SEQ ID NO: 51; (ii) with ( i) substitutions, deletions or additions of one or several amino acids or any combination thereof (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25) compared to the indicated sequence substitutions, deletions or additions of, at most 20, at most 15, at most 10 or at most 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) has at least 80%, at least 85%, at least A sequence of 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and the light chain Comprising: (iv) the sequence shown in SEQ ID NO:52; (v) compared with the sequence shown in (iv), there is one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50 Substitutions, deletions, or additions of, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids, or any combination thereof ; such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or ( vi) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% of the sequence shown in (iv) , sequences with at least 98% or at least 99% sequence identity; preferably, the substitutions described in (ii) or (v) are conservative substitutions.

[0264] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody or a fully human antibody.

[0265] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof is selected from scFv, Fab, Fab', (Fab')2, Fv fragments, disulfide bonded Fv (dsFv), diabodies ( diabody).

[0266] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof is a scFv. In certain embodiments, the scFv of the present disclosure comprises: (a) VH of the sequence shown in SEQ ID NO:49 and VL of the sequence shown in SEQ ID NO:50; (b) heavy chain variable region (VH) and A light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) independently have at least 70% of said VH and VL in group (a) respectively , at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence identity; or (c) heavy chain variable region (VH) and light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) are identical to (a) set Compared with VH and VL described in, independently have one or several amino acid substitutions, deletions or additions or any combination thereof (such as 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof). Preferably, said substitution is a conservative substitution.

[0267] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof is selected from: the 202-2-1 antibody.

[0268] In some embodiments of the present disclosure, the targeting moiety is Cetuximab. Cetuximab is an anti-EGFR monoclonal antibody, its amino acid sequence is known to those with ordinary knowledge in the technical field of the present invention, and its schematic sequence can be found in the antibody shown in IMGT / mAb-DB ID 151 .

[0269] In some embodiments, the ligand drug conjugate is selected from: wherein, Tb1 is an anti-B7H3 antibody or an antigen-binding fragment thereof, such as enoblituzumab, mirzotamab, omburtamab, 1D1-01, 2E3-02 antibody, preferably 1D1-01 or 2E3-02 antibody; q is selected from any value between 0.1-16.0, preferably any value between 2-8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0270] In some embodiments, the ligand drug conjugate is selected from: wherein, Tb2 is an anti-Trop-2 antibody or an antigen-binding fragment thereof, such as datopotamab, sacituzumab, preferably sacituzumab; q is selected from 0.1-16.0 Any value between, preferably any value between 2-8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0271] In some embodiments, the ligand drug conjugate is selected from: wherein Tb3 is an anti-Her2 antibody or an antigen-binding fragment thereof, such as anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, Trastuzumab, Pertuzumab, preferably Trastuzumab or Pertuzumab; q is selected from any value between 0.1-16.0, preferably any value between 2-8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0272] In some embodiments, the ligand drug conjugate is selected from: wherein Tb4 is an anti-Her3 antibody or an antigen-binding fragment thereof, for example, the antibody barecetamab, duligotuzumab, elgemtumab, istiratumab, lumretuzumab, patritumab, seribantumab, zenocutuzumab, 202-2-1 antibody, the antibody shown in CN 103189392 B heavy chain SEQ: 10 and light chain SEQ: 14, the antibody shown in IMGT / mAb-DB ID: 564, 202-2-1 antibody; preferred 202-2-1 antibody; q is selected from any value between 0.1-16.0, preferably any value between 2-8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0273] In some embodiments, the ligand drug conjugate is selected from: wherein Tb5 is an anti-EGFR antibody or an antigen-binding fragment thereof, such as demupitamab, depatuxizumab, futuximab, imgatuzumab, laprituximab, losatuximab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, touzotuximab, zalutumumab, cetuximab; preferably cetuximab; q is selected from any value between 0.1-16.0, preferably any value between 2-8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0274] In some embodiments, the ligand drug conjugate is selected from: wherein Tb6 is an antibody that has no tumor cell endocytosis activity or an antibody that binds non-endocytosis (such as ALCAM / CD166) antigen activity, for example: There is no IgG isotype antibody corresponding to the cell surface antigen in the human body, and the anti-CD166 antibody is preferably an anti-chicken lysozyme human IgG1 isotype antibody; q is selected from any value between 0.1-16.0, preferably any value between 2-8 value. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0275] In some embodiments, the ligand drug conjugate is selected from: wherein, Tb7 is an antibody with weak or no tumor cell endocytic activity, but has tumor cell surface antigen binding activity; q is selected from 0.1- Any value between 16.0, preferably any value between 2-8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0276] In some embodiments, the antibody has an antibody that binds the B7H3 antigen but does not have endocytic activity. For example, INV721 and I7-01 of WO2021168379A1. More specifically, the Anti-B7H3 antibody has the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 1 described in WO2021168379A1.

[0277] In some embodiments, the antibody has an antibody that binds the GD-2 antigen but does not have endocytic activity. For example, INV721 and GD2-5 of WO2021168379A1. More specifically, the Anti-GD-2 antibody has the VH of SEQ ID NO:4 and the VL of SEQ ID NO:3 described in WO2021168379A1.

[0278] In some embodiments, the antibody has an antibody that binds to the Her3 antigen but does not have endocytic activity, such as the ANTI-Her3 21F06 antibody shown in SEQ ID NO: 22 of US10808032B2.

[0279] In some embodiments, the antibody has an antibody that binds the CD20 antigen but does not have endocytic activity. Although so-called "type II" CD20-specific antibodies have been shown to be poorly internalized by CD20-positive target cells, other so-called "type I" CD20-specific antibodies have been found to be internalized to some extent and degraded, depending on Initiates and inhibits FcγR expression levels on target cells it interacts with. In some embodiments, the antibody that binds the CD20 antigen but does not have endocytic activity is a type II "CD20-specific antibody, such as obinutuzumab.

[0280] In some embodiments, the antibody has antibody binding activity to a non-endocytic (eg, ALCAM / CD166) antigen. In some embodiments, the antibody comprises VH of SEQ ID No: 73 and VL of SEQ ID No: 74, VH of SEQ ID No: 75 and VL of SEQ ID No: 76 in EP3911682A1, SEQ ID No: 77 The VH of SEQ ID No:78 and the VL of SEQ ID No:78, or the antibody shown by the VH of SEQ ID No:79 and the VL of SEQ ID No:88.

[0281] In some embodiments, the ligand drug conjugate is selected from: wherein, Tb8 is an antibody that has tumor cell endocytosis activity and tumor cell surface antigen binding activity, such as 1D1-01, 2E3-02 Antibody, sacituzumab, pertuzumab, trastuzumab or cetuximab antibody; q is selected from any value between 0.1-16.0, preferably any value between 2-8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some preferred embodiments, q is 2, 4, 6 or 8.

[0282] In addition, it should be noted that those with ordinary knowledge in the technical field of the present invention can understand that L is related to opening a disulfide bond (for example, the reduction of a disulfide bond by a reducing agent TCEP can open a disulfide bond to generate The sulfhydryl group contained in Tb (such as antibody) itself is connected after the thiol-SH), that is to say, the -S- between L and Tb is not an additional external sulfur atom. For example, -S- is not an additional external sulfur atom, but -S- formed by linking the thiol contained in Tb itself with L after opening the disulfide bond.

[0283] In the second aspect of the disclosure, the disclosure provides the compound shown in formula II: formula II or the stereoisomer of the compound, its prodrug, its pharmaceutically acceptable salt, its pharmaceutically acceptable or its ligand drug conjugate, wherein: R1 and R2 are each independently selected from H, halogen, -OH, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkoxy , or, R1 and R2 form a 5-7-membered carbocyclic ring or a 5-7-membered heterocyclic ring together with the carbon atoms connected to it, and the heterocyclic ring contains 1 or more O, S, N, carbonyl, arylene or Phenyl or any combination thereof; R3 is selected from H, halogen, -OH, -NH2, optionally substituted C1-6 alkyl, optionally substituted C1-6 alkoxy, or, R3 and X and the The carbon atoms together form a 5-7-membered carbocyclic ring or a 5-7-membered heterocyclic ring, and the heterocyclic ring contains 1 or more O, S, N, carbonyl, pyrenylene or pyrenyl groups or any combination thereof; R3 and R2 Form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring together with the carbon atoms connected to it, and the heterocyclic ring contains 1 or more O, S, N, carbonyl, pyrenylene or pyrenyl groups or any combination thereof ; W does not exist or exists, when W exists, W is selected from -OH, -SH, -NHR4,,,,,,,,,, 1 is connected to X; X is selected from direct bonds, optionally substituted -O -(CH2)n3-, -N(R4)-(CH2)n3-, -S-(CH2)n3-, Carbonyl-(CH2)n3, -SO2-(CH2)n3-, -(CH2)n1- ,,,, C3-6 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclic group, the 1st position is connected to the parent ring, and the 2nd position is connected to W; the substituent Selected from one or more C1-4 alkyl groups, C3-6 cycloalkyl groups, or multiple C1-4 alkyl groups and the carbon atoms connected to them at the same time together form a C3-6 cycloalkyl group; each M is independently selected from Direct bond, -CR5aR5b-; R4, R5, R5a, R5b, R6, R7 are each independently selected from H, optionally substituted C1-4 alkyl, optionally substituted C1-4 alkoxy, optionally substituted C3-6 cycloalkyl; n, n', n1, n2, n3 are each independently selected from any integer between 0 and 6; wherein, when R1 and R2 are H at the same time, and X is -(CH2)n1- , when n1 is 1, 2, 3, 4, W is not -OH or -NHR4; and the compound shown in formula II does not include ,,,,,,,,,,,,,,.

[0284] In some embodiments, R1 and R2 are each independently selected from H, halogen, or C1-4 alkyl.

[0285] In some embodiments, R1 and R2 together with the carbon atoms to which they are attached form a 5-6 membered heterocycle containing 1, 2 or 3 O, S or N or any combination thereof.

[0286] In some embodiments, R1 is selected from H and halogen, and R2 is selected from H and C1-4 alkyl.

[0287] In some embodiments, R1 and R2 and the carbon atom to which they are attached form, or, the dashed line indicates where the heterocyclic ring is fused to the benzene ring.

[0288] In some embodiments, R1 is H or F and R2 is H or methyl.

[0289] In some embodiments, R1 and R2 are formed together with the carbon atom to which they are attached.

[0290] In some embodiments, R1 is F and R2 is methyl; or R1 and R2 are formed together with the carbon atom to which they are attached.

[0291] In some embodiments, R1 is F and R2 is methyl.

[0292] In some embodiments, R1 and R2 are formed together with the carbon atom to which they are attached.

[0293] In some embodiments, R3 is selected from H and C1-4 alkyl.

[0294] In some embodiments, R3 is H.

[0295] In some embodiments, W is absent or present, and when W is present, W is selected from -OH, -SH, -NHR4,,,,,,,, and the 1 position is connected to X.

[0296] In some embodiments, W is absent or present, and when W is present, W is selected from the group consisting of -OH, -SH, -NHR4, and, 1 is attached to X.

[0297] In some embodiments, W is selected from the group consisting of -OH, -NHR4 and, 1 is linked to X.

[0298] In some embodiments, W is selected from -OH and -NHR4, and X is attached at position 1.

[0299] In some embodiments, X is selected from optionally substituted -(CH2)n1-,,,, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl, 1 position It is connected to the parent ring, and the 2nd position is connected to W; the substituent is selected from 1 or 2 C1-4 alkyl groups, or 2 C1-4 alkyl groups and the carbon atoms connected to them at the same time form a C3-6 ring alkyl.

[0300] In some embodiments, X is selected from optionally substituted,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 1 position is attached to the parent ring, The 2-position is connected to W; the substituent is selected from 1 or 2 C1-4 alkyl groups (such as methyl), or 2 C1-4 alkyl groups (such as methyl) and the carbon atoms connected to them at the same time Form C3-6 cycloalkyl (such as cyclopropyl).

[0301] In some embodiments, X is selected from the group consisting of ,,,,,,,,,,,,,,,,,,,,,,,,,,, 1 position is connected to the parent ring, and 2 positions are connected to W.

[0302] In some embodiments, X is selected from the group consisting of , , and , with the 1 position attached to the parent ring and the 2 position attached to W.

[0303] In some embodiments, when W does not exist, X is selected from the group consisting of , , and, and the 1 position is connected to the parent ring; when W exists, X is selected from , And, 1 bit is connected to the mother ring, and 2 bits are connected to W.

[0304] In some embodiments, W is selected from -OH, -NHR4 and, 1 position X is connected, 2 positions are connected to L4 or L3; X is selected from , , and, 1 position is connected to the parent ring, 2 positions are connected to W connected. In some embodiments, R4 and R5 are each independently selected from H, C1-4 alkyl, and C3-6 cycloalkyl.

[0305] In some embodiments, each R4 is independently selected from H, C1-4 alkyl, and C3-6 cycloalkyl, and R5 is H.

[0306] In some embodiments, each R4 is independently selected from H, methyl, ethyl, isopropyl, n-propyl, tert-butyl, and cyclopropyl, and R5 is H.

[0307] In some embodiments, R5a and R5b are each independently selected from H and C1-4 alkyl;

[0308] In some embodiments, R5a and R5b are each independently selected from H and methyl;

[0309] In some embodiments, each R7 is independently selected from H and C1-4 alkyl;

[0310] In some embodiments, R7 is H.

[0311] In some embodiments, n is 1, 2 or 3.

[0312] In some embodiments, n is 1.

[0313] In some embodiments, n1 is 1, 2, 3 or 4.

[0314] In some embodiments, n2 is 1;

[0315] In some embodiments, n3 is zero.

[0316] In some embodiments, the compound shown in formula II is selected from any of the following compounds:

[0317] In the third aspect of the present disclosure, the present disclosure provides the drug linker conjugate shown in formula III, the stereoisomer of formula III or the drug linker conjugate, its prodrug, its A pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, wherein: R1 and R2 are each independently selected from H, halogen, -OH, optionally substituted C1-6 alkyl or optionally substituted C1- 6 alkoxy groups, or, R1 and R2 and the carbon atoms connected to them together form a 5-7-membered carbocyclic ring or a 5-7-membered heterocyclic ring, and the heterocyclic ring contains 1 or more O, S, N, carbonyl groups, An arylene group or an aroxyl group or any combination thereof; R3 is selected from H, halogen, -OH, -NH2, optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or, R3 and X Form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring together with the carbon atoms connected to it, and the heterocyclic ring contains 1 or more O, S, N, carbonyl, pyrenylene or pyrenyl groups or any combination thereof , or, R3 and R2 form a 5-7-membered carbocyclic ring or a 5-7-membered heterocyclic ring together with the carbon atoms connected to it, and the heterocyclic ring contains 1 or more O, S, N, carbonyl, arylene or Phenyl or any combination thereof; W does not exist or exists, when W exists, W is selected from -O-, -S-, -NR4-,,,,,,,,, and, 1 position is connected to X, and 2 positions are connected to L4 or L3 are connected; X is selected from a direct bond, optionally substituted -O-(CH2)n3-, -N(R4)-(CH2)n3-, -S-(CH2)n3-, carbonyl-(CH2) n3, -SO2-(CH2)n3-,,,,-(CH2)n1-, C3-6 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl, The 1-position is connected to the parent ring, and the 2-position is connected to W or L4; the substituent is selected from one or more C1-4 alkyl groups, C3-6 cycloalkyl groups, or multiple C1-4 alkyl groups and simultaneously with them The linked carbon atoms together form a C3-6 cycloalkyl group; each M is independently selected from direct bonds, -CR5aR5b-; R4, R5, R5a, R5b, R6 and R7 are each independently selected from H, optionally substituted C1- 4 alkyl, optionally substituted C1-4 alkoxy and optionally substituted C3-6 cycloalkyl; n, n', n1, n2, n3 are each independently selected from any integer between 0 and 6; L1 is selected from: ,,, each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C6-10 aryl group, a 5-10 membered heteroaryl group, an amido group, a sulfonamide group, a Amino group, CF2, Rx, Ry are each independently selected from H, C1-4 alkyl, each m is independently selected from 0, 1, 2, 3, 4, 5, 6, y1, y2, y3, y4 are each independently Ground is selected from any integer between 0-20, 1 position is connected with Lg, 2 positions are connected with L2 or L3; L2 does not exist or exists, when L2 exists, L2 is selected from:, y1, y2, y3, y4 are independent of each other is selected from any integer between 0-20, the 1st position is connected to L1, and the 2nd position is connected to L3; L3 is selected from amino acid residues or short peptides consisting of 2-10 amino acid residues; L4 is not Presence or existence, when L4 exists, L4 is selected from ,,,,,,,,,,, 1 position is connected to L3, 2 positions are connected to W or X; Lg is a leaving group, Lg is selected from halogen, pylori , Tertiary amine base (Me3N+, Et3N+), diazonium base, -OMs, MeSO2-, CF3SO3-.

[0318] In some embodiments, R1, R2 are each independently selected from H, halogen, C1-4 alkyl.

[0319] In some embodiments, R1 and R2 together with the carbon atoms to which they are attached form a 5-6 membered heterocycle containing 1, 2 or 3 O, S or N or any combination thereof.

[0320] In some embodiments, R1 is selected from H, halogen, and R2 is selected from H, C1-4 alkyl.

[0321] In some embodiments, R1 and R2 and the carbon atom to which they are attached form, or, the dashed line indicates where the heterocyclic ring is fused to the benzene ring.

[0322] In some embodiments, R1 is H or F and R2 is H or methyl.

[0323] In some embodiments, R1 is F, R2 is methyl or R1 and R2 are formed together with the carbon atom to which they are attached.

[0324] In some embodiments, R1 is F and R2 is methyl.

[0325] In some embodiments, R1 and R2 are formed together with the carbon atom to which they are attached.

[0326] In some embodiments, R3 is selected from H, C1-4 alkyl.

[0327] In some embodiments, R3 and X, together with the carbon atom to which they are attached, form a 5-6 membered carbocyclic ring.

[0328] In some embodiments, R3 is H or R3 and X are formed together with the carbon atom to which it is attached, and the dashed lines indicate where the carbocycle is fused to the benzene and pyridine rings.

[0329] In some embodiments, R3 is H.

[0330] In some embodiments, R3 and X are formed together with the carbon atom to which they are attached, and the dashed lines indicate where the carbocycles are fused to the benzene and pyridine rings.

[0331] In some embodiments, W does not exist or exists, and when W exists, W is selected from -O-, -S-, -NR4-,,,,,,,,, 1 position is connected to X, 2 positions Connect to L4 or L3.

[0332] In some embodiments, W does not exist or exists, when W exists, W is selected from -O-, -S-, -NR4-,,,,, 1 position is connected to X, 2 positions are connected to L4 or L3 connected.

[0333] In some embodiments, W is selected from -O-, -NR4-, 1 position is linked to X, and 2 positions are linked to L4 or L3.

[0334] In some embodiments, W is selected from -O-, -NR4-, 1 position is linked to X, and 2 positions are linked to L4 or L3.

[0335] In some embodiments, X is selected from optionally substituted -(CH2)n1-,,,, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl, 1 position It is connected to the parent ring, and the 2-position is connected to W or L4; the substituent is selected from 1 or 2 C1-4 alkyl groups, or 2 C1-4 alkyl groups and the carbon atoms connected to them at the same time form a C3- 6 cycloalkyl.

[0336] In some embodiments, X is selected from optionally substituted,,,,,,,,,,,,,,,,,,,, 1 position is connected to the parent ring, and the 2 position is connected to W or L4; The substituent is selected from 1 or 2 C1-4 alkyl groups (such as methyl), or 2 C1-4 alkyl groups (such as methyl) and the carbon atoms connected to them at the same time to form a C3-6 cycloalkane groups (such as cyclopropyl).

[0337] In some embodiments, X is selected from the group consisting of ,,,,,,,,,,,,,,,,,,,,,, the 1st position is connected to the parent ring, and the 2nd position is connected to W or L4.

[0338] In some embodiments, X is selected from the group consisting of , , , and the 1st position is connected to the parent ring, and the 2nd position is connected to W.

[0339] In some embodiments, when W does not exist, X is selected from ,,,, 1 is connected to the parent ring, and 2 is connected to L4; when W exists, X is selected from ,,,,,,,,, ,,,,,,,, 1 bit is connected to the mother ring, and 2 bits are connected to W.

[0340] In some embodiments, W is selected from -O-, -NR4-, 1 position X is connected, 2 positions are connected to L4 or L3; X is selected from ,,, 1 position is connected to the parent ring, 2 positions Connect to W.

[0341] In some embodiments, R4, R5 are each independently selected from H, C1-4 alkyl, C3-6 cycloalkyl.

[0342] In some embodiments, each R4 is independently selected from H, C1-4 alkyl, C3-6 cycloalkyl, and R5 is H.

[0343] In some embodiments, each R4 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, tert-butyl, cyclopropyl, and R5 is H.

[0344] In some embodiments, R5a, R5b are each independently selected from H, C1-4 alkyl.

[0345] In some embodiments, R5a, R5b are each independently selected from H, methyl.

[0346] In some embodiments, each R7 is independently selected from H, C1-4 alkyl.

[0347] In some embodiments, R7 is H.

[0348] In some embodiments, n is selected from 1, 2 or 3.

[0349] In some embodiments, n is 1.

[0350] In some embodiments, n1 is selected from 1, 2, 3 or 4.

[0351] In some embodiments, n2 is 1.

[0352] In some embodiments, n3 is zero.

[0353] In some embodiments, L1 is selected from ,,,,,,,, each Z is independently selected from direct bond, carbon-carbon triple bond, carbon-carbon double bond, C6-10 aryl, 5-10 membered hetero Aryl group, amido group (preferably selected from direct bond, carbon-carbon triple bond, carbon-carbon double bond), Rx, Ry are each independently selected from H, C1-4 alkyl, each m is independently selected from 0, 1, 2, 3, 4, 5, 6, y1 is selected from any integer between 1-6 (such as 4, 5, 6), each y2 is independently selected from any integer between 0-15 (such as 6-15), each y3 is independently selected from 1, 2 or 3, each y4 is independently selected from 0 or 1, the 1 position is connected to Lg, and the 2 position is connected to L2 or L3; for example, each Z is independently selected from a direct bond, a carbon-carbon triple bond, Carbon-carbon double bond, Rx, Ry are each independently selected from H, C1-4 alkyl, each m is independently selected from 0, 1, 2, 3, 4, 5, 6, y1 is selected from any of 1-6 Integers (such as 4, 5, 6), each y2 is independently selected from any integer between 0-15 (such as 6-15), each y3 is independently selected from 1, 2 or 3, each y4 is independently selected from 0 or 1, The 1st position is connected to Lg through the S atom, and the 2nd position is connected to L2 or L3.

[0354] In some embodiments, L1 is selected from ,,,,,,, m is selected from 2, 3, 4, y1 is selected from any integer between 1-6 (such as 4, 5, 6), and each y2 is independently selected from any integer between 0-10 (such as 6-10), each y3 is independently selected from 1 or 2, 1 position is connected to Lg, and 2 positions are connected to L2 or L3.

[0355] In some embodiments, L1 is selected from ,,,,,,,, position 1 is connected to Lg, and position 2 is connected to L2 or L3.

[0356] In some embodiments, L1 is selected from ,,,,,,, position 1 is connected to Lg, and position 2 is connected to L2 or L3.

[0357] In some embodiments, L1 is selected from the group consisting of , , , 1 is linked to Lg, and 2 is linked to L2 or L3.

[0358] In some embodiments, L1 is selected from the group consisting of Lg at position 1 and L2 or L3 at position 2.

[0359] In some embodiments, L2 does not exist or exists, when L2 exists, L2 is selected from ,,,,,,,, y1 is selected from any integer between 1-6 (such as 4, 5, 6), each y2 is independently selected from any integer between 0-10 (such as 6-10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, 1 bit is connected to L1, and 2 bits are connected to L3 .

[0360] In some embodiments, L2 is absent or present, and when L2 is present, L2 is selected from the group consisting of ,,,,, 1 position is connected to L1, and 2 position is connected to L3.

[0361] In some embodiments, L2 is absent or present, and when L2 is present, L2 is selected from the group consisting of ,,,,, position 1 is linked to L1, and position 2 is linked to L3.

[0362] In some embodiments, L2 is absent or present, and when L2 is present, L2 is selected from the group consisting of ,,,, position 1 is linked to L1, and position 2 is linked to L3.

[0363] In some embodiments, L2 is absent.

[0364] In some embodiments, L2 is selected from.

[0365] In some embodiments, L3 is selected from amino acid residues or short peptides consisting of 2-10 amino acid residues; the amino acid residues are selected from natural amino acid residues, Unnatural amino acid residues, amino acid residues shown in AA1 or stereoisomers thereof.

[0366] In some embodiments, L3 is selected from amino acid residues Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, AA1 or by 2 - A short peptide consisting of 10 amino acid residues selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Asp, AAl.

[0367] In some embodiments, L3 is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, AAl, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Ala-Ala, Val-AA1, Ala-AA1, Gly-AA1, AA1-Gly, Ala-Ala-Ala, Ala- Ala-Asn, Ala-Ala-Asp, Val-AA1-Gly, Ala-AA1-Gly, Gly-AA1-Gly, Lys-Ala-Ala-Asn, Lys-Ala-Ala-Asp, Gly-Phe-Gly, Gly-Gly-Phe-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Phe, Val-Lys-Gly, Val-Lys-Gly-Gly, Val- Lys, Lys-Ala-Asn.

[0368] In some embodiments, L3 is selected from AA1, AA1-Gly, Val-Cit, Val-AA1-Gly, AA1-Ala-Asn, Gly-Gly-Phe-Gly.

[0369] In some embodiments, L3 is selected from AA1, Val-AA1-Gly.

[0370] In some embodiments, L3 is selected from Val-AA1-Gly.

[0371] In some embodiments, L3 is selected from the group consisting of,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, X- Selected from halide ion, carboxylate ion, sulfate ion, bisulfate ion, OH-, the 1st position is connected to L1 or L2, and the 2nd position is connected to L4 or W.

[0372] In some embodiments, L3 is selected from ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, X-is selected from halide ion, carboxyl Acid ion, sulfate ion, bisulfate ion, OH-, the 1st position is connected to L1 or L2, and the 2nd position is connected to L4 or W.

[0373] In some embodiments, L3 is selected from ,,,,,,,,,,,,, X-is selected from halide ion, carboxylate ion, sulfate ion, hydrogen sulfate ion, OH-, 1 position Connect to L1 or L2, 2 bits to L4 or W.

[0374] In some embodiments, L3 is selected from ,,,,,,,,,, 1 position is connected to L1 or L2, and 2 positions are connected to L4 or W.

[0375] In some embodiments, L3 is selected from ,,,,,,, 1 position is connected to L1 or L2, and 2 positions are connected to L4 or W.

[0376] In some embodiments, the structure of the amino acid residue shown in AA1 is as follows, AA1 wherein: Ra, Rb are each independently selected from H,,,, and Ra, Rb are not H at the same time; or, Ra and Rb and the carbon atoms connected to them together form a 4-10 membered heterocyclic ring, and the 4-10 membered heterocyclic ring is optionally substituted by one or more R0; r, r1 are each independently selected from 0 Any integer to 20; Rm1, Rn1 are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl, -COORx1; Rx1 is selected from C1-6 alkyl; Or, Rm1 and Rn1 and together with them The connected nitrogen atoms together form a 4-10 membered heterocycle, which is optionally substituted by one or more R0'; Rz is selected from C1-6 alkyl; R0, R0' are independent Rm2, Rn2 are each independently selected from H, C1-6 alkyl.

[0377] In some embodiments, any one of Ra and Rb is H, and the other is selected from , , .

[0378] In some embodiments, either of Ra, Rb is H, and the other is selected from.

[0379] In some embodiments, Ra and Rb, together with the carbon atom to which they are commonly attached, form a 5-6 membered heterocyclic ring substituted with R0.

[0380] In some embodiments, Ra and Rb, together with the carbon atom to which they are commonly attached, form a piperidine or piperidine ring substituted with R0.

[0381] In some embodiments, Ra and Rb, together with the carbon atom to which they are commonly attached, form a piperidine ring substituted with R0.

[0382] In some embodiments, Ra and Rb, together with the carbon atom to which they are commonly attached, form, or, the carbon atom number 1 is the carbon atom to which Ra and Rb are commonly attached.

[0383] In some embodiments, Ra and Rb, together with the carbon atom to which they are commonly attached, form or, the carbon atom number 1 is the carbon atom to which Ra and Rb are commonly attached.

[0384] In some embodiments, r, r1 are each independently selected from 0, 1, 2, 3, 4, 5.

[0385] In some embodiments, r, r1 are each independently selected from 0, 4.

[0386] In some embodiments, either r, r1 is 0 and the other is 4.

[0387] In some embodiments, Rm1, Rn1 are each independently selected from H, methyl, ethyl, n-propyl, n-butyl, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, - COOC(CH3)3, -COOCH2CH2CH2CH3.

[0388] In some embodiments, Rm1, Rn1 are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl, tertiary butoxycarbonyl. In some embodiments, Rm1, Rn1 are each independently selected from H, C1-6 alkyl.

[0389] In some embodiments, Rm1, Rn1 are each independently selected from H, methyl, ethyl, n-propyl.

[0390] In some embodiments, in r and r1, r is 4, and when r1 is 0, Rm1 and Rn1 are each independently selected from H, C1-6 alkyl (such as H, methyl); r is 0, When r1 is 4, Rm1 and Rn1 are each independently selected from C1-6 alkyl (such as methyl, ethyl, n-propyl), preferably selected from C2-6 alkyl (such as ethyl, n-propyl).

[0391] In some embodiments, Rm1 and Rn1, together with the nitrogen atom to which they are jointly attached, form a 5-6 membered heterocyclic ring optionally substituted by R0'.

[0392] In some embodiments, Rm1 and Rn1, together with the nitrogen atom to which they are jointly attached, form a piperidine or piperidine ring optionally substituted with R0'.

[0393] In some embodiments, Rm1 is taken together with Rn1 and the nitrogen atom to which they are jointly attached to form ,,,, nitrogen atom number 1 is the nitrogen atom to which Rm1 and Rn1 are jointly attached.

[0394] In some embodiments, Rz is methyl.

[0395] In some embodiments, R0, R0' are each independently selected from C1-6 alkyl, -NRm2Rn2, 5-6 membered heterocyclyl optionally substituted by C1-6 alkyl.

[0396] In some embodiments, R is selected from C1-6 alkyl, 5-6 membered heterocyclic group substituted by C1-6 alkyl, and said 5-6 membered heterocyclic group is selected from piperidinyl, piperidinyl 𠯤 base.

[0397] In some embodiments, R is selected from methyl, ethyl, 5-6 membered heterocyclyl substituted by methyl, and the 5-6 membered heterocyclyl is piperidinyl.

[0398] In some embodiments, R is selected from methyl, a 5-6 membered heterocyclyl substituted by methyl, and the 5-6 membered heterocyclyl is piperidinyl.

[0399] In some embodiments, R is selected from methyl, ethyl, and

[0400] In some embodiments, R is selected from methyl.

[0401] In some embodiments, R0' is selected from C1-6 alkyl, -NRm2Rn2.

[0402] In some embodiments, R0' is selected from methyl, -NRm2Rn2.

[0403] In some embodiments, Rm2, Rn2 are methyl.

[0404] In some embodiments, the amino acid residue shown in AA1 is selected from ,,,,,,,,,,,,,,,,,, or.

[0405] In some embodiments, the amino acid residue represented by AA1 is selected from the group consisting of ,,,,,,,,,,,,,,.

[0406] In some embodiments, the amino acid residue represented by AA1 is selected from the group consisting of ,,,,,,,,.

[0407] In some embodiments, the amino acid residue shown in AA1 is selected from ,,,,,,.

[0408] In some embodiments, L4 is absent or present, and when L4 is present, L4 is selected from , , , 1 is linked to L3, and 2 is linked to W or X.

[0409] In some embodiments, L4 is absent or present, and when L4 is present, L4 is or, position 1 is linked to L3, position 2 is linked to W or X.

[0410] In some embodiments, L4 is absent.

[0411] In some embodiments, L4 is selected from , , 1 is linked to L3, and 2 is linked to W or X.

[0412] In some embodiments, L4 is selected from the group where position 1 is linked to L3 and position 2 is linked to W or X.

[0413] In some embodiments, Lg is selected from F, Cl, MeSO2-.

[0414] In some embodiments, Lg is selected from F, MeSO2-.

[0415] In some embodiments, the structure of is selected from the following: Among them, 1 bit is connected to Lg, and 2 bits are connected to W.

[0416] In some embodiments, the structure of is selected from the group consisting of: wherein position 1 is linked to L4; when L4 is absent, position 1 is linked to L3.

[0417] In some embodiments, the drug linker conjugate has the formula III-(1) structure: wherein, L1, L2, L3, L4, X, R1, R2, R3, R4 and Lg have the above meaning provided herein and in any embodiment specifically recited herein.

[0418] In some embodiments, the drug linker conjugate has the structure of formula III-(1A) or III-(1B): , wherein, L2, L3, L4, X, R1, R2, R3, R4 and Lg have the meanings provided above and in any embodiment specifically recited herein.

[0419] In some embodiments, the drug linker conjugate has the structure of formula III-(2): wherein, L1, L2, L3, L4, X, R1, R2, R3 and Lg have the above and as provided in any embodiment specifically recited herein.

[0420] In some embodiments, the drug linker conjugate has a structure of formula III-(2A) or III-(2B): , wherein, L2, L3, L4, X, R1, R2, R3 and Lg has the meaning provided above and in any embodiment specifically recited herein.

[0421] In some embodiments, the drug linker conjugate has the structure of formula III-(3): wherein, L1, L2, L3, L4, X, R1, R2, R3, R4, R5, n and Lg have the meanings provided above as well as in any embodiment specifically recited herein.

[0422] In some embodiments, the drug linker conjugate has a structure of formula III-(3A) or III-(3B): wherein, Lg, L2, L3, L4, X, R1, R2, R3 , R4 and Lg have the meanings provided above and in any embodiment specifically recited herein.

[0423] In some embodiments, the drug linker conjugate has the structure of formula III-A: wherein, Lg, X, R1, R2, R3, Ra, Rb and q have the above and specifically described herein Meaning provided in any implementation.

[0424] In some embodiments, the drug linker conjugate has the structure of formula III-B: wherein, Lg, X, R1, R2, R3, Ra, Rb and q have the above and specifically described herein Meaning provided in any implementation.

[0425] In some embodiments, the drug linker conjugate shown in formula III is selected from the following structures: .

[0426] In some embodiments, compounds are provided wherein the L1-L2-L3 unit in the drug linker conjugate of Formula III has been partially cleaved, leaving the amino acid residue to which it is bonded drug part.

[0427] In some embodiments, the free drug partially released is a compound of formula III-(A): or a stereoisomer or a mixture of stereoisomers thereof, or a pharmaceutically acceptable salt thereof, wherein L4 , X, W, R1, R2 and R3 have the meanings provided above and in any embodiment specifically recited herein.

[0428] In some embodiments, the structure of III-(A) is selected from the following: .

[0429] The present disclosure also provides a linker in a ligand-drug conjugate, the structure of which contains fragments as shown below: wherein, the 2-position is connected to the biologically active molecule fragment; (1-position is connected to the ligand end, for example , when it is connected to the linking unit (such as L2), and then connected to the ligand or targeting moiety (Tb) through the extending unit (such as L1); or, when the connecting unit does not exist, directly connected to the extending unit, and then further connected to the Ligand linked); L3, L4 are defined as described in any scheme in the present disclosure.

[0430] In some embodiments, the linker in the ligand drug conjugate has a structure as follows: wherein, the 1st position is connected to the ligand or targeting moiety that binds to the target, and the 2nd position is connected to the The biologically active molecular fragments are connected; the definitions of L1, L2, L3, and L4 are as described in any scheme in the present disclosure; preferably, the target-binding ligand or targeting moiety and the biologically active molecular fragment The definitions of are as described in Tb and D in any scheme of the present disclosure, respectively.

[0431] In some embodiments, the linker is linked at position 1 to an antibody or an antigen-binding fragment thereof, and at position 2 to a biologically active molecule fragment.

[0432] In some embodiments, the 1st position of the linker is connected to cysteine ​​or lysine on the antibody or its antigen-binding fragment, and the 2nd position is connected to a biologically active molecule fragment; Preferably, the The antibody or antigen-binding fragment thereof is as described in any aspect of the present disclosure.

[0433] In the fourth aspect of the present disclosure, the present disclosure also provides the linker shown in formula III-1: Lg, L1, L2, L3 and L4 have the above and any embodiment specifically described herein. Meaning; Lg1 is the leaving group when reacting with drug molecules.

[0434] In some embodiments, Lg1 is preferably -OH, or halogen.

[0435] In the fifth aspect of the present disclosure, the present disclosure also provides a linker shown in formula III-2: Lg, L1, L2 and L3 have the meanings provided above and in any embodiment specifically described herein; Lg2 is a leaving group when reacting with L4 or a fragment containing a drug molecule.

[0436] In some embodiments, Lg2 is preferably -OH, or halo.

[0437] In the sixth aspect of the present disclosure, the present disclosure provides a preparation method of the compound (drug molecule, bioactive molecule) shown in formula II and the drug linker conjugate shown in formula III.

[0438] In one aspect, the present disclosure provides a method for preparing a compound (drug molecule, biologically active molecule) represented by Formula II. Specifically: The compound (VI) can be obtained from the compound (IV) represented by the general formula through an alkylation reaction with the compound (V) under the catalysis of an iron compound or other related Minisci reactions. Alternatively, compound (IV) generates compound (VII) through nitrogen oxidation, and compound (VII) is treated with phosphorus oxyhalide to generate compound (VIII), a halogenated product of compound (IV). Compound (VIII) can be obtained by various forms of reactions, such as Heck reaction, Suzuki reaction, Buchwald reaction, Nigishi reaction, Stille reaction, etc. to obtain compound (VI). R11 in compound (VI) can be synthesized into different or more complex molecules through a variety of chemical transformations, such as oxidation, reduction, substitution and other common methods in textbooks. Alternatively, compound (VI) and compound (IV) can also be obtained by ring-closing reaction of compound (IX) and compound (X) shown below.

[0439] In another aspect, the present disclosure provides methods for preparing the fragments represented by Formula III-1 and Formula III-2. Specifically: compound III-a can obtain compound III-c through its functional group Fg1 and compound III-b's functional group Fg2 reaction, compound III-c can obtain compound III-d through its functional group Fga, similar transformation Compounds of general formula III-2 and III-1 can be obtained. Lga, Lgb, Lg1, Lg2 are reactive leaving groups, such as -OH, or halogen.

[0440] In yet another aspect, the present disclosure provides a method for preparing a drug-linker conjugate represented by formula III. Specifically: The target product can be obtained by coupling the B segment and the C segment as shown in the following reaction formula, and the coupling product can be subjected to some common chemical modifications, such as oxidation, deprotection, etc., to obtain the drug shown in the general formula III Linker conjugates.

[0441] In the seventh aspect of the present disclosure, the present disclosure provides a method for preparing the aforementioned ligand drug conjugate, which includes: Tb and the drug linker conjugate shown in formula III in a suitable solvent and conditions carry out a coupling reaction; wherein: Tb has the meanings provided above and in any embodiment specifically described herein; R1, R2, R3, X, W, L1, L2, L3, L4, Lg have the meanings provided above and specifically herein meaning provided in any embodiment recited.

[0442] In some embodiments, the method includes the step of performing a coupling reaction between Tb and the drug linker conjugate represented by formula III in a suitable solvent and conditions to form a C-S bond.

[0443] In some embodiments, the ratio of the amount of substance of the Tb to the drug linker conjugate is 1:(1-20), such as 1:2, 1:3, 1:4, 1 :5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:(10-20), 1:( 12-20), 1:(14-20), 1:(16-20) or 1:(18-20).

[0444] In some embodiments, the coupling reaction is performed in water and / or an organic solvent.

[0445] In some embodiments, the organic solvent is selected from N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, nitriles (such as acetonitrile), alcohols (such as methanol , ethanol) or any combination thereof.

[0446] In some embodiments, the method further comprises the step of purifying the coupling product.

[0447] In some embodiments, the coupling product is purified by chromatographic methods.

[0448] In some embodiments, the chromatographic method comprises one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, or affinity chromatography.

[0449] In an eighth aspect of the present disclosure, the present disclosure provides an antibody or antigen-binding fragment thereof that binds B7H3, the antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence and a variant of HCDR3 or its sequence contained in the heavy chain variable region (VH) shown in SEQ ID NO: 3 or 23; and / Or LCDR1 or a sequence variant thereof, LCDR2 or a sequence variant thereof, and LCDR3 or a sequence variant thereof contained in the light chain variable region (VL) represented by SEQ ID NO: 13 or 33. In certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, and HCDR3 or a variant thereof contained in the VH set forth in SEQ ID NO: 3 A variant of the sequence; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL set forth in SEQ ID NO: 13.

[0452] In certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, and HCDR3 or a variant thereof contained in the VH set forth in SEQ ID NO: 3 A variant of the sequence; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 33. In certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, and HCDR3 or a variant thereof contained in the VH set forth in SEQ ID NO: 23 A variant of the sequence; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 33. In certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1 or a variant thereof, HCDR2 or a variant thereof, and HCDR3 or a variant thereof contained in the VH set forth in SEQ ID NO: 23 A variant of the sequence; and / or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL set forth in SEQ ID NO: 13.

[0455] In certain preferred embodiments, variants of the sequence are substitutions, deletions or additions (e.g., 1, 2 or 3 amino acids) of one or several amino acids compared to its source CDR substitutions, deletions or additions) of the CDRs.

[0456] In certain preferred embodiments, the substitutions are conservative substitutions.

[0457] Preferably, the CDRs are defined according to the AbM, Chothia, Kabat or IMGT numbering system.

[0458] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof include human immunoglobulin framework regions (FR).

[0459] In certain embodiments, the antibody or antigen-binding fragment thereof binds human B7H3 and / or monkey B7H3. In certain embodiments, the antibody or antigen-binding fragment thereof binds human 2Ig B7H3. In certain embodiments, the antibody or antigen-binding fragment thereof binds human 4Ig B7H3. In certain embodiments, the antibody or antigen-binding fragment thereof binds monkey 4Ig B7H3. In certain embodiments, the antibody or antigen-binding fragment thereof binds human 2Ig B7H3 and human 4Ig B7H3, and more preferentially binds human 4Ig B7H3. In certain embodiments, the antibody or antigen-binding fragment thereof binds human 4Ig B7H3 and does not bind human 2Ig B7H3.

[0460] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof capable of binding B7H3, said antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) and / or a light chain variable region (VL). In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the IMGT numbering system: (a) Heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 10 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence HCDR1, the sequence is SEQ ID NO: 11 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence HCDR2, the sequence is SEQ ID NO: 12 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the HCDR3 sequence; and / or, a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 20 or has one compared thereto or a sequence of substitutions, deletions or additions of several amino acids (e.g. substitutions, deletions or additions of 1, 2 or 3 amino acids) of LCDR1, the sequence being GTF or having one or several amines compared thereto The LCDR2 of the sequence of substitution, deletion or addition of amino acids (such as substitution, deletion or addition of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 22 or has one or several amines compared thereto The LCDR3 of the sequence of amino acid substitution, deletion or addition (such as 1, 2 or 3 amino acid substitution, deletion or addition); (b) heavy chain variable region (VH) comprising the following 3 CDRs : HCDR1 whose sequence is SEQ ID NO: 30 or a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto , the sequence is SEQ ID NO: 31 or the HCDR2 of a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto , the sequence is SEQ ID NO: 32 or HCDR3 having a sequence of one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto and / or, a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 40 or it has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence LCDR1, the sequence is GAS or has one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitution, deletion or addition) sequence LCDR2, the sequence is SEQ ID NO: 42 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitution, deletion or addition) sequence LCDR3; (c) heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 10 or one or more HCDR1 of a sequence of amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions), the sequence is SEQ ID NO: 11 or has one or more compared thereto HCDR2 of a sequence of amino acid substitutions, deletions or additions (for example 1, 2 or 3 amino acid substitutions, deletions or additions), the sequence is SEQ ID NO: 12 or has one or more compared thereto Amino acid substitution, deletion or addition (such as 1, 2 or 3 amino acid substitutions, deletions or additions) sequence HCDR3; and / or, light chain variable region comprising the following 3 CDRs ( VL): the sequence is SEQ ID NO: 40 or a sequence with one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to it LCDR1 whose sequence is GAS or LCDR2 with a sequence of one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to it, the sequence LCDR3 which is SEQ ID NO: 42 or a sequence having a substitution, deletion or addition of one or several amino acids compared thereto (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids); or (d) A heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 30 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence HCDR1, the sequence is SEQ ID NO: 31 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence HCDR2, the sequence is SEQ ID NO: 32 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the HCDR3 sequence; and / or, a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 20 or has one compared thereto or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) sequence LCDR1, LCDR2 sequence is GTF or has one or several amino acids compared thereto A sequence of amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) of SEQ ID NO: 22 or having one or several amines compared thereto The LCDR3 of the sequence of amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions).

[0462] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the IMGT numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 whose sequence is SEQ ID NO:10, HCDR2 whose sequence is SEQ ID NO:11, and HCDR3 whose sequence is SEQ ID NO:12; and / or Or, a light chain variable region (VL) comprising the following 3 CDRs: LCDR1 whose sequence is SEQ ID NO: 20, LCDR2 whose sequence is GTF, and LCDR3 whose sequence is SEQ ID NO: 22; (b) includes the following 3 The heavy chain variable region (VH) of the CDR: HCDR1 whose sequence is SEQ ID NO: 10, HCDR2 whose sequence is SEQ ID NO: 11, and HCDR3 whose sequence is SEQ ID NO: 12; and / or, comprising the following 3 CDRs The light chain variable region (VL): the sequence is LCDR1 of SEQ ID NO: 40, the sequence is LCDR2 of GAS, the sequence is LCDR3 of SEQ ID NO: 42; (c) the heavy chain variable region comprising the following 3 CDRs (VH): HCDR1 whose sequence is SEQ ID NO: 30, HCDR2 whose sequence is SEQ ID NO: 31, and HCDR3 whose sequence is SEQ ID NO: 32; and / or, a light chain variable region comprising the following 3 CDRs ( VL): the sequence is LCDR1 of SEQ ID NO:40, the sequence is LCDR2 of GAS, the sequence is LCDR3 of SEQ ID NO:42; or (d) the heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is HCDR1 of SEQ ID NO: 30, HCDR2 of SEQ ID NO: 31, HCDR3 of SEQ ID NO: 32; and / or, a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 32 LCDR1 with ID NO:20, LCDR2 with sequence GTF, and LCDR3 with sequence SEQ ID NO:22.

[0463] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Chothia numbering system: (a) A heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 4 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence HCDR1, the sequence is SEQ ID NO: 5 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence HCDR2, the sequence is SEQ ID NO: 6 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the HCDR3 sequence; and / or, a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 14 or has one compared thereto or LCDR1 of a sequence of substitutions, deletions or additions of several amino acids (such as substitutions, deletions or additions of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 15 or has one compared thereto or LCDR2 of a sequence of substitutions, deletions or additions of several amino acids (such as substitutions, deletions or additions of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 16 or has one compared thereto or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) sequence LCDR3; (b) heavy chain variable comprising the following 3 CDRs Region (VH): the sequence is SEQ ID NO: 4 or has one or several amino acid substitutions, deletions or additions compared thereto (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) HCDR1 of the sequence, the sequence is SEQ ID NO: 5 or has one or several amino acid substitutions, deletions or additions compared thereto (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) The sequence of HCDR2, the sequence is SEQ ID NO: 6 or has one or several amino acid substitutions, deletions or additions compared thereto (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) HCDR3 of the sequence; and / or, the light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 34 or it has one or several amino acid substitutions, deletions or additions ( Such as 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR1 of the sequence, the sequence is SEQ ID NO: 35 or it has one or several amino acid substitutions, deletions or additions ( Such as 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2 of the sequence, the sequence is SEQ ID NO: 36 or has one or several amino acid substitutions, deletions or additions ( For example, 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR3 sequence; (c) heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 24 or Compared with HCDR1 having a sequence of one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions), the sequence is SEQ ID NO: 25 or Compared with HCDR2 having a sequence of one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions), the sequence is SEQ ID NO: 26 or HCDR3 with a sequence of one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to it; and / or, comprising the following 3 Light chain variable region (VL) of the CDR: the sequence is SEQ ID NO: 34 or has one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2 or 3 amino acid Substitutions, deletions or additions) of the LCDR1 sequence of SEQ ID NO: 35 or a substitution, deletion or addition of one or several amino acids compared thereto (e.g. 1, 2 or 3 amino acids) substitutions, deletions or additions) of the LCDR2 sequence of SEQ ID NO: 36 or a substitution, deletion or addition of one or several amino acids compared thereto (e.g. of 1, 2 or 3 amino acids) Substitution, deletion or addition) of the LCDR3 of the sequence; or (d) the heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 24 or one or several amino acids compared thereto HCDR1 of a sequence of substitutions, deletions or additions (such as substitutions, deletions or additions of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 25 or one or several amino acids compared thereto HCDR2 of a sequence of substitutions, deletions or additions (such as substitutions, deletions or additions of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 26 or one or several amino acids compared thereto HCDR3 with a sequence of substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions); and / or, a light chain variable region (VL) comprising the following 3 CDRs: sequence LCDR1 which is SEQ ID NO: 14 or a sequence having a substitution, deletion or addition of one or several amino acids (for example a substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, the sequence LCDR2 of SEQ ID NO: 15 or a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, the sequence LCDR3 which is SEQ ID NO: 16 or a sequence having one or several amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.

[0464] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the chothia numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 whose sequence is SEQ ID NO:4, HCDR2 whose sequence is SEQ ID NO:5, and HCDR3 whose sequence is SEQ ID NO:6; and / or Or, a light chain variable region (VL) comprising the following 3 CDRs: LCDR1 whose sequence is SEQ ID NO: 14, LCDR2 whose sequence is SEQ ID NO: 15, and LCDR3 whose sequence is SEQ ID NO: 16; (b) A heavy chain variable region (VH) comprising the following three CDRs: HCDR1 whose sequence is SEQ ID NO:24, HCDR2 whose sequence is SEQ ID NO:25, and HCDR3 whose sequence is SEQ ID NO:26; and / or, comprising The light chain variable region (VL) of the following three CDRs: LCDR1 whose sequence is SEQ ID NO:34, LCDR2 whose sequence is SEQ ID NO:35, and LCDR3 whose sequence is SEQ ID NO:36; (c) comprising the following 3 The heavy chain variable region (VH) of a CDR: the sequence is HCDR1 of SEQ ID NO: 4, the sequence is HCDR2 of SEQ ID NO: 5, and the sequence is HCDR3 of SEQ ID NO: 6; and / or, comprising the following three The light chain variable region (VL) of the CDR: LCDR1 whose sequence is SEQ ID NO:34, LCDR2 whose sequence is SEQ ID NO:35, and LCDR3 whose sequence is SEQ ID NO:36; or (d) contains the following 3 CDRs The heavy chain variable region (VH): the sequence is HCDR1 of SEQ ID NO: 24, the sequence is HCDR2 of SEQ ID NO: 25, the sequence is HCDR3 of SEQ ID NO: 26; and / or, comprising the following 3 CDRs Light chain variable region (VL): the sequence is LCDR1 of SEQ ID NO:14, the sequence is LCDR2 of SEQ ID NO:15, and the sequence is LCDR3 of SEQ ID NO:16.

[0465] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the kabat numbering system: (a) Heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 7 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence HCDR1, the sequence is SEQ ID NO: 8 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence HCDR2, the sequence is SEQ ID NO: 9 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the HCDR3 sequence; and / or, a light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 17 or has one compared thereto or LCDR1 of a sequence of substitutions, deletions or additions of several amino acids (such as substitutions, deletions or additions of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 18 or has one compared thereto or LCDR2 of a sequence of substitutions, deletions or additions of several amino acids (such as substitutions, deletions or additions of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 19 or has one compared thereto or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) sequence LCDR3; (b) heavy chain variable comprising the following 3 CDRs Region (VH): the sequence is SEQ ID NO: 27 or has one or several amino acid substitutions, deletions or additions compared thereto (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) HCDR1 of the sequence, the sequence is SEQ ID NO: 28 or has one or several amino acid substitutions, deletions or additions compared thereto (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) HCDR2 of the sequence, the sequence is SEQ ID NO: 29 or it has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) HCDR3 of the sequence; and / or, the light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 37 or it has one or several amino acid substitutions, deletions or additions ( Such as 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR1 of the sequence, the sequence is SEQ ID NO: 38 or has one or several amino acid substitutions, deletions or additions ( For example, LCDR2 of a sequence of 1, 2 or 3 amino acid substitutions, deletions or additions), the sequence is SEQ ID NO: 39 or has one or several amino acid substitutions, deletions or additions ( For example, 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR3 sequence; (c) heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 7 or Compared with HCDR1 having a sequence of one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions), the sequence is SEQ ID NO: 8 or Compared with HCDR2 having a sequence of one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions), the sequence is SEQ ID NO: 9 or HCDR3 with a sequence of one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to it; and / or, comprising the following 3 The light chain variable region (VL) of the CDR: the sequence is SEQ ID NO: 37 or has one or several amino acid substitutions, deletions or additions compared thereto (e.g. 1, 2 or 3 amino acid Substitutions, deletions or additions) of the sequence LCDR1, the sequence is SEQ ID NO: 38 or a substitution, deletion or addition of one or several amino acids compared thereto (e.g. 1, 2 or 3 amino acids Substitutions, deletions or additions) of the LCDR2 sequence of SEQ ID NO: 39 or a substitution, deletion or addition of one or several amino acids compared thereto (e.g. 1, 2 or 3 amino acids Substitution, deletion or addition) of the LCDR3 of the sequence; or (d) the heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 27 or one or several amino acids compared thereto HCDR1 of a sequence of substitutions, deletions or additions (such as substitutions, deletions or additions of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 28 or one or several amino acids compared thereto HCDR2 of a sequence of substitutions, deletions or additions (such as substitutions, deletions or additions of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 29 or one or several amino acids compared thereto HCDR3 with a sequence of substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acid substitutions, deletions, or additions); and / or, a light chain variable region (VL) comprising the following 3 CDRs: sequence LCDR1 which is SEQ ID NO: 17 or a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, the sequence LCDR2 of SEQ ID NO: 18 or a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto, the sequence LCDR3 which is SEQ ID NO: 19 or a sequence having one or several amino acid substitutions, deletions or additions (eg, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto. In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows, wherein the CDRs are defined by the kabat numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 whose sequence is SEQ ID NO: 7, HCDR2 whose sequence is SEQ ID NO: 8, and HCDR3 whose sequence is SEQ ID NO: 9; and / or Or, a light chain variable region (VL) comprising the following 3 CDRs: LCDR1 whose sequence is SEQ ID NO: 17, LCDR2 whose sequence is SEQ ID NO: 18, and LCDR3 whose sequence is SEQ ID NO: 19; (b) A heavy chain variable region (VH) comprising the following three CDRs: HCDR1 whose sequence is SEQ ID NO:27, HCDR2 whose sequence is SEQ ID NO:28, and HCDR3 whose sequence is SEQ ID NO:29; and / or, comprising The light chain variable region (VL) of the following 3 CDRs: LCDR1 whose sequence is SEQ ID NO:37, LCDR2 whose sequence is SEQ ID NO:38, and LCDR3 whose sequence is SEQ ID NO:39; (c) comprising the following 3 The heavy chain variable region (VH) of a CDR: the sequence is HCDR1 of SEQ ID NO: 7, the sequence is HCDR2 of SEQ ID NO: 8, and the sequence is HCDR3 of SEQ ID NO: 9; and / or, comprising the following three The light chain variable region (VL) of the CDR: LCDR1 whose sequence is SEQ ID NO:37, LCDR2 whose sequence is SEQ ID NO:38, and LCDR3 whose sequence is SEQ ID NO:39; or (d) contains the following 3 CDRs The heavy chain variable region (VH): the sequence is HCDR1 of SEQ ID NO: 27, the sequence is HCDR2 of SEQ ID NO: 28, the sequence is HCDR3 of SEQ ID NO: 29; and / or, comprising the following 3 CDRs Light chain variable region (VL): the sequence is LCDR1 of SEQ ID NO:17, the sequence is LCDR2 of SEQ ID NO:18, and the sequence is LCDR3 of SEQ ID NO:19.

[0467] In certain embodiments, an antibody of the present disclosure or an antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein, with the aforementioned IMGT, chothia or Compared with the CDR defined by kabat, at least one CDR in the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation, and the mutation is a substitution or deletion of one or several amino acids Or addition or any combination thereof (eg substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof).

[0468] Preferably, the substitutions described in this disclosure are conservative substitutions.

[0469] In certain embodiments, the VH of an antibody of the present disclosure or an antigen-binding fragment thereof comprises a framework region (FR) derived from a heavy chain variable region (VH) of a human immunoglobulin, and / or the antibody The VL of an antigen-binding fragment thereof comprises a framework region (FR) derived from a light chain variable region (VL) of a human immunoglobulin. Accordingly, in certain embodiments, the disclosed antibodies or antigen-binding fragments thereof are humanized. In certain embodiments, the disclosed antibodies or antigen-binding fragments thereof are fully human.

[0470] In certain embodiments, the VH of an antibody of the present disclosure or an antigen-binding fragment thereof comprises a framework region (FR) derived from a heavy chain variable region (VH) of a human immunoglobulin, and / or the antibody The VL of an antigen-binding fragment thereof comprises a framework region (FR) derived from a light chain variable region (VL) of a human immunoglobulin. Accordingly, in certain embodiments, the disclosed antibodies or antigen-binding fragments thereof are humanized. In certain embodiments, the disclosed antibodies or antigen-binding fragments thereof are fully human.

[0471] In certain embodiments, an antibody of the disclosure, or antigen-binding fragment thereof, comprises: (a) a heavy chain framework region of a human immunoglobulin, or a variant thereof, associated with the germline antibody from which it is derived Conservative substitutions of up to 20 amino acids compared to the amino acid sequence encoded by the gene (e.g. conservative substitutions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions); and / or (b) a light chain framework region of a human immunoglobulin or a variant thereof The variant has a conservative substitution of up to 20 amino acids (e.g., up to 20, up to 15, up to 10, or up to 5 amines) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived A conservative substitution of amino acids; for example, a conservative substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids). In certain embodiments, the degree of humanization of an antibody or antigen-binding fragment thereof of the present disclosure is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0473] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (i) SEQ ID NO : the sequence shown in 3 or 23; (ii) compared with the sequence shown in SEQ ID NO: 3 or 23, there is one or several amino acid substitutions, deletions or additions or any combination thereof (such as 1, 2 , 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) with SEQ ID NO :3 or 23 compared to the sequence shown in at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% , a sequence of at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of: ( iv) the sequence shown in SEQ ID NO: 13 or 33; (v) compared with the sequence shown in SEQ ID NO: 13 or 33, there is one or several amino acid substitutions, deletions or additions or any combination thereof ( For example, a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi ) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% compared to the sequence shown in SEQ ID NO: 13 or 33 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0474] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a VH set forth in SEQ ID NO:3, and / or, a VL set forth in SEQ ID NO:13.

[0475] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a VH set forth in SEQ ID NO:23, and / or, a VL set forth in SEQ ID NO:33.

[0476] In certain embodiments, an antibody of the present disclosure or an antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO:3, and / or, a VL set forth in SEQ ID NO:33.

[0477] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a VH set forth in SEQ ID NO:23, and / or, a VL set forth in SEQ ID NO:13.

[0478] In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) VH of the sequence shown in SEQ ID NO:3 and VL of the sequence shown in SEQ ID NO:13; (b) SEQ ID NO: The VH of the sequence shown in ID NO:23 and the VL of the sequence shown in SEQ ID NO:33; (c) the VH of the sequence shown in SEQ ID NO:3 and the VL of the sequence shown in SEQ ID NO:33; (d) SEQ ID NO:33 VH of the sequence shown in ID NO:23 and VL of the sequence shown in SEQ ID NO:13. (e) heavy chain variable region (VH) and light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) independently have at least 70%, at least 80%, at least 85%, at least 90% compared with the VH and VL described in any group (a) to (f). %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (f) heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) are the same as the VH described in any one of (a) to (d) Compared with VL respectively, there are independently one or several amino acid substitutions, deletions or additions or any combination thereof (such as 1, 2, 3, 4, 5, 6, 7, 8 1, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof). Preferably, said substitution is a conservative substitution.

[0479] In certain embodiments, the heavy chain of an antibody of the present disclosure, or antigen-binding fragment thereof, comprises a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof that is associated with the wild-type Conservative substitutions of up to 50 amino acids (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 A conservative substitution of amino acids; for example, a conservative substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). In certain embodiments, the light chain of an antibody or antigen-binding fragment thereof of the present disclosure comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof that is similar to the wild-type sequence from which it was derived. Conservative substitutions with up to 50 amino acids (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amine groups Conservative substitutions of acids; for example, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).

[0480] In some embodiments, the constant region is altered, e.g., mutated, to modify the properties of the anti-B7H3 antibody molecule (e.g., alter one or more of the following properties: Fc receptor binding, antibody glycosylation, cysteine amino acid residues, effector cell function or complement function). Effector function can be altered by substituting at least one amino acid residue in the constant region of the antibody with a different residue, e.g., altering the affinity of the antibody for an effector ligand such as FcR or complement C1q ( e.g. lower). The Fc region of an antibody mediates several important effector functions such as ADCC, phagocytosis (ADCP), CDC, etc. In certain embodiments, an antibody of the disclosure, or antigen-binding fragment thereof, has a heavy chain constant region (CH) selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE. Heavy chain constant region; in particular a heavy chain constant region selected from eg IgGl, IgG2, IgG3 and IgG4, more particularly a heavy chain constant region selected from IgGl (eg human IgGl). In some embodiments, the human IgG1 heavy chain constant region is set forth in SEQ ID NO:43. In some embodiments, an antibody of the disclosure, or an antigen-binding fragment thereof, has a light chain constant region selected from, for example, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (eg, a human kappa light chain constant region). In some embodiments, the light chain constant region has the sequence set forth in SEQ ID NO:44. In some embodiments, the antibody or antigen-binding fragment thereof comprises CH set forth in SEQ ID NO: 43 or a variant thereof having up to 20 amine groups compared to SEQ ID NO: 43 Conservative substitutions of acids (e.g. conservative substitutions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0483] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region or variant thereof. In some embodiments, the light chain constant region comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises a light chain constant region (CL) set forth in SEQ ID NO: 44, or a variant thereof having up to 20 amines compared to SEQ ID NO: 44 Conservative substitutions of amino acids (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 amino acid conservative substitutions), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% compared to SEQ ID NO: 16 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

[0484] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO:43 and a light chain constant region (CL) set forth in SEQ ID NO:44.

[0485] In certain embodiments, the disclosed antibody or antigen-binding fragment thereof comprises: (a) a heavy chain comprising an amino acid sequence selected from the group consisting of: (i) comprising SEQ ID NO:3 The sequence of the VH sequence and the CH sequence shown in SEQ ID NO:43; (ii) compared with the sequence shown in (i), there is one or several amino acid substitutions, deletions or additions or any combination thereof (for example, at most 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions or any of them Combinations; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence of at least 97%, at least 98%, or at least 99% sequence identity; and (b) a light chain comprising an amino acid sequence selected from the group consisting of: (iv) comprising the VL sequence shown in SEQ ID NO:13 and the sequence of the CL sequence shown in SEQ ID NO:44; (v) has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50 amino acids) compared to the sequence shown in (iv) , up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; For example, a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi ) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% compared to the sequence shown in (iv) %, at least 98%, or at least 99% sequence identity.

[0486] In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0487] In certain embodiments, an antibody of the present disclosure or an antigen-binding fragment thereof comprises: (a) a heavy chain comprising an amino acid sequence selected from the group consisting of: (i) comprising the amino acid sequence shown in SEQ ID NO:23 The sequence of the VH sequence and the CH sequence shown in SEQ ID NO:43; (ii) compared with the sequence shown in (i), there is one or several amino acid substitutions, deletions or additions or any combination thereof (for example, at most 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions, or additions or any of them Combinations; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence of at least 97%, at least 98%, or at least 99% sequence identity; and (b) a light chain comprising an amino acid sequence selected from the group consisting of: (iv) comprising the VL sequence shown in SEQ ID NO:33 and the sequence of the CL sequence shown in SEQ ID NO:44; (v) has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50 amino acids) compared to the sequence shown in (iv) , up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; For example, a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi ) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% compared to the sequence shown in (iv) %, at least 98%, or at least 99% sequence identity.

[0488] In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0489] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a heavy chain and a light chain, the heavy chain comprising: (i) the sequence shown in SEQ ID NO: 45; (ii) with ( i) substitutions, deletions or additions of one or several amino acids or any combination thereof (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25) compared to the indicated sequence substitutions, deletions or additions of, at most 20, at most 15, at most 10 or at most 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) has at least 80%, at least 85%, at least A sequence of 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and the light chain Comprising: (iv) the sequence shown in SEQ ID NO:46; (v) compared with the sequence shown in (iv), there is one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50 Substitutions, deletions, or additions of, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids, or any combination thereof ; such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or ( vi) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% of the sequence shown in (iv) , sequences with at least 98% or at least 99% sequence identity; preferably, the substitutions described in (ii) or (v) are conservative substitutions.

[0490] In certain embodiments, an antibody of the present disclosure, or an antigen-binding fragment thereof, comprises a heavy chain and a light chain, the heavy chain comprising: (i) the sequence shown in SEQ ID NO: 47; (ii) with ( i) substitutions, deletions or additions of one or several amino acids or any combination thereof (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25) compared to the indicated sequence substitutions, deletions or additions of, at most 20, at most 15, at most 10 or at most 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) has at least 80%, at least 85%, at least A sequence of 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and the light chain Comprising: (iv) the sequence shown in SEQ ID NO:48; (v) compared with the sequence shown in (iv), there is one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50 Substitutions, deletions, or additions of, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids, or any combination thereof ; such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or ( vi) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% of the sequence shown in (iv) , sequences with at least 98% or at least 99% sequence identity; preferably, the substitutions described in (ii) or (v) are conservative substitutions.

[0491] In certain embodiments, antibodies of the present disclosure are chimeric antibodies, humanized antibodies or fully human antibodies. In certain embodiments, the disclosed antibodies or antigen-binding fragments thereof are selected from scFv, Fab, Fab', (Fab')2, Fv fragments, disulfide-linked Fv (dsFv), diabodies.

[0492] In certain embodiments, an antibody of the disclosure is a scFv. In certain embodiments, the scFv of the present disclosure comprises: (a) VH of the sequence shown in SEQ ID NO:3 and VL of the sequence shown in SEQ ID NO:13; (b) VL of the sequence shown in SEQ ID NO:23 VH and the VL of the sequence shown in SEQ ID NO:33; (c) the VH of the sequence shown in SEQ ID NO:3 and the VL of the sequence shown in SEQ ID NO:33; (d) the sequence shown in SEQ ID NO:23 VH and VL of the sequence shown in SEQ ID NO: 13. (e) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable Region (VL) independently has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% compared with VH and VL described in any group (a) to (d). %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or (f) the heavy chain variable region (VH) and the light chain may Variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) are compared with VH and VL described in any one of (a) to (d), respectively, independently Substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amines amino acid substitutions, deletions or additions or any combination thereof). Preferably, said substitution is a conservative substitution.

[0493] In certain embodiments, an antibody of the disclosure is a scFv. In certain embodiments, the scFv of the present disclosure comprises: (i) a sequence shown in SEQ ID NO: 1 or 2; (ii) a sequence having one or more amino acids compared to the sequence shown in (i). Substitutions, deletions or additions or any combination thereof (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 Substitution, deletion or addition of amino acids or any combination thereof; for example, substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, deletion or addition or any combination thereof); or (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% of the sequence shown in (i) %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; preferably, the substitutions described in (ii) are conservative substitutions.

[0494] In certain embodiments, an antibody of the disclosure is a scFv.

[0495] In certain embodiments, a scFv of the disclosure comprises the sequence set forth in SEQ ID NO: 1 or 2.

[0496] Antibody derivatives

[0497] An antibody or antigen-binding fragment thereof of the disclosure can be derivatized, eg, linked to another molecule (eg, another polypeptide or protein). Typically, derivatization (eg, labeling) of an antibody or antigen-binding fragment thereof will not adversely affect its binding to B7H3, particularly human B7H3. Accordingly, the disclosed antibodies or antigen-binding fragments thereof are also intended to include such derivatized forms. For example, an antibody of the disclosure, or an antigen-binding fragment thereof, can be linked (by chemical coupling, genetic fusion, non-covalent linkage, or otherwise) to one or more other molecular moieties, such as another antibody (e.g., to form a bispecific Antibodies), detection reagents, pharmaceutical reagents, and / or proteins or polypeptides that mediate the binding of an antibody or antigen-binding fragment thereof to another molecule (eg, avidin or polyhistidine tags).

[0498] One type of derivatized antibody (eg, a bispecific antibody) is produced by cross-linking two or more antibodies (of the same type or of different types). Methods for obtaining bispecific antibodies are well known in the art, examples of which include, but are not limited to, chemical cross-linking methods, cell engineering methods (hybridoma methods) or genetic engineering methods.

[0499] Another type of derivatized antibody is a labeled antibody. For example, an antibody of the disclosure, or an antigen-binding fragment thereof, can be linked to a detectable label. A detectable label as described in the present disclosure can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclide Chlorine (for example, 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), luciferin, tetramethylrhodamine isothiocyanate (TRITC), algal Erythropoin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g. Cy7, Alexa 750), acridinium esters, magnetic beads (e.g. Dynabeads®), calorimetric labels Substances such as colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the above-mentioned label-modified avidin (eg, streptavidin). Patents that teach the use of such markers include, but are not limited to, US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; Detectable labels as described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation counters, and fluorescent labels can be detected using photodetectors to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and thermometric labels are detected by simply visualizing a colored label. In certain embodiments, such labels can be adapted for use in immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.). In certain embodiments, a detectable label as described above can be attached to an antibody or antigen-binding fragment thereof of the present disclosure via linkers of various lengths to reduce potential steric hindrance.

[0500] In addition, the disclosed antibodies or antigen-binding fragments thereof may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life. Antigen-binding fragments of the present disclosure can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 ( 1985)). Alternatively, these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000 )). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). In addition, Fv, Fab or F(ab')2 fragments can also be directly isolated from the culture medium of recombinant host cells. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

[0502] The IgG isotype control antibodies of the present disclosure are well known and can be purchased or prepared by those of ordinary skill in the art. For example, human anti-Hen Egg Lysozyme IgG (anti-HEL, such as human IgG1, referred to as hIgG1), the sequence of which comes from Affinity maturation increases the stability and plasticity of the Fv domain of Variable region sequence of Fab F10.6.6 sequence from anti-protein antibodies study (Acierno et al. J Mol Biol. 2007; 374(1):130-46.). The preparation method is as follows: entrust Nanjing GenScript to optimize the amino acid codons and gene synthesis of the heavy and light chain (full sequence or variable region) genes of human IgG antibodies, refer to the "Molecular Cloning Experiment Guide (Third Edition)" "Standard techniques introduced in ", use standard molecular cloning techniques such as PCR, enzyme digestion, DNA gel recovery, ligation transformation, colony PCR or enzyme digestion identification to subclone the heavy and light chain genes into the antibody heavy chain expression vector of the mammalian expression system and antibody light chain expression vector, and further sequenced and analyzed the heavy and light chain genes of the recombinant expression vector. After the sequence verification was correct, a large number of endotoxin-free expression plasmids were prepared, and the heavy and light chain expression plasmids were transiently co-transfected into HEK293 cells for expression of recombinant antibodies. After 7 days of culture, the cell culture fluid was collected and subjected to affinity purification with rProtein A column (GE). The quality of the harvested antibody samples was identified by SDS-PAGE and SEC-HPLC standard analysis techniques.

[0503] In a ninth aspect, the disclosure provides a multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of the eighth aspect of the disclosure, and an additional antibody or fragment or antibody analog thereof .

[0504] In certain embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

[0505] Accordingly, in a tenth aspect, the present disclosure provides an isolated nucleic acid molecule comprising an antibody encoding the disclosure, or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof, or The nucleotide sequence of one or more CDRs thereof. According to the codon degeneracy known in the art, in some embodiments, the nucleotide sequence can be substituted according to the codon degeneracy. In certain embodiments, the nucleotide sequence is codon optimized. In certain embodiments, the isolated nucleic acid molecules of the present disclosure comprise: (i) a first nucleic acid encoding a heavy chain variable region and a light chain variable region, respectively, of an antibody of the disclosure or an antigen-binding fragment thereof and a second nucleic acid, or (ii) a first nucleic acid encoding a heavy chain variable region and a heavy chain constant region of an antibody of the present disclosure or an antigen-binding fragment thereof, respectively, and a second nucleic acid that encodes a light chain variable region and a light chain constant region nucleic acid, or (iii) a first nucleic acid and a second nucleic acid encoding a heavy chain and a light chain, respectively, of an antibody of the present disclosure or an antigen-binding fragment thereof. In some embodiments, the first nucleic acid and the second nucleic acid comprise degenerate sequences or substantially identical sequences to any of the first nucleic acid and the second nucleic acid in (i)-(iii) above. In certain embodiments, the degenerate sequence or substantially identical sequence refers to having at least about 85%, 90%, 95%, 99% or more of the nucleic acid molecules described in (i)-(iii) Sequences with sequence identity or sequences with one or more nucleotide substitutions, or sequences that differ by no more than 3, 6, 15, 30 or 45 nucleotides.

[0507] In an eleventh aspect, there is provided a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present disclosure. In certain embodiments, vectors of the present disclosure are, for example, plasmids, cosmids, phage, lentiviruses, and the like. In certain embodiments, the vector is capable of expressing an antibody of the disclosure or an antigen-binding fragment thereof in a subject (eg, a mammal, eg, a human).

[0508] In a twelfth aspect, a host cell comprising an isolated nucleic acid molecule of the present disclosure or a vector of the present disclosure is provided. Host cells can be eukaryotic cells (eg, mammalian cells, insect cells, yeast cells) or prokaryotic cells (eg, E. coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, a host cell of the present disclosure is a mammalian cell, such as a CHO (eg, CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0509] In a thirteenth aspect, there is provided a method for preparing an antibody or an antigen-binding fragment thereof, or a multispecific antibody of the present disclosure, comprising, in a method that allows expression of the antibody or an antigen-binding fragment thereof, or a multispecific antibody conditions, a host cell of the disclosure is cultured, and the antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.

[0510] In a fourteenth aspect, an antibody-drug conjugate is provided, wherein the antibody is the above-mentioned anti-B7H3 antibody, which is connected to the coupling moiety through a linker. The coupling part is selected from: detectable labels, radioactive isotopes, fluorescent substances, luminescent substances, colored substances, enzymes, polyethylene glycol (PEG), nuclides, nucleic acids, small molecule toxins, polypeptides with binding activity, Proteins, receptors, ligands, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

[0511] In some embodiments, the antibody-drug conjugate comprises an anti-B7H3 antibody-drug conjugate (B7H3-ADC) of the formula: Ab-(L-D)n, wherein: Ab is the antibody of the eighth aspect or an antigen binding fragment, and; D is a small molecule toxin drug moiety; L is a bond or linker molecule that covalently links Ab and D; n is an integer between 1-16 and represents the number of L-Ds covalently linked to Ab .

[0512] In some embodiments, L of the antibody-drug conjugate comprises an amino acid residue or a short peptide consisting of 2-10 amino acid residues; the amino acid residue is selected from From natural amino acid residues, unnatural amino acid residues, amino acid residues shown in AA1 or their stereoisomers; amino acid residues shown in AA1: Ra1 and Rb are each independently selected from H,, and; Or, Ra1 and Rb and the carbon atoms connected to them together form a 4-10 membered heterocyclic ring, and in the 4-10 membered heterocyclic ring, the heteroatom is selected from 1 of O, N and S 1, 2, 3 or 4; the 4-10 membered heterocycle is optionally substituted by one or more R0; r, r1, r1a and r1b are each independently any integer from 0 to 20; Rm1, Rn1, Rm1a, Rn1a, Rm1b and Rn1b are each independently H, C1-6 alkyl, C3-6 cycloalkyl or -COORx1, wherein Rx1 is C1-6 alkyl; or, Rm1 and Rn1, Rm1a Together with Rn1a, Rm1b and Rn1b and the nitrogen atoms connected to them, a 4-10 membered heterocyclic ring is formed, and in the 4-10 membered heterocyclic ring, the heteroatom is selected from one of O, N and S, 2, 3 or 4; the 4-10 membered heterocycle is optionally substituted by one or more R0'; Rz is selected from C1-6 alkyl; R0, R0' are each independently selected from C1 -6 alkyl, C3-6 cycloalkyl, -NRm2Rn2 and 4-10 membered heterocyclic group optionally substituted by C1-6 alkyl; in the 4-10 membered heterocyclic ring, the heteroatom is selected from O, 1, 2, 3 or 4 of N and S; Rm2 and Rn2 are each independently selected from H and C1-6 alkyl.

[0513] In some embodiments, in the amino acid residue shown in AA1, Ra1 and Rb and the carbon atoms connected to them together form a 5-6 membered heterocycle, and in the 5-6 membered heterocycle, The heteroatom is N, and the number of heteroatoms is 1 or 2; the 5-6 membered heterocycle is optionally substituted by one or more R0. The 5-6 membered heterocyclic ring is preferably a piperidine ring or a piperidine ring, more preferably a piperidine ring, for example or, the carbon atom No. 1 is the carbon atom connected to Ra1 and Rb. In some embodiments, r, r1, r1a and r1b are each independently 0, 1, 2, 3, 4 or 5; preferably, r, r1, r1a and r1b are each independently 0 or 4 ; More preferably, r is 0, r1, r1a and r1b are 4; or r is 4, r1, r1a and r1b are 0.

[0515] In some embodiments, r, r1, r1a, and r1b are not all zero at the same time.

[0516] In some embodiments, Rm1, Rn1, Rm1a, Rn1a, Rm1b and Rn1b are each independently H, C1-6 alkyl or -COORx1, wherein Rx1 is C1-6 alkyl; preferably each independently is H, methyl, ethyl, n-propyl, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC(CH3)3 or -COOCH2CH2CH2CH3.

[0517] In some embodiments, Rm1 and Rn1, Rm1a and Rn1a, and Rm1b and Rn1b form a 5-6 membered heterocyclic ring together with the nitrogen atom to which they are jointly attached, and in said 5-6 membered heterocyclic ring, hetero The atoms are selected from 1 or 2 N atoms; the 5-6 membered heterocyclic ring is optionally substituted by one or more R0'; the 5-6 membered heterocyclic ring is preferably a piperidine ring or a piperidine ring Ring; more preferably,,, or, the nitrogen atom No. 1 is the nitrogen atom jointly connected with Rm1 and Rn1.

[0518] In some embodiments, Rz is preferably C1-6 alkyl; more preferably methyl.

[0519] In some embodiments, R0 and R0' are each independently C1-6 alkyl, -NRm2Rn2 or a 5-6 membered heterocyclyl optionally substituted by C1-6 alkyl; the 5-6 In the membered heterocycle, the heteroatom is selected from 1 or 2 N atoms.

[0520] In some embodiments, R is preferably a C1-6 alkyl group or a 5-6 membered heterocyclic group substituted by a C1-6 alkyl group, and the 5-6 membered heterocyclic group is piperidinyl or piperidinyl Methyl; more preferably methyl or piperidinyl substituted by methyl; for example methyl or .

[0521] In some embodiments, R01' is each independently preferably C1-6 alkyl, -NRm2Rn2 or a 5-6 membered heterocyclic group optionally substituted by C1-6 alkyl; the 5-6 membered In the heterocycle, the heteroatom is 1 or 2 N; more preferably C1-6 alkyl or -NRm2Rn2; more preferably methyl or -NRm2Rn2, wherein Rm2 and Rn2 are each independently preferably H or C1-6 Alkyl, more preferably methyl.

[0522] In some embodiments, R0' is preferably C1-6 alkyl or -NRm2Rn2; Rm2 and Rn2 are each independently preferably H or C1-6 alkyl (e.g. methyl).

[0523] In some embodiments, in the amino acid residue shown in AA1, any one of Ra1 and Rb is H, and the other is and; Preferably, any one of Ra1 and Rb is H, and the other selected from.

[0524] In some embodiments, the amino acid residue shown in AA1 is preferably,,,,,,,,,,,,,,, or; more preferably,,,, or; more preferably,, ,,,; Most preferably, ,,.

[0525] In some embodiments, the antibody-drug conjugate consists of an antibody-linker-drug, wherein the drug is selected from the group consisting of microtubule blocking drugs, DNA damaging drugs, Bcl-xL inhibitors, and the like.

[0526] In some embodiments, the antibody-drug conjugate consists of an antibody-linker-drug, wherein the drug is selected from the group consisting of: Auristatins (e.g. MMAF, MMAE), maytansine derivatives (e.g. DM1, DM4), tubulysin, cryptocolistin, anti-mitotic inhibitors, pyrrole and indochlorobenzazepines, ducamycin, camptothecin, calicheamicin, etc. .

[0527] In some embodiments, the antibody-drug conjugate consists of antibody-linker-drug, wherein the drug is selected from camptothecin (CPT) and derivatives thereof.

[0528] In some embodiments, the antibody-drug conjugate consists of an antibody-linker-drug, wherein the drug is selected from a topoisomerase I inhibitor.

[0529] In some embodiments, the antibody-drug conjugate consists of an antibody-linker-drug, wherein the drug is selected from the group consisting of: SN-38, DXd.

[0530] In some embodiments, in the antibody-drug conjugate, D is a drug unit having formula II: , wherein, R1 is H or F, R2 is H or methyl; or R1 and R2 Formed together with the carbon atom connected to it; R3 is H; R4 is selected from H, -(C1-C4 alkyl)-OH, -(C1-C4 alkenyl)-OH, -(C1-C4 alkyl)-NH2 , or -(C1-C4 alkenyl)-NH2; wherein L is linked via a hydroxyl or amine group present on D.

[0531] In some embodiments, L of the antibody-drug conjugate is selected from; Wherein, 1 position is connected with Ab, 2 positions are connected with D; AA1 is as defined above.

[0532] In addition, it should be noted that those with ordinary knowledge in the technical field of the present invention can understand that L is related to the opening of the disulfide bond (for example, the reduction of the disulfide bond by the reducing agent TCEP can open the disulfide bond to generate The sulfhydryl group contained in the Ab (such as an antibody) itself is connected after the sulfhydryl-SH), that is to say, the -S- between L and Ab is not an additional external sulfur atom. For example, in, -S- is not an additional external sulfur atom, but -S- formed after the sulfhydryl group contained in Tb itself after opening the disulfide bond is connected with L.

[0533] In some preferred embodiments, the antibody-drug conjugate is selected from: Where Ab is B7H3 antibody 1D1-01 or 2E3-02, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, q is preferably 1 , 2, 3, 4, 5, 6, 7, 8.

[0534] In the fifteenth aspect of the present disclosure, the present disclosure provides a ligand-drug conjugate comprising the aforementioned ligand-drug conjugate or the aforementioned antibody-drug conjugate in the present disclosure, said The ligand drug conjugate has two or more q values, and the antibody-drug conjugate has two or more n values. The Ligand Drug Conjugate refers to a Ligand Drug Conjugate or Antibody-Drug Conjugate (ADC) comprising a heterogeneous distribution of DARs.

[0535] In some embodiments, when the ligand-drug conjugate has a q-value or n-value in the majority, the q-value or n-value is close to DAR.

[0536] In some embodiments, the drug-to-antibody ratio (DAR) in the Ligand Drug Conjugate is selected from integers or decimals from 1-10.

[0537] In some embodiments, the drug-to-antibody ratio (DAR) in the Ligand Drug Conjugate is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, and 9. In some embodiments, the Ligand Drug Conjugate ADC comprises ADCs having a distribution of DARs from 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, and 8 drug-loaded species). It is worth noting that degradation products can be produced, so that the DARs of 1, 3, 5 and 7 can also be included in the ligand drug conjugate. In addition, the ADC in the ligand drug conjugate may also have a DAR greater than 8. The ligand-drug conjugate is produced by reducing interchain disulfides and then coupling. In some embodiments, the ligand drug conjugate comprises the following two: the ligand drug conjugate with a DAR of 4 or lower (that is, the drug-loaded species is 4 or lower) and the DAR The ligand drug conjugate is 6 or higher (ie, the drug-loaded species is 6 or higher).

[0539] In another aspect, there is provided the use of an antibody of the present disclosure or an antigen-binding fragment thereof in the preparation of a kit for detecting the presence or level of B7H3 in a sample. In another aspect, the disclosure provides a diagnostic or therapeutic kit comprising one or more of the following: an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, Conjugates or pharmaceutical compositions. Optionally, the diagnostic or therapeutic kit also includes instructions for use.

[0540] In the sixteenth aspect of the disclosure, the disclosure provides a pharmaceutical composition, which includes substance A, and optionally one or more pharmaceutical excipients; the substance A is the aforementioned ligand drug couple The conjugate, or the stereoisomer of the ligand-drug conjugate, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or the aforementioned antibody or its antigen-binding fragment, Nucleic acid molecules, vectors, host cells, multispecific antibodies, and / or ligand-drug conjugates, conjugates, or the aforementioned antibody-drug conjugates, or the aforementioned compounds, or stereoisomers of the compounds body, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate. Said substance A may be in a therapeutically effective amount.

[0541] In certain embodiments, a pharmaceutical composition of the disclosure comprises an antibody of the disclosure, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0542] In certain embodiments, the pharmaceutical composition of the present disclosure comprises the host cell of the present disclosure, and a pharmaceutically acceptable carrier and / or excipient, wherein the host cell comprises the isolated Nucleic acid molecule or vector.

[0543] In certain embodiments, a pharmaceutical composition of the disclosure comprises a multispecific antibody of the disclosure, and a pharmaceutically acceptable carrier and / or excipient.

[0544] In certain embodiments, a pharmaceutical composition of the disclosure comprises a conjugate of the disclosure and a pharmaceutically acceptable carrier and / or excipient.

[0545] In the seventeenth aspect of the present disclosure, the present disclosure provides the use of the aforementioned substance A or the aforementioned pharmaceutical composition in the preparation of medicines for the treatment and / or prevention of diseases related to abnormal cell activity (such as cancer diseases) . The substance A or the aforementioned pharmaceutical composition may be in a therapeutically effective amount.

[0546] In some embodiments, there is provided an antibody of the present disclosure or an antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, an antibody-drug conjugate, or a use of a multispecific antibody in the preparation of a medicament for Modulates (inhibits or blocks) the activity of B7H3.

[0547] In some embodiments, there is provided an antibody of the present disclosure or an antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, an antibody-drug conjugate, or a use of a multispecific antibody in the preparation of a medicament for Treating or preventing diseases associated with the activity of B7H3.

[0548] In some embodiments, there is provided an antibody of the present disclosure or an antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, an antibody-drug conjugate, or a use of a multispecific antibody in the preparation of a medicament for Treating or preventing tumors associated with B7H3 activity. In some embodiments, the cancerous disease is selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, or lung adenocarcinoma) ), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer (such as human colon adenocarcinoma), rectal cancer, Colorectal cancer, liver cancer, renal cancer, urothelial cancer, epidermal cancer, non-Hodgkin's lymphoma, central nervous system tumor (eg, glioma, glioblastoma multiforme, glioma, or sarcoma) , prostate or thyroid cancer.

[0550] In some embodiments, the cancer disease is a cancer disease associated with Trop-2, Her2, B7H3, Her3, EGFR. In some embodiments, the cancer disease is a Trop-2 or Her2-related cancer disease.

[0551] In some embodiments, the cancer disease is a Trop-2 associated cancer disease.

[0552] In some embodiments, the cancer disease is a Her3-associated cancer disease.

[0553] In some embodiments, the cancer disease is a cancer disease associated with EGFR.

[0554] In some embodiments, the cancer disease is a B7H3-related cancer disease.

[0555] In some embodiments, the cancerous disease is a solid tumor.

[0556] In some embodiments, the cancerous disease is breast cancer or lung cancer (preferably non-small cell lung cancer).

[0557] In the seventeenth aspect of the present disclosure, the present disclosure provides the aforementioned substance A or the aforementioned pharmaceutical composition, which is used for treating and / or preventing diseases related to abnormal cell activity (such as cancer diseases). In some embodiments, the cancer disease is selected from esophageal cancer (such as esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer, or lung adenocarcinoma) ), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer (such as human colon adenocarcinoma), rectal cancer, Colorectal cancer, liver cancer, renal cancer, urothelial cancer, epidermal cancer, non-Hodgkin's lymphoma, central nervous system tumor (eg, glioma, glioblastoma multiforme, glioma, or sarcoma) , prostate or thyroid cancer.

[0559] In some embodiments, the cancer disease is a cancer disease associated with Trop-2, Her2, B7H3, Her3, EGFR. In some embodiments, the cancer disease is a Trop-2 or Her2-related cancer disease.

[0560] In some embodiments, the cancer disease is a Trop-2 associated cancer disease.

[0561] In some embodiments, the cancer disease is a Her3-associated cancer disease.

[0562] In some embodiments, the cancer disease is a cancer disease associated with EGFR.

[0563] In some embodiments, the cancer disease is a B7H3-related cancer disease.

[0564] In some embodiments, the cancerous disease is a solid tumor. In some embodiments, the cancer disease is breast cancer or lung cancer (preferably non-small cell lung cancer).

[0565] In the eighteenth aspect of the present disclosure, the present disclosure provides a method for preventing and / or treating a disease (such as a cancer disease) related to abnormal cell activity, which includes: giving an individual in need to prevent and / or treat an effective amount of the aforementioned substance A, or the aforementioned pharmaceutical composition. In some embodiments, the cancer disease is selected from esophageal cancer (such as esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer or lung adenocarcinoma) ), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer (such as human colon adenocarcinoma), rectal cancer, Colorectal cancer, liver cancer, renal cancer, urothelial cancer, epidermal cancer, non-Hodgkin's lymphoma, central nervous system tumor (eg, glioma, glioblastoma multiforme, glioma, or sarcoma) , prostate or thyroid cancer.

[0567] In some embodiments, the cancer disease is a cancer disease associated with Trop-2, Her2, B7H3, Her3, EGFR.

[0568] In some embodiments, the cancer disease is a Trop-2 or Her2-associated cancer disease.

[0569] In some embodiments, the cancer disease is a Trop-2 associated cancer disease.

[0570] In some embodiments, the cancer disease is a Her3-associated cancer disease.

[0571] In some embodiments, the cancer disease is a cancer disease associated with EGFR.

[0572] In some embodiments, the cancer disease is a B7H3-related cancer disease.

[0573] In some embodiments, the cancerous disease is a solid tumor.

[0574] In some embodiments, the cancerous disease is breast cancer or lung cancer (preferably non-small cell lung cancer).

[0575] In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. Moreover, the laboratory operation steps of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all routine steps widely used in the corresponding fields. Meanwhile, for a better understanding of the present disclosure, definitions and explanations of related terms are provided below.

[0576] As used herein, examples of the term "pharmaceutically acceptable salt" are organic acid addition salts formed from organic acids that form pharmaceutically acceptable anions, including but not limited to formate, acetate , propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, alkyl Sulfonate or arylsulfonate; preferably, the alkylsulfonate is methanesulfonate or ethylsulfonate; the arylsulfonate is benzenesulfonate or p-toluenesulfonic acid salt. Suitable inorganic salts may also be formed including, but not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, bicarbonate, and carbonate, sulfate or phosphate salts, and the like.

[0577] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient Agents, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ions Strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.

[0578] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid affording a pharmaceutically acceptable anion.

[0579] In this disclosure, the pharmaceutical excipients refer to the excipients and additives used in the production of medicines and prescriptions, and refer to the active ingredients, which have been reasonably evaluated in terms of safety, and include Substances in pharmaceutical preparations. In addition to shaping, acting as a carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubility enhancement, and sustained and controlled release. They are important components that may affect the quality, safety, and effectiveness of drugs. According to its source, it can be divided into natural products, semi-synthetics and full synthetics. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, Glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surface active Agents, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.; For oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration and eye administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different administration routes, and has different functions and uses.

[0580] The pharmaceutical composition can be made into various suitable dosage forms according to the route of administration. such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants Agents, aerosols, powders, sprays, etc. Wherein, the pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound of the present disclosure or a pharmaceutically acceptable salt or conjugate thereof, suitably containing 0.1 mg to 800 mg, preferably containing 0.5-500 mg, preferably Contains 0.5 to 350 mg, particularly preferably 1-250 mg.

[0581] The pharmaceutical composition can be administered in the form of injection, including injection, sterile powder for injection and concentrated solution for injection. Among them, usable vehicles and solvents include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, such as mono- or diglycerides, can be employed as a solvent or suspending medium.

[0582] The term "treating" as used herein generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms; and / or therapeutic in terms of partial or complete stabilization or cure of the disease and / or side effects due to the disease. "Treatment" as used herein encompasses any treatment of a disease in a patient, including: (a) prophylaxis of a disease or condition in a patient susceptible to the disease or condition but not yet diagnosed; (b) suppressing the symptoms of the disease, ie arresting its development; or (c) alleviating the symptoms of the disease, ie causing regression of the disease or symptoms.

[0583] In this disclosure, the term "individual" includes a human or a non-human animal. Exemplary human subjects include human subjects suffering from a disease (eg, a disease described herein) (referred to as a patient) or normal subjects. The term "non-human animal" in this disclosure includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0584] In this disclosure, the term "effective dose" refers to the amount of a compound which, when administered, alleviates to some extent one or more symptoms of the condition being treated.

[0585] In this disclosure, the term "ligand drug conjugate" refers to a substance obtained by linking a biologically active molecule (drug molecule) to a targeting moiety. In some embodiments of the present disclosure, the biologically active molecule is connected to the targeting moiety through a linker. The linker can be cleaved in a specific environment (such as intratumoral hydrolytic enzymes and / or low pH environment) or under a specific action (such as the action of lysosomal proteases), thereby separating the biologically active molecule from the targeting moiety. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the bioactive molecule is directly connected to the targeting moiety through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the ligand drug conjugate comprises a targeting moiety, a linker and a fragment of the compound of formula II of the present disclosure.

[0586] In the present disclosure, the term "biologically active substance", "bioactive molecule" or "drug molecule" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. In some embodiments of the present disclosure , the bioactive substance, bioactive molecule or drug molecule in the conjugate is a molecule with anti-tumor bioactivity. For example: radioisotopes such as those of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and Lu; metal complexes such as metal platinum complexes, metal gold complexes, oxaliplatin etc.; glycopeptide antibiotics, such as bleomycin, pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors, camptothecin, hydroxycamptothecin, 9-aminocamptothecin , SN-38, irinotecan, topotecan, belotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, doxorubicin, doxorubicin, docarmycin, Daunorubicin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fluoride Darabine, cladribine, nelarabine, etc.; drugs acting on structural proteins, such as tubulin inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel etc.; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; also include proteasome inhibitors, Histone deacetylase inhibitors, tumor angiogenesis inhibitors, cyclin inhibitors, maytansine derivatives, calicheamicin derivatives, auristatin derivatives, pyrrolobenzodiazepines (PBD) derivatives, Melphalan, mitomycin C, chlorambucil, or other active substances that inhibit tumor cell growth, promote tumor cell apoptosis and necrosis; enzymes and their fragments, such as nucleolytic enzymes; antibiotics; toxins, such as small Molecular toxins or enzymatically active toxins of bacterial, fungal, vegetable or animal origin, including fragments and / or variants thereof; growth inhibitors; pharmaceutical modules. The term "toxin" refers to a substance capable of having deleterious effects on the growth or proliferation of cells.

[0587] In this disclosure, the term "small molecule" refers to a small molecule drug that has biological activity. The term small-molecule toxin has cell-damaging activity, that is, the state of causing pathological changes in cells in some form, and cell damage is not limited to direct damage, including all kinds of damage to the structure and function of cells, such as DNA cleavage, base - Dimer formation, cleavage of chromosomes, damage to cell division machinery and decrease of different enzymatic activities.

[0588] In this disclosure, the term "linker" refers to a fragment that joins a biologically active molecule (drug molecule) with a targeting moiety.

[0589] In the present disclosure, the term "targeting moiety" refers to a portion of a conjugate that is capable of specifically binding to a target (or portion of a target) on the surface of a cell. Through the interaction of the targeting moiety with the target, the conjugate can be delivered to a specific cell population.

[0590] In this disclosure, when the targeting moiety in the conjugate is an antibody, the conjugate may be referred to as a "drug-antibody conjugate."

[0591] In the present disclosure, an antibody or antigen-binding fragment thereof includes a derivatized antibody or antigen-binding fragment thereof, such as an antibody or antigen-binding fragment thereof having a sulfhydryl group, wherein the derivatization allows the antibody to have a drug-linker conjugate Reactive group or ability. The sulfhydryl-SH can be derivatized by opening disulfide bonds (eg, by reduction with the reducing agent TCEP).

[0592] As used herein, the terms "cancer" and "tumor" are used with the same meaning.

[0593] The term "gene" as used herein includes not only DNA but also its mRNA, its cDNA and its cRNA.

[0594] As used herein, the term "polynucleotide" is used in the same sense as nucleic acid, and also includes DNA, RNA, probes, oligonucleotides, and primers.

[0595] The term "Tb" used herein is an abbreviation of "target binding", including an antibody or any molecule that binds to a target, and the term "Ab" is an abbreviation of "antibody", and is used indiscriminately from "antibody".

[0596] As used herein, the terms "polypeptide" and "protein" are used indiscriminately.

[0597] As used herein, the term "cell" also includes cells in an animal subject and cells in culture.

[0598] The B7H3 involved in the anti-B7H3 antibody described in the present disclosure can be conventional B7H3 in the art, such as soluble B7H3, membrane form B7H3, etc., and also represent B7H3 variant 1 and / or B7H3 variant 2.

[0599] KD refers to the ratio obtained from Kd (dissociation rate of specific binding molecule-target protein interaction) and Ka (on-rate of specific binding molecule-target protein interaction) (or Kd / Ka, in molar concentration ( M ) represents the dissociation constant of ). KD values ​​can be determined using methods well established in the art. A preferred method of determining the KD of a binding molecule is through the use of surface plasmon resonance, eg a biosensor system such as the Biacore™ (GE Healthcare Life Sciences) system.

[0600] The term "B7H3 variant" refers to a polypeptide that has a similar or identical function to a B7H3 polypeptide, B7H3 fragment, anti-B7H3 antibody or antibody fragment thereof, but does not necessarily comprise a similar or identical B7H3 polypeptide, B7H3 fragment , the amino acid sequence of an anti-B7H3 antibody or a fragment thereof, or have a structure similar or identical to a B7H3 polypeptide, a B7H3 fragment, an anti-B7H3 antibody or a fragment thereof.

[0601] The term "Her3 variant" refers to a polypeptide having similar or identical functions to Her3 polypeptides, Her3 fragments, anti-Her3 antibodies or antibody fragments thereof, but not necessarily comprising similar or identical Her3 polypeptides, Her3 fragments , the amino acid sequence of an anti-Her3 antibody or a fragment thereof, or have a structure similar or identical to a Her3 polypeptide, a Her3 fragment, an anti-Her3 antibody or a fragment thereof.

[0602] As used herein, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences. The percent sequence identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and each The length of the gap that needs to be introduced for optimal alignment of the two sequences. Sequence comparisons and determination of percent identity between sequences can be performed using methods generally known in the art, and can be accomplished using mathematical algorithms. For example, the algorithm of Meyers and Miller, 1988 Comput. Appl. Biosci. 4: 11-17 (integrated into the ALIGN program (version 2.0)) can be used to determine the distance between amino acid sequences and / or between nucleotide sequences. percent identity. In addition, the GAP program (using its default parameters) in the GCG software suite available online from Accelrys can be used to determine percent identity between amino acid sequences or between nucleotide sequences. In one embodiment, the two sequences are of equal length.

[0603] The term "epitope" refers to a part of a B7H3 polypeptide or protein that has antigenic or immunogenic activity in an animal body, preferably a mammal. B7H3 The epitope of the antibody of the present disclosure or its antigen-binding fragment can be determined by existing techniques, such as synthetic peptide method, immunoinformatics prediction, determination of polypeptide activity, epitope peptide scanning method, phage display technology, X-ray diffraction and NMR analysis, antibody homology modeling protein docking prediction method. The phrase "antibodies that bind the same epitope" as used herein means that different antibodies bind a common epitope. If the second antibody binds part of the peptide or part of the tertiary structure to which the first antibody binds, it can be determined that the first antibody and the second antibody bind the same epitope.

[0604] In this disclosure, the term "antibody" is used in its broadest sense to include intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies) formed from at least two intact antibodies, provided that They possess the desired biological activity. In this disclosure, "antibody" and "immunoglobulin" are used interchangeably. An "antibody molecule" or "antibody" as described herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds an antigen. Thus, the term antibody broadly encompasses not only whole antibody molecules, but also fragments of said antibodies and variants (including derivatives) of said antibodies and antibody fragments. When "antibody molecule" or "antibody" is used in the same context as an antigen-binding fragment, "antibody molecule" or "antibody" refers to an intact antibody molecule or a full-length antibody. The term antibody molecule used in this specification includes, but is not limited to, single chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') 2, disulfide bonded Fv (sdFv), Fv, and intact antibody or full-length antibodies. The term "single-chain Fv" or "scFv" refers to a polypeptide comprising the VL domain of an antibody linked to an antibody VH domain. For example, antibodies that immunospecifically bind B7H3 may cross-react with other antigens. Preferably, antibodies that immunospecifically bind B7H3 do not cross-react with other antigens. Antibodies that immunospecifically bind B7H3 can be identified, for example, by immunoassays or other methods known to those of ordinary skill in the art to which the invention pertains. "Intact" antibody or "full-length" antibody refers to a protein comprising two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, the protein comprising: ( 1) for a heavy chain, comprising a variable region (abbreviated herein as "VH") and a heavy chain constant region comprising three domains CH1, CH2, CH3; and (2) for a light chain, comprising a light chain The variable region (abbreviated herein as "VL") and the light chain constant region contains one domain, CL. Antibodies of the disclosure include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, for example, antibodies of the disclosure anti-Id antibody), and epitope-binding fragments of any of the above antibodies. Immunoglobulin molecules of the present disclosure may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin. Preferably, the antibody of the present disclosure comprises or consists of a VH domain having any amino acid sequence or a fragment or variant thereof described in the sequence and its specific information table, VH CDR (represented by HCDR herein), VL domain, Or VL CDR (in this paper, LCDR is often used).

[0605] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for minor natural mutations that may be present. Monoclonal antibodies have high specificity against one determinant (epitope) of an antigen, as opposed to polyclonal antibodies, which contain different antibodies directed against different determinants (epitopes). In addition to specificity, the advantage of monoclonal antibodies is that they can be synthesized without contamination from other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized as being from a substantially homogeneous antibody population, and should not be construed as having to be prepared by a specific method.

[0606] In some embodiments of the present disclosure, monoclonal antibodies also specifically include chimeric antibodies, that is, a part of the heavy chain and / or light chain is identical or homologous to a certain, a certain class or a certain subclass of antibodies, and the rest are then identical or homologous to another, another class or another subclass of antibodies, so long as they possess the desired biological activity (see for example US 4,816,567; and Morrison et al., 1984, PNAS, 81: 6851-6855) . Chimeric antibodies useful in the present disclosure include primatized antibodies comprising variable region antigen-binding sequences from a non-human primate (eg, archaic monkey, orangutan, etc.) and human constant region sequences.

[0607] The term "antigen-binding fragment" refers to a portion of an antibody, preferably the antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region fragments, diabodies, linear antibodies and single chain antibody molecules. The term "antigen-binding fragment" as used herein refers to a partial fragment of an antibody having antigen-binding activity, wherein the fragment has full or partial function of the antibody, including but not limited to single-chain Fv (scFv), Fab, Fab', F( ab')2, disulfide-linked Fv (sdFv), Fv, di-scFv, etc. The term also includes Fab', which is a monovalent fragment of the variable region of an antibody obtained by treatment of F(ab')2 under reducing conditions. However, the term is not limited to these molecules so long as the fragment has binding affinity for the antigen. In addition, these functional fragments include not only fragments obtained by treating full-length molecules of antibody proteins with appropriate enzymes, but also proteins produced in appropriate host cells using genetically modified antibody genes.

[0608] The term "Fab'" as used herein denotes a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions as described above. However, Fab' of the present disclosure also includes Fab' produced using genetically modified antibody genes.

[0609] As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker or directly (see, e.g., Bird et al. Human, Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-Linker-VH-COOH or NH2-VH-Linker-VL-COOH. Suitable prior art linkers consist of the repeating GGGGS amino acid sequence or variants thereof. For example, a linker with the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001 ), Cancer Immunol. In some cases, there may also be a disulfide bond between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.

[0610] As used herein, even if the antibody or antigen-binding fragment thereof contains a variant, amino acid substitution, deletion or addition, it still has the activity of binding the antigen.

[0611] The term "bispecific antibody", also known as "bifunctional antibody conjugate", refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a coupling arm, the The conjugates retain the activity of their respective antibodies, so they have dual functions and dual specificities.

[0612] The term "multispecific antibody" includes, for example, trispecific antibodies, which are antibodies with three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen-binding specificities.

[0613] The term "intact antibody" or "full-length antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2 and CH3). The constant region may be a native sequence (eg, a human native constant region sequence) or an amino acid sequence variant thereof. An intact antibody is preferably an intact antibody with one or more effector functions.

[0614] The term "probody" is a modified antibody, including an antibody or an antibody fragment, capable of specifically binding to its target, capable of coupling with a masking group, wherein the masking group refers to the antibody or The ability of the antibody fragment to bind to its target has a cleavage constant that is at least 100-fold or 1000-fold, or 10,000-fold greater than the cleavage constant for the ability of an antibody or antibody fragment not coupled to a masking group to bind to its target.

[0615] In the present disclosure, "humanized" forms of non-human (eg, murine) antibodies refer to chimeric antibodies that contain minimal non-human immunoglobulin sequence. Most humanized antibodies are human recipient immunoglobulin hypervariable region residues substituted with non-human (e.g. mouse, rat, rabbit or non-human primate) with desired specificity, affinity and function Hypervariable region residues (donor antibody). In some embodiments, framework region (FR) residues of the human immunoglobulin are also substituted with non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further optimize antibody performance. Humanized antibodies generally comprise at least one, and usually two, variable regions in which all or nearly all hypervanable loops correspond to those of a non-human immunoglobulin, while the FRs are entirely or almost entirely human immunoglobulin protein sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc). For details see, eg, Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-329; and Presta, 1992, Curr Op Struct Bwl 2: 593-596.

[0616] Intact antibodies can be assigned to different "classes" based on the amino acid sequence of the heavy chain constant region. The five main classes are IgA, IgD, IgE, IgG, and IgM, and several of these classes can also be divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant regions of the different classes of antibodies are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art.

[0617] As used herein, the CDRs contained in the disclosed antibodies or antigen-binding fragments thereof can be identified according to various numbering systems known in the art. In certain embodiments, the disclosed antibodies or antigen-binding fragments thereof contain CDRs preferably identified by the Kabat, Chothia or, AbM or IMGT numbering systems. According to the IMGT numbering system, the corresponding numbers of CDR and FR are as follows: FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT FR3-IMGT For germline V-GENE (*) CDR3-IMGT For rearranged V-J-GENEs and V-D-J-GENEs Germline CDR3-IMGT Rearranged CDR3-IMGT FR4-IMGT Amino acid number 1 --> 26 (C 23) 27 --> 38 39 --> 55 (W41) 56 --> 65 66 --> 104 (C 104) 105 --> 116 105 --> 117 (112.1, 111.1, 112.2, 111.2, etc.) 118 --> 129 Number of amino acids 25-26 aa 5-12 aa 16-17 aa 0-10 aa 36-39 aa 2-12 aa 2-13 aa (more than 13 aa) 10-12 aa

[0618] As used herein, the term "framework residue region" or "FR residues" refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0619] As used herein, the term "germline antibody gene" is a gene encoding an immunoglobulin expressed by a non-lymphoid cell that has not undergone a maturation process leading to genetic rearrangement and maturation leading to expression of a specific immunoglobulin. One advantage provided by various embodiments of the present disclosure stems from the recognition that the amino acid sequences encoded by germline antibody genes are more characteristic of individual animal species than the amino acid sequences encoded by mature antibody genes Sexually important amino acid sequence structure. It is therefore less recognized as foreign by the species when applied therapeutically to that species.

[0620] As amino acid substitutions, conservative amino acid substitutions are preferred in this specification. Conservative amino acid substitutions refer to substitutions that occur within a group of amino acids related to the amino acid side chain. Preferred sets of amino acids are as follows: acidic set (aspartic acid and glutamic acid); basic set (lysine, arginine, and histidine); nonpolar set (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan); and the uncharged polar family (glycine, asparagine, glutamine, semi cystine, serine, threonine and tyrosine). More preferred sets of amino acids are as follows: aliphatic hydroxyl groups (serine and threonine); amide-containing sets (asparagine and glutamine); aliphatic sets (alanine, valine, leucine and isoleucine); and aromatic groups (phenylalanine, tryptophan, and tyrosine). Such amino acid substitution is preferably performed within a set that does not impair the properties of the substance having the original amino acid sequence.

[0621] Furthermore, it is known that the deletion of the lysine residue at the carboxy-terminal of the heavy chain of an antibody produced in cultured mammalian cells (Journal of Chromatography A, 705: 129-134 (1995)), has also been It is known that two amino acid residues (glycine and lysine) at the carboxy-terminal of the heavy chain of an antibody produced in cultured mammalian cells are deleted, and a proline residue newly located at the carboxy-terminal It is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antibody's antigen-binding affinity and effector functions (activation of complement, antibody-dependent cellular cytotoxicity, etc.).

[0622] The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, eg, Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Herein, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in this disclosure, amino acids are generally referred to by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala; arginine can be represented by R or Arg; glycine can be represented by G or Gly; glutamine can be represented by Q or Gln.

[0623] As used herein, the term "prevention" refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom (eg, tumors and infectious diseases) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, relief of symptoms, reduction of the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delay or slowing of the progression of the disease, amelioration or palliation of the disease status, and relief of symptoms (whether partial or total), whether detectable or not. Additionally, "treating" can also refer to prolonging survival as compared to expected survival if not receiving treatment.

[0624] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a non-human primate mammal or a human. In certain embodiments, the subject (eg, a human) has a tumor and an infectious disease, or is at risk of having one of these diseases.

[0625] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing diseases (such as tumors and infectious diseases) refers to an amount sufficient to prevent, arrest, or delay the occurrence of diseases (such as tumors and infectious diseases); an effective amount for treating diseases refers to an amount sufficient to cure or at least partially The amount of disease and its complications that are prevented in patients who already have the disease. Determining such an effective amount is well within the ability of one of ordinary skill in the art to which this invention pertains. For example, amounts effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.

[0626] As used herein, the term "effector function" refers to those biological activities attributable to an antibody Fc region (native sequence Fc region or amino acid sequence variant Fc region) , and it varies with antibody isotype.

[0627] The term "pharmaceutically acceptable" means that the molecular entities, molecular fragments or compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid and the like.

[0628] The in vivo therapeutic effects of antibodies and antibody-drug conjugates on cancer can be determined using experimental animals, for example, administering antibodies to nude mice implanted with a B7H3-expressing tumor cell line and measuring any changes in cancer cells.

[0629] In the present disclosure, although amino acid substitutions in antibodies are in most cases substituted with L-amino acids, it is not limited thereto. In some embodiments, one or more D-amino acids may be included in the antibody peptide chain. Peptides comprising D-amino acids are more stable and less prone to degradation in the oral cavity, intestinal tract or plasma than peptides comprising only L-amino acids.

[0630] Monoclonal antibodies used in the present disclosure can be produced by a number of methods. For example, monoclonal antibodies useful in the present disclosure can be obtained by the hybridoma method, using cells from a number of species, including mouse, hamster, rat, and human (see, e.g., Kohler et al., 1975, Nature, 256: 495), Either produced by recombinant DNA technology (see, for example, US 4,816,567), or isolated from a phage antibody library (see, for example, Clackson et al., 1991, Nature, 352: 624-628; and Marks et al., 1991, Journal of Molecular Biology , 222: 581-597).

[0631] In this article, unless it is clearly stated otherwise, the descriptions "each...independently selected from" and "...independently selected from" used throughout this article can be interchanged, and should be understood in a broad sense. It can mean that in different groups, the specific options expressed between the same or different symbols do not affect each other, and it can also mean that in the same group, the specific options expressed between the same or different symbols do not affect each other.

[0632] Herein, the term "direct bond" means that the groups on both sides are directly connected. For example, in the compound shown in formula II, if X is a direct bond, its structural formula is. Other direct keys can be understood with reference to the foregoing content.

[0633] Herein, the term "absence" means that the group does not exist, for example, in the compound shown in formula II, if W does not exist, then its structural formula is. In the compound shown in formula II, R1 and R2 and the carbon atoms connected to them form ,, or, and the "dotted line" bond indicates the position where the heterocyclic ring is fused with the benzene ring, for example, forming ,, or.

[0634] Herein, in the drug linker conjugate shown in formula III, when L4 is, the labels 1 and 2 represent the connection positions of L4 and the remaining groups, specifically, position 1 is connected to L3, position 2 is When it is connected with drug molecules, it is formed. The remaining labels 1 and 2 can be understood with reference to the foregoing content.

[0635] Herein, in the compound shown in formula II, R3 and X are formed together with the carbon atom connected to it, and the dotted line indicates the position where the carbocycle is fused with the benzene ring and the pyridine ring.

[0636] Herein, the definition of X is, for example, "X is selected from optionally substituted, and the substituent is selected from 1 or 2 C1-4 alkyl (such as methyl)", then X, for example, can be or . Other similar definitions of X can be understood with reference to the foregoing.

[0637] Herein, the definition of X is, for example, "X is selected from optionally substituted, said substituents are selected from 2 C1-4 alkyl (such as methyl) and together with the carbon atoms attached to them to form C3 -6 cycloalkyl (such as cyclopropyl)", then X can be, for example. Other similar definitions of X can be understood with reference to the foregoing.

[0638] In the structure of the amino acid residue shown in AA1, if r is 0, those skilled in the art of the present invention can understand that the structure of the amino acid residue shown in AA1 will become.

[0639] In the structure of the amino acid residue shown in AA1, if Ra and Rb and the carbon atoms connected to them together form a 4-10 membered heterocyclic ring, the 4-10 membered heterocyclic ring is optionally surrounded by a or more R0, wherein the term "the 4-10 membered heterocycle is optionally substituted by one or more R0" means that the 4-10 membered heterocycle can be unsubstituted or is replaced by one or more R0, and among the multiple R0, the definition of each R0 may be the same or different. Other similar definitions can be understood with reference to the foregoing.

[0640] Herein, for example, L3 is selected from Lys, Val-Cit, Ala-Ala-Asn, Ala-Ala-Asp, Gly-Gly-Phe-Gly, Val-Lys-Gly, Val-Ala, Lys-Ala -Asn, "the remote amino group of the lysine (Lys) is optionally replaced by 1, 2 or 3 groups selected from tertiary butoxycarbonyl, C1-6 alkyl (preferably methyl), O The substituent substituted by " means that the remote amino group of Lys in each option of L3 is optionally 1, 2 or 3 selected from tertiary butoxycarbonyl, C1-6 alkyl (preferably Methyl), O substituents are substituted. Wherein, "the remote amino group of Lys" refers to the exposed amino group-NH2 in the lysine residue. "The remote amino group of the lysine (Lys) is optionally replaced by 1, 2 or 3 substituents selected from tertiary butoxycarbonyl, C1-6 alkyl (preferably methyl), O "Substituted" means that the remote amino group of the lysine (Lys) can be unsubstituted, or can be replaced by 1, 2 or 3 groups selected from tertiary butoxycarbonyl, C1-6 alkyl (preferably methyl) , O substituents, for example, can be substituted by 1 third butoxycarbonyl group, that is, become, or can be substituted by 2 methyl groups, that is, become, or can be substituted by 2 methyl groups and 1 O at the same time To replace, to become. It should be noted that "the remote amino group of the lysine (Lys) is substituted by O" means that the remote amino group of the lysine (Lys) is substituted with oxygen, that is, if the remote The terminal amino group is further substituted by two methyl groups, and it becomes .

[0641] In various parts of this specification, substituents of the disclosed compounds are disclosed by type or range of groups. It is specifically noted that this disclosure includes each individual subcombination of each member of these radical classes and ranges. For example, the term "C1-6 alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl.

[0642] As used herein, the term "C1-6 alkyl" means a linear or branched alkyl group containing 1-6 carbon atoms, including for example "C1-3 alkyl" or "C1-4 alkyl" , methyl, ethyl, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, second butyl, third butyl, pentyl, Hexyl.

[0643] Herein, the term "C1-4 alkyl" means a linear or branched alkyl group containing 1-4 carbon atoms, including for example "C1-3 alkyl", methyl, ethyl, etc., Specific examples include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, second-butyl, third-butyl.

[0644] As used herein, the term "C2-6 alkenyl" refers to a linear, branched or cyclic alkenyl group containing at least one double bond and having 2 to 6 carbon atoms, including for example "C2-4 alkenyl Base" and so on. Examples include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl Alkenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hex Dienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl and the like.

[0645] Herein, the term "C2-6 alkynyl" refers to a straight-chain or branched alkynyl group containing at least one triple bond and a carbon number of 2-6, including for example "C2-4 alkynyl" etc. . Examples include, but are not limited to: ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3 -hexynyl, 5-methyl-2-hexynyl and the like.

[0646] As used herein, the term "halogen" includes fluorine, chlorine, bromine, iodine.

[0647] As used herein, the term "3-6 membered cycloalkyl" or "C3-6 cycloalkyl" refers to a saturated cyclic alkyl group containing 3-6 carbon atoms, including cyclopropyl (i.e. cyclopropyl base), cyclobutanyl (i.e. cyclobutyl), cyclopentyl (i.e. cyclopentyl), cyclohexyl.

[0648] As used herein, the term "3-7 membered carbocycloalkyl" or "C3-7 cycloalkyl" refers to a saturated cyclic alkyl group containing 3-7 carbon atoms, including cyclopropyl, cyclobutyl Alkyl, cyclopentyl, cyclohexyl, cycloheptyl.

[0649] As used herein, the term "C1-6 alkoxy" means an alkyl group, as defined above, attached to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, Oxygen, Hexyloxy, etc.

[0650] As used herein, the term "C1-4 alkoxy" means an alkyl group, as defined above, attached to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, and the like.

[0651] As used herein, the term "4-10 membered heterocyclic group" refers to a cyclic group containing 4-10 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom) group. The term "4-6 membered heterocyclic group" refers to a cyclic group containing 4-6 ring atoms (wherein at least one ring atom is a heteroatom, such as nitrogen atom, oxygen atom or sulfur atom). Optionally, ring atoms (such as carbon, nitrogen or sulfur atoms) in the ring structure may be oxo substituted. "4-8 membered heterocyclic group" includes, for example, "4-8 membered nitrogen-containing heterocyclic group", "4-8 membered oxygen-containing heterocyclic group", "4-7 membered heterocyclic group", "4-7 membered Oxygen-containing heterocyclic group", "4-7-membered heterocyclic group", "4-6-membered heterocyclic group", "5-7-membered heterocyclic group", "5-6-membered heterocyclic group", "5- "6-membered nitrogen-containing heterocyclic group", including but not limited to oxocyclobutanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperyl, tetrahydropyranyl, homopiperyl, etc.

[0652] As used herein, the term "4-10 membered heterocyclic ring" refers to a ring containing 4-10 ring atoms (wherein at least one ring atom is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "5-6 membered heterocyclic ring" refers to a ring containing 5-6 ring atoms (wherein at least one ring atom is a heteroatom, such as nitrogen atom, oxygen atom or sulfur atom), including but not limited to pyrrolidine, tetrahydrofuran, Piperidine, piperidine, tetrahydropyran and other rings.

[0653] As used herein, the term "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon group, such as a 6-10 membered aryl group, a 5-8 membered aryl group, and the like. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like. The "6-10 membered aryl" refers to an aryl group containing 6-10 ring atoms. The "C6-10 aryl" refers to an aryl group containing 6-10 carbon atoms.

[0654] As used herein, the term "heteroaryl" refers to an aromatic cyclic group in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, ring atoms (such as carbon, nitrogen or sulfur atoms) in the ring structure may be oxo substituted. Specific examples include, but are not limited to, 5-10-membered heteroaryl, 5-6-membered heteroaryl, 5-10-membered nitrogen-containing heteroaryl, 6-10-membered oxygen-containing heteroaryl, 6-8-membered nitrogen-containing heteroaryl group, 5-8 membered oxygen-containing heteroaryl group, etc., such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazole Base, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1, 2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxinyl , 2H-1,2-㗁𠯤 group, 4H-1,2-㗁𠯤 group, 6H-1,2-㗁𠯤 group, 4H-1,3-㗁𠯤 group, 6H-1,3-㗁𠯤 group , 4H-1,4-㗁𠯤yl, da-𠯤yl, pyrylyl, 1,2,3-tri-𠯤yl, 1,3,5-tri-𠯤yl, 1,2,4,5-tetra-𠯤yl , azacycloheptatrienyl, 1,3-diazepanyl, azacyclohexatetraenyl, etc.

[0655] A bond in a structural formula represented by a wavy line "~~" herein is intended to indicate that the structure represents a cis or trans isomer, or a mixture of cis and trans isomers in any proportion.

[0656] The term "drug to antibody ratio" or "DAR" refers to the amount of drug, eg, a small molecule toxin, attached to an antibody of an ADC. The DAR of an ADC can range from 1 to 16, but depending on the number of attachment sites on the antibody, higher loadings (eg 20) are also possible. The term DAR may be used when referring to the amount of drug loaded onto a single antibody, or alternatively, the average or mean DAR of a group of ADCs.

[0657] Beneficial effects of the invention

[0658] This disclosure obtains bioactive drugs (bioactive molecules or payloads) with high activity and high cell permeability through a large number of studies, linkers with high in vivo cycle stability and high chemical stability, and by adjusting the physical and chemical properties of linkers (such as basicity and lipophilicity) to realize the enrichment of antibody drug conjugates (ADC) on the tumor microenvironment, the unique in vivo enzyme cleavage properties of the linker and the coupling method with the targeting moiety, combined with the screening and verification of a large number of in vivo and in vitro drug effects , to obtain a class of novel antibody bioactive molecule conjugates. Using the conjugates obtained in the above manner can achieve a variety of following surprising technical effects:

[0659] The conjugate obtained according to the above method has better solubility and excellent chemical stability, such as the reversible Michael addition reaction caused by the maleimide linking method in the traditional ADC does not occur, so high drug- Antibody ratio, in some embodiments, the DAR value of the conjugate can reach 6-8; has extremely high coupling efficiency, in some embodiments, the coupling efficiency can reach or exceed 90%; by A large number of studies have found a class of linkers with high plasma stability, but at the same time can be cleaved in the tumor microenvironment (both inside the tumor cell and outside the tumor cells), so it can be used in tumors with low antigen expression or antigen non-expression Produce good anti-tumor effect; The conjugate (ADC) obtained according to the above method increases the exposure of the entire ADC molecule in a relatively acidic tumor environment by adjusting the physical and chemical properties of the linker and the overall ADC molecule, so ADC has Better tumor tissue targeting, that is, the enrichment ability in the tumor microenvironment, increases the concentration ratio of bioactive molecules in the tumor and blood, and reduces the mechanism-related toxicity of ADC molecules (ADC binds to cells in non-tumor tissues surface antigen and endocytosis, or also called "on-target toxicity"), so it has a higher therapeutic index; the conjugate obtained according to the above method has high stability in the circulation in the body, It reduces the shedding of drug molecules in non-target tissues and reduces the "off-target" toxicity caused by toxin shedding in non-target tissues; the bioactive molecules of the conjugates have higher anti-tumor cell activity, so they have excellent By-stander effect, ADC can more effectively kill tumor cells with high antigen expression and tumor cells with low antigen expression or no antigen expression in tumor tissue; the toxin-linker of the present disclosure uses its linker The extracellular lysis ability in the tumor microenvironment can form an antibody-conjugated drug with an antibody that has no endocytic ability. This type of antibody-conjugated drug still has high anti-tumor activity; The extracellular lysis ability of the body in the tumor microenvironment and its enrichment ability in the tumor microenvironment can form antibody conjugated drugs with antibodies that have no endocytosis ability and antibodies that have no ability to bind tumor extracellular antigens. Antibody-drug conjugates still have high anti-tumor activity; the antibodies provided by the present disclosure have a very high degree of humanization or fully human antibodies, so they can be safely administered to human subjects without causing immunogenic reactions. Molecules with a high ability to bind human and non-human B7H3, especially related to antibodies with cross-reactivity to B7H3 of non-human primates (such as cynomolgus monkeys); In summary, the linker, antibody of the present invention and ADC have significant clinical value.

Implementation

[0661] The present disclosure will be further illustrated through the description of specific embodiments below, but this is not a limitation of the present disclosure. Those skilled in the technical field to which the present invention pertains can make various modifications or improvements based on the teachings of the present disclosure without departing from the basic idea and scope of the present disclosure. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

[0662] Abbreviations in this disclosure have the following meanings: OMs mesylate group FA formic acid OTs p-toluenesulfonate group ACN Acetonitrile OTf Triflate CCK8 reagent 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfobenzene)-2H-tetrazole monosodium salt TBS tertiary butyldimethylsilyl FBS fetal bovine serum MMT p-methoxyphenylbenzhydryl DMSO DMSO PB / PBS Phosphate buffer HBTU O-Benzotriazole-tetramethyluronium hexafluorophosphate HOBT 1-Hydroxybenzotriazole HATU 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate NBS N-Bromosuccinimide TFA Trifluoroacetate PPTS Pyridine p-toluenesulfonate DCM Dichloromethane THF Tetrahydrofuran DMF N,N-Dimethylformamide EDC 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride IgG immunoglobulin G CDR Complementarity Determining Regions in Immunoglobulin Variable Regions HRP Horseradish peroxidase FR Antibody framework region: the amino acid residues in the antibody variable region other than the CDR residues hFc Human IgG Antibody Fc Fragment VH Antibody heavy chain variable region KD equilibrium dissociation constant VL Antibody light chain variable region HCDR1 Complementarity Determining Region 1 in Immunoglobulin Heavy Chain Variable Regions AbM The definition of AbM CDR comes from the related research of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272), this definition method integrates Kabat and Chothia is a partial definition of both. HCDR2 Complementarity Determining Region 2 in Immunoglobulin Heavy Chain Variable Regions Kabat Immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) . HCDR3 Complementarity Determining Region 3 in Immunoglobulin Heavy Chain Variable Region Chothia The immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). LCDR1 Complementarity Determining Region 1 in Immunoglobulin Light Chain Variable Regions IMGT Based on the numbering system of The international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. LCDR2 Complementarity Determining Region 2 in Immunoglobulin Light Chain Variable Regions mAb Monoclonal antibodies LCDR3 Complementarity-determining region 3 in the variable region of an immunoglobulin light chain EC50 Concentration that produces 50% efficacy or binding SEC-HPLC Size Exclusion High Performance Liquid Chromatography ELISA ELISA HC immunoglobulin heavy chain PCR polymerase chain reaction LC immunoglobulin light chain

[0663] Sequence and its specific information: SEQ ID NO sequence name amino acid sequence 1 scFv 1D1 EGQLVESGGGLVQPGGSLRLSCAASGFTISSYWMNWVRQAPGKGLEWVANIKQDGSEKYYVDSVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRMYSSGWYWGAFDMWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLLSPGERATLSCRASQSVSSIYLAWNQQK PGQAPRLLIYGTFKRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDTSSITFGQGTRLEIK 2 scFv 2E3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISGSGGSTYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKARSGYDYFFDYWGQGTLVTVSSGGGGSGGGSGGGSEIVLTQSPGTLLSPGERATLSCRASQRISSSFLAWYQQKPGQAPSL LIYGASSRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVFYCQQYSNSPLTFGGGTKVEIK 3 1D1-01 VH EGQLVESGGGLVQPGGSLRLSCAASGFTISSYWMNWVRQAPGKGLEWVANIKQDGSEKYYVDSVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRMYSSGWYWGAFDMWGQGTMVTVSS 4 1D1-01 chothia HCDR1 GFTISSY 5 1D1-01 chothia HCDR2 KQDGSE 6 1D1-01 chothia HCDR3 RMYSSGWYWGAFDM 7 1D1-01 kabat HCDR1 SYWMN 8 1D1-01 kabat HCDR2 NIKQDGSEKYYVDSVQG 9 1D1-01 kabat HCDR3 RMYSSGWYWGAFDM 10 1D1-01 IMGT HCDR1 GFTISSYW 11 1D1-01 IMGT HCDR2 IKQDGSEK 12 1D1-01 IMGT HCDR3 ARRMYSSGWYWGAFDM 13 1D1-01 VL EIVLTQSPGTLLSPGERATLSCRASQSVSSIYLAWNQQKPGQAPRLLIYGTFKRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDTSSITFGQGTRLEIK 14 1D1-01 chothia LCDR1 RASQSVSSIYLA 15 1D1-01 chothia LCDR2 GTFKRAT 16 1D1-01 chothia LCDR3 QQYDTSSIT 17 1D1-01 kabat LCDR1 RASQSVSSIYLA 18 1D1-01 kabat LCDR2 GTFKRAT 19 1D1-01 kabat LCDR3 QQYDTSSIT 20 1D1-01 IMGT LCDR1 QSVSSIY \ 1D1-01 IMGT LCDR2 GTF twenty one 202-2-1 kabat / IMGT LCDR3 QQLNSYPLT twenty two 1D1-01 IMGT LCDR3 QQYDTSSIT twenty three 2E3-02 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISGSGGSTYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKARSGYDYFFDYWGQGTLVTVSS twenty four 2E3-02 chothia HCDR1 GFTFSSY 25 2E3-02 chothia HCDR2 SGSGGS 26 2E3-02 chothia HCDR3 ARSGYDYFFDY 27 2E3-02 kabat HCDR1 SYAMN 28 2E3-02 kabat HCDR2 GISGSGGSTYYADSVQG 29 2E3-02 kabat HCDR3 ARSGYDYFFDY 30 2E3-02 IMGT HCDR1 GFTFSSYA 31 2E3-02 IMGT HCDR2 ISGSGGST 32 2E3-02 IMGT HCDR3 AKARSGYDYFFDY 33 2E3-02 VL EIVLTQSPGTLLSPGERATLSCRASQRISSSFLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVFYCQQYSNSPLTFGGGTKVEIK 34 2E3-02 chothia LCDR1 RASQRISSSFLA 35 2E3-02 chothia LCDR2 GASSRAT 36 2E3-02 chothia LCDR3 QQYSNSPLT 37 2E3-02 kabat LCDR1 RASQRISSSFLA 38 2E3-02 kabat LCDR2 GASSRAT 39 2E3-02 kabat LCDR3 QQYSNSPLT 40 2E3-02 IMGT LCDR1 QRISSSF \ 2E3-02 IMGT LCDR2 GAS 41 202-2-1 IMGT LCDR1 QGISSY 42 2E3-02 IMGT LCDR3 QQYSNSPLT 43 IgG1 CH, IgG1 constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 44 Kappa CL, the kappa constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 45 1D1-01 HC, namely 1D1-01 heavy chain EGQLVESGGGLVQPGGSLRLSCAASGFTISSYWMNWVRQAPGKGLEWVANIKQDGSEKYYVDSVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRMYSSGWYWGAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 46 1D1-01 LC, namely 1D1-01 light chain EIVLTQSPGTLLSPGERATLSCRASQSVSSIYLAWNQQKPGQAPRLLIYGTFKRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDTSSITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC 47 2E3-02 HC, namely 2E3-02 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSGISGSGGSTYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKARSGYDYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 48 2E3-02 LC, namely 2E3-02 light chain EIVLTQSPGTLSLPGERATLSCRASQRISSSFLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFLTISSLEPEDFAVFYCQQYSNSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC 49 202-2-1 VH EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWYDVSHKYYADSVKGRFTISRDNSKNTLSLQMNSLRAEDTAVYYCARDWGDPDAFDIWGQGTMVTVSS 50 202-2-1 VL DIQLTQSPSSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIK 51 202-2-1 HC, ie 202-2-1 heavy chain EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWYDVSHKYYADSVKGRFTISRDNSKNTLSLQMNSLRAEDTAVYYCARDWGDPDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 52 202-2-1 LC, ie 202-2-1 light chain DIQLTQSPSSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC 53 202-2-1 kabat HCDR1 TYGMH 54 202-2-1 kabat HCDR2 VIWYDVSHKYYADSVKG 55 202-2-1 kabat HCDR3 DWGDPDAFDI 56 202-2-1 IMGT HCDR1 GFTFSTYG 57 202-2-1 IMGT HCDR2 IWYDVSHK 58 202-2-1 IMGT HCDR3 ARDWGDPDAFDI 59 202-2-1 kabat LCDR1 RASQGISSYLA 60 202-2-1 kabat LCDR2 AASTLQS \ 202-2-1 IMGT LCDR2 AAS

[0664] The present disclosure will now be described with reference to the following examples, which are intended to illustrate, not limit, the disclosure.

[0665] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this disclosure are basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Refinement Molecular Biology Laboratory Guide, 3rd Edition, John Wiley & Sons, Inc., 1995 described in the method. Those skilled in the art to which the present invention pertains are well aware that the embodiments describe the present disclosure by way of example and are not intended to limit the scope of protection claimed by the present disclosure.

[0666] Preparation scheme

[0667] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (1H NMR) or mass spectroscopy (MS).

[0668] The measuring instrument of nuclear magnetic resonance (1H NMR) uses Bruker 400 MHz nuclear magnetic resonance instrument; The measuring solvent is deuterated methanol (CD3OD), deuterated chloroform (CDCl3) or six deuterated dimethyl sulfide (DMSO-d6) ; The internal standard substance is tetramethylsilane (TMS).

[0669] Abbreviations in nuclear magnetic resonance (NMR) spectra used in the examples are shown below.

[0670] s: singlet (singlet), d: doublet (doublet), t: triplet (triplet), q: quartet (quartet), dd: double doublet (double doublet), qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet (multiplet), br: broad peak (broad), J: coupling constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfide. The delta values ​​are expressed in ppm.

[0671] The measuring instrument of mass spectrometry (MS) uses Agilent (ESI) mass spectrometer, model is Agilent 6120B.

[0672] 1. Synthesis of biologically active molecules and intermediates used in the process of synthesizing "drug-linker compounds"

[0673] A, synthesis of biologically active molecules

[0674] Embodiment A1.1: (S)-4-ethyl-8-fluoro-4-hydroxyl-9-methyl-11-(1H-pyrazol-4-yl)-1,12-dihydro Synthesis step 1 of -14H-pyrano[3',4':6,7]indolo[1,2-b]quinoline-3,14(4H)-dione (A1.1): 4-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (4.6g) was dissolved in anhydrous tetrahydrofuran (50ml), and the above solution was cooled to - 78oC, then dropwise added n-butyllithium (12ml, 2M), the reaction solution was stirred at -78oC for 20 minutes, then added 2-amino-4-fluoro-5-methylbenzoic acid methyl ester (1.83g), the addition was complete Afterwards, the reaction was naturally raised to room temperature, and the reaction was continuously stirred for 5 hours. Add ethyl acetate (200ml) after methanol (3ml) was quenched, the solution was washed with water (100ml x 3), the organic phase was dried and the organic solvent was removed, and the target product (2-amino-4 -Fluoro-5-methylphenyl)(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanone, ESI-MS (m / z): 304 [ M+H]+. Step 2: (S)-4-Ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indeno-3,6,10(4H)-trione (2.63g), (2-Amino-4-fluoro-5-methylphenyl)(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanone (3.03g), p-toluenesulfonic acid (1.74g) was dissolved in dichloromethane (50ml), and then the solvent was removed, and the mixture was heated to 120oC for 4 hours under the protection of nitrogen. The mixture was dissolved in ethyl acetate (300ml), the organic phase was washed with water (100ml x 2), and the organic solvent was removed after drying. The residue was separated by silica gel column chromatography to obtain the target product (S)-4-ethyl-8-fluoro- 4-Hydroxy-9-methyl-11-(1H-pyrazol-4-yl)-1,12-dihydro-14H-pyrano[3',4':6,7]indole 𠯤[ 1 ,2-b]quinoline-3,14(4H)-dione (A1.1). ESI-MS (m / z): 447 [M+H]+.

[0675] Embodiment A1.2: (S)-7-ethyl-7-hydroxyl-14-(1H-pyrazol-4-yl)-10,13-dihydro-11H-[1,3]di Oxo[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione (A1.2 ) was synthesized using the same method and reaction conditions as in Example A1.1, replacing 2-amino-4 with 6-aminobenzo[d][1,3]dioxolane-5-carboxylic acid methyl ester - Methyl fluoro-5-methylbenzoate to obtain the target product (S)-7-ethyl-7-hydroxyl-14-(1H-pyrazol-4-yl)-10,13-dihydro-11H- [1,3]dioxo[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)- Diketones (A1.2). ESI-MS (m / z): 459 [M+H]+.

[0676] Embodiment A1.3: (S)-14-(3-aminophenyl)-7-ethyl-7-hydroxyl-10,13-dihydro-11H-[1,3]dioxa Cyclopentano[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione (A1 .3) Synthesis of compound (A1.3-A) (1.4g), compound (A1.3-B) (2.2g), Pd2(DBA)3 (300 mg), tricyclohexylphosphine (300 mg), Potassium acetate (1.1g) was sequentially added to a mixed solvent of dioxane (30ml) and water (5ml), and the mixture was heated and stirred at 100oC under the protection of nitrogen for 12 hours. After cooling, add ethyl acetate (200ml), wash with water (100ml), dry, and separate by silica gel column chromatography to obtain the target product (S)-14-(3-aminophenyl)-7-ethyl-7-hydroxy - 10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indole 𠯤[1, 2-b] Quinoline-8,11(7H)-dione (A1.3). ESI-MS (m / z): 484 [M+H]+.

[0677] Embodiment A1.4: (S)-14-(4-aminophenyl)-7-ethyl-7-hydroxyl-10,13-dihydro-11H-[1,3]dioxa Cyclopentano[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione (A1 .4) Synthesis of compound (A1.4-A) (1.4g), compound (A1.4-B) (2.2g), Pd2(DBA)3 (300mg), tricyclohexylphosphine (300mg), potassium acetate (1.1g) was sequentially added to a mixed solvent of dioxane (30ml) and water (5ml), and the mixture was heated and stirred at 100oC under the protection of nitrogen for 12 hours. After cooling, add ethyl acetate (200ml), wash with water (100ml), dry, and separate by silica gel column chromatography to obtain the target product (S)-14-(4-aminophenyl)-7-ethyl-7-hydroxy - 10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indole 𠯤[1, 2-b] Quinoline-8,11(7H)-dione (A1.4). ESI-MS (m / z): 484 [M+H]+; 1H NMR (400 MHz, DMSO) δ 7.56 (s, 1H), 7.34 (d,J= 8.0 Hz, 2H), 7.27 (s, 1H ), 7.14 (s, 1H), 6.89 (d,J= 7.8 Hz, 2H), 6.26 (s, 2H), 5.40 (s, 2H), 5.05 (s, 2H), 1.96 – 1.78 (m, 2H) , 0.88 (t,J= 7.2 Hz, 3H).

[0678] Embodiment A1.5: (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxyl-10,13-dihydro-11H-[1,3]dioxa Cyclopentano[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione (A1 .5) Synthesis step 1: (S)-14-(3-chloropropyl)-7-ethyl-7-hydroxyl-10,13-dihydro-11H-[1,3]dioxolane Synthesis of alkano[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione in ice bath conditions, to compound (S) 7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclo[4,5-g]pyrano[3',4 ': 6,7]indeno[1,2-b]quinoline-8,11(7H)-dione (A1.5-A, 500 mg) in 75% sulfuric acid solution (5 mL) was added heptahydrate Ferrous sulfate (570 mg ferrous sulfate heptahydrate dissolved in 1 mL water), and 4,4-dimethoxychlorobutane (3.89 g), the reaction solution was stirred for three minutes and hydrogen peroxide (29%, 2.5 mL) was added dropwise . The reaction solution was stirred and reacted at 0°C for 5 minutes, then rose to room temperature, and stirred and reacted for 3 hours. The reaction solution was diluted with water (50 mL), extracted with ethyl acetate (80 mL×2), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product, which was used on a C18 column (acetonitrile / 0.05% Aqueous formic acid: 5%-60%) was further purified to obtain the target compound (yellow solid, 400 mg, yield: 67%). LCMS (ESI) [M+H]+: 468.9; 1H NMR (400 MHz, DMSO-d6) δ 7.65 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.50 (s, 1H ), 6.30 (s, 2H), 5.42 (s, 2H), 5.26 (s, 2H), 3.81 (d,J= 5.9 Hz, 2H), 3.22 (s, 2H), 1.98 (d,J= 6.7 Hz , 4H), 0.88 (t,J= 7.2 Hz, 3H). Step 2: (S)-14-(3-azidopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[ Synthesis of 4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione to compound (S) -14-(3-Chloropropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyran N,N-Dimethylformamide of [3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione (200 mg) Sodium azide (284 mg) was added to the solution (3 mL), and the reaction solution was stirred and reacted at 100 °C for 1 hour. Water (30 mL) was added to the reaction solution, extracted with ethyl acetate (60 mL×2), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the target compound (S)-14-(3- Azidopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3' , 4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione (yellow solid, 180 mg, yield: 88%). LCMS (ESI) [M+H]+: 476; 1H NMR (400 MHz, DMSO-d6) δ 7.65 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H ), 6.29 (s, 2H), 5.42 (s, 2H), 5.25 (s, 2H), 3.53 – 3.49 (m, 2H), 3.16 – 3.12 (m, 2H), 1.93 – 1.80 (m, 4H), 0.88 (t,J= 7.2 Hz, 3H). Step 3: (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4 ,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione synthesis to compound (S)- 14-(3-Azidopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyridine Synthesis of Furo[3',4':6,7]indolo[1,2-b]quinoline-8,11(7H)-dione (130 mg, 0.274 mmol) in THF (3 mL ) and water (1 mL) were added triphenylphosphine (108 mg, 0.411 mmol), and reacted at 55 °C for 16 hours. LCMS showed the reaction was complete. Water (5 mL) was added to the reaction solution, acidified with 2N hydrochloric acid (3 mL), and extracted with ethyl acetate (10 mL). The aqueous phase was purified by HPLC (acetonitrile / 0.05% formic acid in water) to obtain the target compound (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro -11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline- Synthesis of 8,11(7H)-dione (A1.5, yellow solid, 15 mg, yield: 12%). LCMS (ESI) [M+H]+: 449.9; 1H NMR (400 MHz, DMSO-d6) δ 7.72 (s, 3H), 7.53 (s, 1H), 7.25 (s, 1H), 6.50 (s, 1H ), 6.30 (s, 2H), 5.43 (s, 2H), 5.24 (s, 2H), 3.15 (d,J= 6.4 Hz, 2H), 3.03 (t,J= 6.9 Hz, 2H), 1.96 – 1.81 (m, 4H), 0.88 (t,J= 7.3 Hz, 3H).

[0679] Embodiment A1.6: (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxyl-8,11-dioxo-7,8,11,13-tetra Hydrogen-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline -14-yl)ethyl)-2-hydroxyacetamide (A1.6) Synthesis step 1: (S)-2-(ethyl(2-(7-ethyl-7-hydroxyl-8,11 -Dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indole𠯤 And[1,2-b]quinolin-14-yl)ethyl)amino)-2-oxoethyl acetate (A1.6-B) preparation: compound (S)-7-ethyl -14-(2-(ethylamino)ethyl)-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclo[4,5-g]pyrano[3' , 4':6,7]indole ? 73 mg), triethylamine (50 mg) were sequentially added to dichloromethane (5 mL), and stirred at room temperature for 1 hour. The solvent was removed by rotary evaporation of the reaction solution to obtain 75 mg of crude oily compound. LCMS (ESI) [M+H]+: 564.2. Step 2: (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1 ,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinolin-14-yl)ethyl Synthesis of (S)-2-hydroxyacetamide (A1.6) The crude compound (S)-2-(ethyl (2-(7-ethyl-7-hydroxy-8,11-dioxo-7 ,8,11,13-tetrahydro-10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indole 𠯤[1,2-b ]quinolin-14-yl)ethyl)amino)-2-oxoethyl acetate (75 mg, 0.13 mmol) was dissolved in a mixed solution of concentrated hydrochloric acid and absolute ethanol (1 / 2 volume ratio, 3 mL) , and then the reaction solution was refluxed at 85°C for 1 hour. LCMS showed the reaction was complete. The reaction solution was concentrated, and the crude product was purified by preparative chromatography (0.01% trifluoroacetic acid in water, acetonitrile) to obtain the target compound (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxyl-8, 11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6, 7] Indolo[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyacetamide (A1.6) (15 mg, yield: 26%) was a white solid. LCMS (ESI) [M+H]+: 522.0; 1H NMR (400 MHz, DMSO) δ 7.92 (s, 1H), 7.53 (m, 1H), 7.25 (s, 1H), 6.50 (s, 1H), 6.31 (s, 2H), 5.43 (s, 2H), 5.34 (m, 2H), 4.65 (m, 1H), 4.07 (m, 2H), 3.52 (m, 2H), 3.41 (m, 2H), 2.00 (m, 2H), 1.88 (m, 2H), 1.16 – 1.04 (m, 3H), 0.89 – 0.83 (m, 3H).

[0680] Embodiment A1.7: (S)-N-methyl-N-(2-(7-ethyl-7-hydroxyl-8,11-dioxo-7,8,11,13-tetra Hydrogen-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinoline -14-yl)ethyl)-2-hydroxyacetamide (A1.7) Synthesis Step 1: The compound methylbenzyl (3-(6-nitrobenzo[d][1,3]dioxo Preparation of Heterocyclyl-5-yl)-3-oxopropyl)carbamate The compound 1-(6-nitrobenzo[d][1,3]dioxin-5-yl)ethane -1-ketone (A1.7-A, 500 mg), methylamine hydrochloride (1.6 g) and paraformaldehyde (714 mg) were dissolved in ethanol (8 mL), and reacted at 100°C for 16 hours . The reaction solution was lowered to room temperature and concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane (80 mL), and the organic phase was extracted with water (50 mL × 3). The obtained aqueous phase was adjusted to pH=9 with sodium bicarbonate, then benzyl chloroformate (513 mg, 3.0 mmol) was added, and the reaction was carried out at room temperature for 16 hours. The reaction solution was extracted with ethyl acetate (30 mL × 3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction, and concentrated to obtain the crude product, which was separated and purified by C18 (acetonitrile / water containing 0.05% formic acid= 5 ~95%) to obtain the target compound (A1.7-B, 180 mg, yield 23%). LCMS (ESI) [M+H]+: 387.1. 1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 7.35 (m, 5H), 7.32 (m, 1H), 6.17 (s, 2H), 5.13 (s, 2H), 3.71 (m, 2H), 3.03 (m, 3H), 2.99 – 2.86 (m, 2H). Step 2: Compound benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxopropyl)(methyl)carbamate (A1 .7-C) Preparation of compound A1.7-B methylbenzyl (3-(6-nitrobenzo[d][1,3]dioxan-5-yl)-3-oxopropane Base) carbamate (180 mg, 0.47 mmol) was dissolved in a mixed solution of saturated aqueous ammonium chloride (8 mL) and ethanol (8 mL), then iron powder (130 mg) was added to the reaction solution, and the reaction was Stir at 80°C for 2 hours. The reaction solution was filtered, the filtrate was concentrated under reduced pressure into a solid, and the crude product was separated and purified by TLC (petroleum ether: ethyl acetate = 2 / 1) to obtain the target compound A1.7-C (76 mg). LCMS (ESI) [M+H]+: 357.0. Step 3: Benzyl (S)-(2-(7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxy Heterocyclo[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quinolin-14-yl)ethyl)(methyl)amino Preparation of formate (A1.7-D) -3-oxopropyl)(methyl)carbamate (38 mg), compound (S) -4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3, 4-f]indole-3,6,10(4H)-trione (29 mg) and p-toluenesulfonic acid (23 mg) were dissolved in dichloromethane (5 mL) solution, the solution was clear and mixed Concentrate under reduced pressure, evacuate to vacuum with an oil pump, and react under vacuum at 120°C for 2 hours. The reaction solution was cooled down to room temperature, water (30 mL) was added and extracted with dichloromethane (30 mL × 3). The combined organic phases were sequentially dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound A1.7-D (50 mg). LCMS (ESI) [M+H] += 584.0. Step 4: (S) 7-ethyl-7-hydroxy-14-(2-(methylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxane[4, 5-g]pyrano[3',4':6,7]indole[1,2-b]quinoline-8,11(7H)-dione (A1.7-E) preparation room Compound A1.7-D benzyl (S)-(2-(7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H- [1,3]dioxacyclo[4,5-g]pyrano[3',4':6,7]indole[1,2-b]quinolin-14-yl)ethyl) (Methyl) carbamate (50 mg) was dissolved in dichloromethane (5 mL) solution, trimethyl iodosilane (51 mg, 0.26 mmol) was added at 0°C, and reacted for 3 hours. The reaction solution was concentrated to remove the solvent, and purified by HPLC (acetonitrile / water containing 0.05% formic acid) to obtain compound A1.7-E (15 mg) as a brown solid. LCMS (ESI) [M+H]+: 450.0. Step 5: Compound (S) -2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3] Dioxyalcohol[4,5-g]pyrano[3',4':6,7]indole[1,2-b]quinolin-14-yl)ethyl)(methyl)amino )-2-Oxoethyl acetate (A1.7-F) Preparation of compound A1.7-E (S) 7-ethyl-7-hydroxy-14-(2-(methylamino)ethyl )-10,13-dihydro-11H-[1,3]dioxacyclo[4,5-g]pyrano[3',4':6,7]indolo[1,2- b] Quinoline-8,11(7H)-dione (15 mg) and triethylamine (15 mg) were dissolved in dichloromethane (5 mL) and acetyloxyacetyl was added at 0°C Chlorine (20 mg) was reacted for 1 hour. The reaction solution was concentrated to remove the solvent to obtain a crude product, and the obtained solid was directly used in the next step without further purification. LCMS (ESI) [M5+H]+= 550.1. Step 6: Compound (S) -N-(2-(7-Ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]di Oxyheterocyclo[4,5-g]pyrano[3',4':6,7]indole[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N - Preparation of methyl acetamide (A1.7) Compound A1.7-F(S)-2-((2-(7-ethyl-7-hydroxyl-8,11-dioxo-7, 8,11,13-Tetrahydro-10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolo[1,2-b ]quinolin-14-yl)ethyl)(methyl)amino)-2-oxoethyl acetate (15 mg) was dissolved in ethanol (5 mL) solution, concentrated hydrochloric acid (1.5 mL) was added to React at 80°C for 2 hours. The reaction solution was concentrated to remove the solvent, and purified by HPLC (acetonitrile / water containing 0.05% formic acid) to obtain compound A1.7 (1.6 mg) as a white solid. LCMS (ESI) [M+H]+: 508.2.

[0681] Embodiment A1.8: (S)-N-isopropyl-N-(2-(7-ethyl-7-hydroxyl-8,11-dioxo-7,8,11,13- Tetrahydro-10H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolo[1,2-b]quino[1,2-b]quino Synthesis of phen-14-yl)ethyl)-2-hydroxyacetamide (A1.8) Step 1: Compound isopropylbenzyl (3-(6-nitrobenzo[d][1,3] Preparation of dioxin-5-yl)-3-oxypropyl)carbamate (A1.8-B) to compound (A1.8-A) 3-(isopropylamino)-1-(6 -Nitrobenzo[d][1,3]dioxin-5-yl)propan-1-one (900 mg) in dichloromethane solution (10 mL), followed by adding triethylamine (1.8 g) and benzyloxycarbonyl chloride (734 mg, 4.3 mmol) at room temperature for 2 hours. The reaction solution was diluted with water (50 mL), extracted with dichloromethane (50 mL×3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was separated and purified by column chromatography (petroleum ether : ethyl acetate=3 / 1) to obtain the target compound (A1.8-B) (600 mg). LCMS (ESI) [M+H]+: 414.9; 1H NMR (400 MHz, DMSO-d6) δ 7.72 (s, 1H), 7.34 (br s, 6H), 6.31 (s, 2H), 5.08 (s, 2H), 4.14 – 4.10 (m, 1H), 3.48 (d, J= 7.9 Hz, 2H), 3.03 (s, 2H), 1.13 – 1.11 (m, 6H). Step 2: Compound benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxopropyl)(isopropyl)carbamate ( A1.8-C) Preparation of compound (A1.8-B) isopropylbenzyl (3-(6-nitrobenzo[d][1,3]dioxin-5-yl)-3 -Oxypropyl) carbamate (200 mg, 0.48 mmol) was dissolved in a mixed solution of saturated ammonium chloride (3 mL) and ethanol (3 mL), then iron powder (135 mg) was added to the reaction solution, The reaction was stirred at room temperature for 2 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to a solid. The crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 3 / 1) to obtain the target compound (A1.8-C) (65 mg). LCMS (ESI) [M+H]+: 395.2. Step 3: (S) 7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxane[4, 5-g]pyrano[3',4':6,7]indole[1,2-b]quinoline-8,11(7H)-dione (A1.8-D) preparation room Add compound (A1.8-C) benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxopropyl)(isopropyl base) carbamate (65 mg), compound (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indole-3 , 6,10(4H)-triketone (45 mg) and p-toluenesulfonic acid (33 mg, 0.17 mmol) were dissolved in dichloromethane (10 mL) solution, the solution was clarified and mixed, then concentrated under reduced pressure, pumped with an oil pump to vacuum and the reaction was run at 120°C under vacuum for 2 hours. The reaction solution was lowered to room temperature, added water (50 mL), extracted with dichloromethane (30 mL×3), combined organic phases were sequentially dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the crude product was subjected to column chromatography Separation and purification (dichloromethane / methanol=10 / 1) gave the target compound (A1.8-D) (40 mg). LCMS (ESI) [M+H]+: 478.0; 1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.67 (s, 1H), 7.57 (s, 1H), 7.27 (s, 1H ), 6.52 (s, 1H), 6.33 (s, 2H), 5.44 (s, 2H), 5.35 (s, 2H), 3.41 (br s, 2H), 3.23 (br s, 3H), 1.94 – 1.78 ( m, 2H), 1.25 (d,J= 6.4 Hz, 6H), 0.88 (t,J= 7.3 Hz, 3H). Step 4: Compound ((S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3 ]dioxa[4,5-g]pyrano[3',4':6,7]indole[1,2-b]quinolin-14-yl)ethyl)(isopropyl) Preparation of amino)-2-oxoethyl acetate (A1.8-E) under ice bath, to compound (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl base)-10,13-dihydro-11H-[1,3]dioxacyclo[4,5-g]pyrano[3',4':6,7]indole 𠯤[1,2- b] To a solution of quinoline-8,11(7H)-dione (A1.8-D) (40 mg) in dichloromethane (2 mL) was added triethylamine (35 mg), Oxyethyl acetate (57 mg) was reacted at zero for 30 minutes. The reaction solution was concentrated under reduced pressure to obtain compound A1.8-E, which was directly used in the next step. LCMS (ESI) [M+H]+: 578.0 Step 5: Compound (S) -N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3] Dioxo[4,5-g]pyrano[3',4':6,7]indole[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N - Preparation of isopropylacetamide (A1.8) to compound (S) -2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11, 13-tetrahydro-10H-[1,3]dioxa[...

Claims

1. A ligand-drug conjugate of Formula I or a stereoisomer of said ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein: Formula I Tb is the ligand or targeting moiety that binds to the target; q is the drug-ligand coupling ratio; q is any value between 1 and 8; the structure is selected from the following: where position 1 is connected to L4; when L4 is absent, position 1 is connected to L3; L1 is selected from and, position 1 is connected to Tb through the S atom, and position 2 is connected to L3; L2 is absent; L3 is selected from Val-AA1-Gly; the amino acid residues shown in AA1 are selected from,, and; L4 is absent or present, when L4 is present, L4 is, position 1 is connected to L3, and position 2 is connected to W.

2. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, q is any value between 2 and 8.

3. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, q is selected from 1, 2, 3, 4, 5, 6, 7 and 8.

4. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, L3 is selected from, with 1 bit connected to L1 and 2 bits connected to L4 or W.

5. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, L4 is configured such that bit 1 is connected to L3 and bit 2 is connected to W.

6. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, L4 does not exist.

7. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The structure is selected from the following structural fragments: .

8. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein the structure is selected from the following:

9. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The structure is selected from the following: , , , , , and; where 1 bit is connected to L4; when L4 does not exist, 1 bit is connected to L3.

10. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The structure is selected from the following: , , and; where 1 bit is connected to L4.

11. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, Tb is an antibody or its antigen-binding fragment.

12. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, Tb includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, non-human antibodies, humanized antibodies, chimeric antibodies, fully human antibodies, proantibodies, monoclonal antibodies, bispecific antibodies, or multispecific antibodies.

13. A stereoisomer of the ligand-drug conjugate as described in claim 1, or a pharmaceutically acceptable salt thereof, wherein, Tb is a monoclonal antibody.

14. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, Tb is an anti-Trop-2 antibody or its antigen-binding fragment, an anti-Her2 antibody or its antigen-binding fragment, an anti-EGFR antibody or its antigen-binding fragment, or an IgG1 isotype antibody against chicken lysin.

15. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, Tb is any of the following: (i) Tb is an anti-Her2 antibody or its antigen-binding fragment, wherein the anti-Her2 antibody is anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, Trastuzumab, Pertuzumab or its antigen-binding fragment; (ii) Tb is an antibody against Trop-2 or its antigen-binding fragment, wherein the antibody against Trop-2 is datopotamab, sacituzumab or its antigen-binding fragment; or (iii) Tb is an antibody against EGFR or its antigen-binding fragment, wherein the antibody against EGFR is demupitamab, depatuxizumab, futuximab, imgatuzumab, lapatuximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, zalutumumab, cetuximab or its antigen-binding fragment.

16. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The ligand-drug conjugate is selected from the following: wherein Tb and q are as defined in any one of claims 1-15.

17. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The ligand-drug conjugate is selected from the following: where Tb2 is an anti-Trop-2 antibody or its antigen-binding fragment; q is selected from any value between 1 and 8.

18. The ligand-drug conjugate as claimed in claim 17, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, Tb2 is an antibody against datopotamab and sacituzumab.

19. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The ligand-drug conjugate is selected from the following: where Tb3 is an anti-Her2 antibody or its antigen-binding fragment; q is selected from any value between 1 and 8.

20. The ligand-drug conjugate as claimed in claim 19, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, Tb3 is an antibody against anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, or trastuzumab.

21. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The ligand-drug conjugate is selected from the following: where Tb5 is an anti-EGFR antibody or its antigen-binding fragment; q is selected from any value between 1 and 8.

22. The ligand-drug conjugate as claimed in claim 21, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, Tb5 is a demupitamab, depatuxizumab, futuximab, imgatuzumab, laprituximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomuzotuximab, zalutumumab or Cetuximab antibody.

23. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The ligand-drug conjugate is selected from the following: wherein Tb6 is an antibody that has no tumor cell endocytic activity and no tumor cell surface antigen binding activity, or an antibody that has non-endocytic antigen binding activity; q is selected from any value between 1 and 8.

24. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The ligand-drug conjugate is selected from the following: Tb7 is an antibody with weak or no tumor cell endocytic activity, but with tumor cell surface antigen binding activity; q is selected from any value between 1 and 8.

25. The ligand-drug conjugate as claimed in claim 24, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, Tb7 contains antibodies that bind to GD-2, Trop-2, EGFR, CD19, CD30, GPNMB, CD20, CD79b, and BCMA antigens but do not have endocytic activity.

26. The ligand-drug conjugate as claimed in claim 1, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, The ligand-drug conjugate is selected from the following: wherein Tb8 is an antibody with tumor cell endocytosis activity and tumor cell surface antigen binding activity; q is selected from any value between 1 and 8.

27. The ligand-drug conjugate as claimed in claim 26, or a stereoisomer of the ligand-drug conjugate, or a pharmaceutically acceptable salt thereof, wherein, Tb8 is an antibody against sacituzumab, pertuzumab, trastuzumab, or cetuximab.

28. A linker in a ligand-drug conjugate, comprising the following fragment: wherein, Position 1 is linked to the ligand or target portion that binds to the target, and position 2 is linked to the bioactive molecular fragment; L1, L2, L3, and L4 are defined as described in any of claims 1-26.

29. A compound of Formula II: Formula II or a stereoisomer of the compound, or a pharmaceutically acceptable salt thereof, wherein, The compounds shown in Formula II are selected from the following: and.

30. A drug linker conjugate of Formula III or a stereoisomer of said drug linker conjugate, or a pharmaceutically acceptable salt thereof, wherein: Formula III R1, R2, R3, X, L1, L2, L3, L4 and W are defined as described in any of claims 1-26; L1 is connected to Lg at position 1; Lg is a leaving group.

31. The drug linker conjugate as claimed in claim 30, or a stereoisomer of the drug linker conjugate, or a pharmaceutically acceptable salt thereof, wherein, Lg is selected from halogens, ternary ammonium salts, diazonium salts, -OMs, MeSO2- and CF3SO3-.

32. The drug linker conjugate as claimed in claim 31, or a stereoisomer of the drug linker conjugate, or a pharmaceutically acceptable salt thereof, wherein, The drug linker conjugate is selected from the following:

33. A method for preparing the ligand-drug conjugate according to any one of claims 1-27, comprising: The drug linker conjugate shown in Formula III is coupled with Tb in a suitable solvent and under suitable conditions; wherein: Tb is as defined in any of claims 1-27; L1, L2, L3, L4, R1, R2, R3, X, W, Lg are as defined in any of claims 30-32.

34. The method as described in claim 33, wherein, The molar ratio of Tb to the drug linker conjugate is 1:(1-20); the coupling reaction is carried out in water and / or an organic solvent; the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles, alcohols, or any combination thereof.

35. A ligand-drug conjugate comprising any one of claims 1-27, wherein the ligand-drug conjugate has two or more q values.

36. The ligand-drug conjugate as claimed in claim 35, wherein, The drug-to-antibody ratio (DAR) in the ligand-drug conjugate is selected from an integer or decimal number from 1 to 8.

37. The ligand-drug conjugate as claimed in claim 36, wherein, The drug-to-antibody ratio (DAR) in the ligand-drug conjugate is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8.

0.

38. A pharmaceutical composition comprising substance A and optionally one or more pharmaceutical excipients, wherein substance A is a ligand-drug conjugate as described in any one of claims 1-27, or a stereoisomer of said ligand-drug conjugate, a pharmaceutically acceptable salt thereof, or a drug linker conjugate as described in any one of claims 30-32, or a stereoisomer of said drug linker conjugate, a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate as described in any one of claims 35-37.

39. Use of a substance A or the pharmaceutical composition of claim 38 in the preparation of a medicament for treating and / or preventing diseases associated with abnormal cell activity; wherein the substance A is a ligand-drug conjugate as described in any one of claims 1-27, or a stereoisomer of the ligand-drug conjugate, a pharmaceutically acceptable salt thereof, or a drug linker conjugate as described in any one of claims 30-32, or a stereoisomer of the drug linker conjugate, a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate as described in any one of claims 35-37.

40. The use as described in claim 39, wherein, The diseases associated with abnormal cell activity are cancerous diseases; the cancerous diseases are selected from esophageal cancer, brain tumor, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer, rectal cancer, colorectal cancer, liver cancer, kidney cancer, urothelial carcinoma, epidermal cancer, non-Hodgkin's lymphoma, central nervous system tumors, prostate cancer, or thyroid cancer.

41. The use as described in claim 40, wherein, The esophageal cancer is esophageal adenocarcinoma or esophageal squamous cell carcinoma; the lung cancer is small cell lung cancer, non-small cell lung cancer, or lung adenocarcinoma; the central nervous system tumor is glioma, glioblastoma multiforme, glioma, or sarcoma; and the colon cancer is human colon adenocarcinoma.

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