Linker, antibody drug conjugate and use thereof

Abstract

The present application provides a linker, an antibody drug conjugate and a preparation method thereof, and the use thereof in preventing and / or treating diseases related to abnormal cell activities, including but not limited to preventing or and / or treating tumor diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a linker, an antibody-drug conjugate and its preparation method, as well as its use in the prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to its use in the prevention and / or treatment of tumor diseases. Background Technology

[0002] CD38 is a transmembrane glycoprotein that functions as a cell surface receptor and extracellular enzyme. As a cell surface receptor, CD38 participates in leukocyte adhesion and migration, and promotes lymphocyte growth, survival, and activation. The enzymatic activity of CD38 involves catalyzing the substrate NAD+. + The catalytic process transforms the cells into products such as ADPR and cADPR. ADPR / cADPR, as important second messengers in cells, can influence cell growth or activation by regulating cellular calcium ions. The catalytically generated ADPR / cADPR products can be further processed by other enzymes to produce adenosine, which exerts its anti-tumor immunosuppressive function by recruiting MDSCs and Treg cells. (Malavasi F, Deaglio S, Funaro A, et al. Evolution and function of the ADP ribosyl cyclase / CD38 gene family in physiology and pathology. Physiol Rev. 2008;88(3):841-886. Wo YJ, Gan ASP, Lim X, et al. The Roles of CD38 and CD157 in the Solid Tumor Microenvironment and Cancer Immunotherapy. Cells. 2019;9(1):26. Published 2019 Dec 20. CD38 is expressed in multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and Burkitt's lymphoma. (Calabretta E, Carlo-Stella C. The Many Facets of CD38 in Lymphoma: From Tumor-Microenvironment Cell Interactions to Acquired Resistance to Immunotherapy. Cells. 2020;9(4):802. )。

[0003] Daratumumab is a monoclonal antibody targeting CD38 and has been approved by the National Medical Products Administration (NMPA) for the treatment of adult patients with relapsed and refractory multiple myeloma (MM). However, most MM patients develop primary resistance to daratumumab monotherapy, and a small percentage of responsive MM patients eventually acquire resistance as well. (Saltarella I, Desantis V, Melaccio A, et al. Mechanisms of Resistance to Anti-CD38 Daratumumab in Multiple Myeloma. Cells. 2020;9(1):167. ) Clinical trials have shown that, compared to patients with high CD38 expression in multivariate myeloma (MM), daratumumab monotherapy in patients with intermediate to low CD38 expression resulted in a lower objective response rate (ORR). (Nijhof IS, Casneuf T, van Velzen J, et al. CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma. Blood. 2016;128 （7）:959-970.）。 Furthermore, daratumumab monotherapy in CD38-expressing NHL patients, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL), had an objective response rate (ORR) of only approximately 8.3%. Salles G, Gopal AK, Minnema MC, et al. Phase 2 Study of Daratumumab in Relapsed / Refractory Mantle-Cell Lymphoma, Diffuse Large B-Cell Lymphoma, and Follicular Lymphoma. Clin Lymphoma Myeloma Leuk. 2019;19(5):275-284.). Therefore, there is an urgent need in the field for antibody drugs targeting CD38 with higher biological activity, especially antibody-drug conjugates (ADCs) that are effective against primary or acquired resistance to daratumumab, in order to further improve efficacy and reduce side effects.

[0004] Antibody-drug conjugates (ADCs) consist of an antibody, a payload, and a linker. The bioactive molecular payload (e.g., monomethylaurestatin E (MMAE)) is covalently coupled to the antibody via the linker. The antibody (e.g., a monoclonal antibody) specifically recognizes a target site on the surface of tumor cells, guiding the ADC to the cancer cell surface and into the cancer cells via endocytosis. The linker then cleaves within the cell under conditions such as low pH or lysosomal enzymatic reactions, releasing the payload and thus specifically killing cancer cells without damaging normal tissue cells. An ideal linker should ensure sufficient stability and integrity of the ADC drug in the bloodstream, while efficiently releasing the payload upon reaching the therapeutic target (tumor cells). 。 (Chau CH, Steeg PS, Figg WD. Antibody-drug conjugates for cancer. Lancet. 2019;394(10200):793-804. Drago JZ, Modi S, Chandarlapaty S. Unlocking the potential of antibody-drug conjugates for cancer therapy. Nat Rev Clin Oncol. 2021;18（6）:327-344.）。

[0005] Compared to daratumumab antibody drugs, the CD38-targeting ADC of this invention uses an innovative cleavable linker to connect the CD38-targeting antibody to a potent payload, specifically killing CD38-expressing cancer cells without damaging normal tissue cells, thus exhibiting stronger anti-tumor activity. Currently, no CD38-targeting ADCs using MMAE as a payload have entered clinical trials; only one ADC, STI-6129, using an MMAF analogue as a payload, has entered Phase I clinical trials. However, MMAF payloads are prone to causing clinical ocular toxicity. (Mahalingaiah PK, Ciurlionis R, Durbin KR, et al. Potential mechanisms of target-independent uptake and toxicity of antibody-drug conjugates. Pharmacol Ther. 2019;200:110-125.) This clearly does not meet medical needs.

[0006] This invention aims to develop novel antibody drugs targeting CD38, novel linkers, and antibody-drug conjugates containing said linkers to meet clinical needs.

[0007] Invention Summary

[0008] This invention provides a novel linker, an antibody-drug conjugate comprising the linker, and its preparation and use. This invention also provides a novel anti-CD38 antibody binding to a CD38 molecule or its antigen-binding fragment, an antibody-drug conjugate comprising the same, and its preparation and use.

[0009] In a first aspect, the present invention provides a drug conjugate of formula (III),

[0010] Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof. Wherein, Ab represents the target portion, such as an antibody or its antigen-binding fragment. p is an integer selected from 1 to 16, for example, an integer selected from 1 to 10, 1 to 9, 2 to 8, 4 to 10, 6 to 8, 3 to 7, 4 to 8, 2 to 6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12; D represents the active pharmaceutical molecule; L a Selected from -C 1-12 alkylene-, -C 1-12 Alkylene -C(O)-, -C 1-12 Alkylene-CONH-(CH2CH2O) n -CH2-C(O)-, -(CH2CH2O) n -CH2-C(O)-, where n is an integer selected from 1 to 18, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; "Cycle A" is an optional substituted 3-8 membered heterocyclic alkyl or 6-10 membered heterocyclic aryl; L1 is selected from -CH2-CHR1-, -O-CH2-, and is absent; R1 is -OH, -NH2, or -C. 1-4 alkylene -OH or -C 1-4 Alkylene-NH2; L2 is a linker unit, selected from short peptide residues composed of 2-10 amino acids, wherein the amino acids are natural or non-natural amino acids; L3 is a spacing unit; and m can be 1, 2, 3 or 4.

[0011] In a second aspect, the present invention provides a compound represented by formula (I): , Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein: L a Rings A, L1, L2, and m are as defined in this paper; L 3a For interval units.

[0012] Thirdly, the present invention provides a compound represented by formula (II),

[0013] Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein the variables are as defined herein.

[0014] Fourthly, the present invention provides a novel anti-CD38 antibody or antigen-binding fragment thereof that binds to CD38 molecules, which is an isolated anti-CD38 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises three heavy chain complementarity-determining regions (HCDRs) as in SEQ ID NO:7 or 9, and the light chain variable region comprises three light chain complementarity-determining regions (LCDRs) as in SEQ ID NO:8 or 10.

[0015] In another aspect, the present invention provides an isolated nucleic acid that encodes any anti-CD38 antibody or its antigen-binding fragment provided by the present invention.

[0016] In another aspect, the present invention provides a recombinant vector or expression vector comprising one or more nucleic acids provided by the present invention, wherein the vector is suitable for recombinant generation of any anti-CD38 antibody or antigen-binding fragment provided by the present invention.

[0017] In another aspect, the present invention provides a host cell comprising one or more nucleic acids, recombinant vectors or expression vectors provided by the present invention.

[0018] In another aspect, the present invention provides an immunoconjugate or immunofusion compound comprising the anti-CD38 antibody or its antigen-binding fragment provided by the present invention.

[0019] In another aspect, the present invention provides a drug conjugate comprising the anti-CD38 antibody or its antigen-binding fragment provided by the present invention.

[0020] In another aspect, the present invention provides a pharmaceutical composition comprising the compounds of the present invention, such as the anti-CD38 antibody or its antigen-binding fragment provided by the present invention, nucleic acid, recombinant vector or expression vector, host cell, immunoconjugate or immunofusion, drug conjugate, and optionally comprising at least one pharmaceutically acceptable excipient (e.g., pharmaceutical carrier or pharmaceutical excipient).

[0021] In another aspect, the present invention provides a pharmaceutical combination comprising the compounds of the present invention, such as the anti-CD38 antibody or its antigen-binding fragment provided by the present invention, nucleic acid, recombinant vector or expression vector, host cell, immunoconjugate or immunofusion compound, drug conjugate, and one or more other therapeutic agents.

[0022] In another aspect, the present invention also provides the use of the anti-CD38 antibody or its antigen-binding fragment, nucleic acid, recombinant vector or expression vector, host cell, immunoconjugate or immunofusion compound, and drug conjugate of the present invention in the preparation of a medicament for treating cancer.

[0023] Fifthly, the present invention provides intermediates represented by formulas (IV-1), (V-1), and (VI-1): ; ; ; R5 is an amino protecting group; the variables such as La, ring A, L1, X, m, R3, and R4 are as defined in this paper. Invention Details

[0025] definition

[0026] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0027] The term "amino acid residue" refers to an amino acid unit in a peptide chain, that is, the portion remaining after the amino acids linked by peptide bonds have lost water. The abbreviation for amino acid residue is a standard 3-letter and / or 1-letter code used in the art to refer to one of the commonly used amino acids. For example, the amino acids forming the residue may be selected from phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), glycine (Gly), alanine (Ala), valine (Val), lysine (Lys), citrulline (Cit), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), and glutamine (Gln), etc. The amino acid may be an L or D isomer, preferably an L-isomer.

[0028] Unless otherwise expressly stated, the singular form used in this application (including the claims) includes its corresponding plural form.

[0029] When the term “and / or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0030] As used herein, the terms "comprising" or "including" mean including the mentioned elements, integers, or steps, but do not exclude any other elements, integers, or steps. As used herein, when the terms "comprising" or "including" are used, unless otherwise stated, instances consisting of the mentioned elements, integers, or steps are also covered. For example, when referring to an antibody variable region that "comprising" a particular sequence, it is also intended to cover the antibody variable region consisting of that particular sequence.

[0031] In this invention, the term "individual" includes humans or non-human animals. Exemplary human individuals include individuals suffering from a disease (such as those described herein) (referred to as patients) or normal individuals. The term "non-human animal" in this invention 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.). The terms "individual" or "patient" are not limited to a specific age or sex. In some embodiments, the individual or patient is a human.

[0032] In this invention, the term "effective amount" refers to the amount of a compound that, when administered, will alleviate the treated condition or one or more symptoms to a certain extent.

[0033] The term "alkyl" as used herein refers to a fully saturated straight-chain or branched hydrocarbon group. Alkyl groups preferably contain 1-16 carbon atoms (C1-C2). 1-16 For example, 1-12 carbon atoms (C 1-12 ), 1-10 carbon atoms (C 1-10 ), 1-6 carbon atoms (C 1-6 ) or 1-4 carbon atoms (C 1-4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0034] The term "alkylene" refers to an alkyl group as defined above, but which is divalent, meaning it has two single bonds attached to two other groups. Examples of alkylene groups include, but are not limited to, -CH2-, -CH2 CH2-, -CH2 CH2 CH2-, -CH2 CH2CH2 CH2-, -CH2 CH2 CH2 CH2 CH2-, -CH2 CH2 CH2 CH2 CH2 CH2-, -CH2 CH2 CH2 CH2 CH2-CH2 CH2, -CH(-CH2CH3)-, or -CH2CH(-CH3)-.

[0035] The terms "heterocyclic alkyl" or "heterocyclic group" are used interchangeably to refer to a fully saturated or unsaturated non-aromatic cyclic group, having, for example, 3-8 ring atoms, such as 4-7 ring atoms (4-7-membered heterocyclic alkyl), 5-6 ring atoms (5-6-membered heterocyclic alkyl), 5 ring atoms (5-membered heterocyclic alkyl), or 6 ring atoms (6-membered heterocyclic alkyl). The ring atoms include one or more (e.g., 1, 2, or 3, preferably 1 or 2) heteroatoms independently selected from N, O, and S, with the remaining ring atoms being carbon atoms; it may have 1 or 2 rings, preferably 1 ring. N and S may optionally be oxidized to various oxidation states. The junction of the heterocyclic alkyl group can be on a heteroatom or a carbon atom. "Heterocyclic alkyl" includes 5- or 6-membered saturated heterocyclic alkyl groups containing 1 or 2 nitrogen atoms. Exemplary heterocyclic groups include, but are not limited to, piperidinyl, piperazine, pyrrolidinyl, or dihydropyridinyl.

[0036] The term "heterocyclic alkylene" refers to a heterocyclic alkyl group as defined above, but which is divalent, meaning it has two single bonds attached to two other groups. Examples of heterocyclic alkylene include, but are not limited to, piperidinyl, piperazine, pyrrolidinyl, or dihydropyridinyl such as 1,2-dihydropyridinyl.

[0037] The term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic aromatic ring system having 5-14 ring atoms (5-14-membered heteroaryl), wherein the ring atoms include one or more, for example, 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, and the remaining ring atoms are carbon atoms, wherein at least one ring is an aromatic ring, and wherein N and S may optionally be oxidized to various oxidation states. When the total number of S and O atoms in the heteroaryl exceeds 1, these S and O heteroatoms are not adjacent to each other. Preferably, the heteroaryl is a 6-10-membered heteroaryl, more preferably a 6-membered heteroaryl. Examples of heteroaryl include, but are not limited to, imidazolyl, pyridyl, pyrazinyl, or pyrimidinyl.

[0038] The term "heteroaryl" refers to a heteroaryl group as defined above, but which is divalent, meaning it has two single bonds attached to two other groups. Examples of heteroaryl groups include, but are not limited to, imidazolyl, pyridinyl, pyrazinyl, or pyrimidinyl.

[0039] The terms “optional,” “optional,” or “optionally” mean that the event or situation described below may or may not occur, and the description includes both scenarios in which the event or situation occurs and scenarios in which the event or situation does not occur. For example, when a group or structure is “optionally substituted,” the group or structure may or may not be substituted.

[0040] The terms “substituted,” “replaced,” or “substituted with” mean that one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on a given atom or group are replaced by one or more (e.g., 1, 2, 3, or 4) substituents, preferably selected from a given group of substituents or groups, provided that the substitution does not exceed the normal valence of the given atom, and the substituents may be the same as or different from each other. The terms “replaced by one or more groups selected from” or “substituted with one or more groups” as used herein mean that one or more hydrogen atoms on a given atom or group are independently replaced by one or more groups selected from a given group of substituents or groups, wherein the groups may be the same as or different from each other. Optional substituents can be various groups, provided that the combination of substituents and / or variables produces a chemically correct and stable compound. A chemically correct and stable compound means that the compound is stable enough to be isolated from the reaction mixture. Preferably, the substituents are those implemented in the example compounds of this application.

[0041] Unless otherwise stated, substituents are named within the core structure. For example, it should be understood that when a (cycloalkyl)alkyl group is listed as a possible substituent, it indicates that the substituent is attached to the core structure at the alkyl moiety.

[0042] An "amino protecting group" is a group that protects the amino group from unwanted reactions during a reaction and can be easily removed after the reaction. Examples of amino protecting groups include Fmoc, Boc, and Cbz.

[0043] A "carboxyl protecting group" is a group that protects the carboxyl group from unwanted reactions during a reaction and can be easily removed after the reaction. Examples of carboxyl protecting groups include benzyl, methyl, ethyl, or tert-butyl.

[0044] In this invention, the term "conjugate" refers to a substance obtained by linking a payload or active molecule to a target moiety. In some embodiments of the invention, the payload or active molecule is linked to the target moiety via a linker. Preferably, the linker is cleavable under specific conditions (e.g., intracellular low pH) or specific action (e.g., action of lysosomal proteases), thereby separating the active molecule from the target moiety. In some embodiments of the invention, the linker comprises a cleavable unit, such as a peptide or a disulfide bond.

[0045] In some embodiments, the payload or active molecule is a pharmaceutically active molecule. In this invention, the term "pharmaceutically active molecule" refers to a substance capable of regulating biological processes, particularly altering or preventing pathological processes, especially substances that inhibit or prevent cell function and / or cause cell death or damage. In some embodiments of this invention, the bioactive substance or pharmaceutically active molecule in the conjugate is a molecule with antitumor biological activity. "Pharmaceutically active molecule" includes "small molecule drugs."

[0046] In this invention, the term "small molecule drug" refers to a low molecular weight pharmaceutically active molecule capable of regulating biological processes, particularly altering or preventing pathological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa, more preferably less than 800 kDa, less than 600 kDa, less than 500 kDa, or less than 300 kDa. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0047] Non-limiting examples of “small molecule drugs” and / or “pharmaceutical active molecules” include platinum complexes (e.g., oxaliplatin), gold complexes; glycopeptide antibiotics, such as bleomycin or pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (e.g., Dxd, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotetan, or rubotecan), and topoisomerase II inhibitors (e.g., actinomycin D, doxorubicin, docarmicin, daunorubicin, mitoxetine). Anthraquinones, podophyllotoxin, or etoposide; auristatin compounds such as monomethylaurestatin E (MMAE) and monomethylaurestatin F (MMAF); DNA synthesis interfering drugs such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, or nelabine; drugs acting on structural proteins such as microtubule inhibitors, vinblastine alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, or cabazitaxel; maytansine derivatives; calichimycin derivatives; Pyrrolobenzodiazepine dimers (PBD) derivatives; melphalan; mitomycin C; chlorambucil; and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis, or necrosis.

[0048] In this invention, the term "linker" refers to a segment that connects a pharmaceutically active molecule to a target moiety. It should be understood that a linker has functional groups that can form bonds with functional groups of the target moiety before being linked to it. Non-limiting examples of linkers include those involved in the embodiments and examples of this invention.

[0049] As used herein, a "spacer unit" refers to a component that helps to separate a portion of the linker from other groups of the payload compound described in this invention. The spacer unit can lead to more efficient coupling and / or more efficient cleavage / lysis of the antibody and payload compound. In some embodiments, the spacer unit is capable of self-degradation after efficient cleavage / lysis; in some embodiments, the spacer unit can also stabilize the antibody-drug conjugate and result in improved overall antibody-drug conjugate properties.

[0050] The term "targeting portion" refers to the part of a conjugate that specifically binds to a target (or a portion of a target) on the cell surface. Through the interaction between the targeting portion and the target, the conjugate can be delivered to a specific cell population. Examples of non-limiting cell surface targets include ACTA2, ADGRE1, AGS-16, AIF1, AKR1C1, AKR1C2, ANGPTL4, ApoE, ASLG659, B7-H3, BAFF-R, BCMA, BMPR1B, BNIP3, Brevican, C1QA, C1QB, CA6, CADM1, CCL5, CCR5, CD11b, CD11c, CD123, CD138, and CD... 19. CD19, CD20, CD21, CD22, CD223, CD30, CD33, CD37, CD38, CD40, CD45(PTPRC), CD49D(ITGA4), CD56, CD 66e, CD70, CD72, CD74, CD79a, CD79b, CD79b, CD80, CDCP1, CDH11, CEACAM5, CEACAM5 / 6, CEACAM6, CLDN18.2. c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTGF, CTSD, CTSS, CXCL10, CXCL11, CXCR5, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGFRvIII, EGLN, EGLN3, EMR2, Endothelin receptor, ENPP3, EpCAM, EphA5, EphB2R, ETBR, FcRH1, FcRH2, FGF2, FGFR2, Fibronectin extra domain B, Folatereceptor1, FR alpha, GEDA, GPNMB, Guanylyl cyclase C, GZMB, HER2, Her3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3, IL10RA1, IL20Rα, IL-6, IRTA2, K ISS1R, KRT33A, LIV1, LOX, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MCPT8, MDP, Mesothelin, MFI2, MM P10, MMP14, MMP16, MPF, MS4A7, MSG783, Mucin-1, Mucin16, NaPi2b, Napi3b, NCA, Nectin-4, NOG, P2 X5, pCAD, P-cadherin, PDGFRA, PDK1, PD-L1, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCA hlg, PSMA, RNF43, ROR1, Sema5b, SERPINEl, SLC39A6, SLITRK6, SLTRK6, Sodiumphosphate cotransporter 2B, STAT1, STC2, STEAP1, STEAP2, TACSTD2, TCF4, TENB2, TGF, TGFB1, TGFB2, TGFBR1, Tissue factor, TNFRSF21, TNFSF9, Trf1, Trop-2, Trophoblastglycoprotein, TrpM4, Tyro7, UPK1B, VEGFA, WNT5A, integrins α5β6, α4β7, 0772P and 5T4. .

[0051] In this invention, when the targeting portion of the conjugate is an antibody or its antigen-binding fragment, the conjugate is referred to as an "immunoconjugate".

[0052] In this invention, when the active molecule in the immunoconjugate is a pharmaceutical active molecule, the immunoconjugate may be referred to as an "antibody-drug conjugate (ADC)". In this invention, "drug conjugate" and "antibody-drug conjugate" can be used interchangeably.

[0053] In this invention, when an antibody or its antigen-binding fragment is covalently linked to one or more other peptides or polypeptides, it is referred to as an "immunofusion".

[0054] The term "linker-payload" refers to a compound formed by linking a payload, such as a drug active molecule (e.g., a small molecule drug), to a linker.

[0055] The term “DAR” refers to the ratio of the drug fraction (D) conjugated to the target portion (e.g., the Ab portion, preferably an antibody) described herein to the target portion (e.g., the Ab portion, preferably an antibody). In some embodiments described herein, the DAR may be determined by p in formula (III), for example, the DAR may be 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The DAR may also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug fraction (D) conjugated to the Ab portion described herein to the Ab portion in the product as determined by a detection method (e.g., by conventional methods such as mass spectrometry and / or HIC-HPLC), this DAR is referred to herein as the average DAR or the determined DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 16, for example 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, for example 1.0-8.0, 2.0-6.0, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, and a range with any two of these values ​​as endpoints. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population or mixture of ADC molecules containing ADC molecules of the present invention having the same and / or different DAR values.

[0056] The term "solvate" refers to an association formed by one or more solvent molecules with the ADC antibody-drug conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0057] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0058] As used herein, the term "cancer" refers to a cellular disorder characterized by uncontrolled or disordered cell proliferation, reduced cell differentiation, inappropriate invasion of surrounding tissues, and / or the ability to establish new growth sites in other locations. The term "cancer" includes, but is not limited to, solid tumors and hematologic malignancies (e.g., leukemia, lymphoma, or myeloma). The term "cancer" includes cancers of the skin, tissues, organs, bones, cartilage, blood, and blood vessels. The term "cancer" includes both primary cancers and metastatic, recurrent, and refractory cancers. Examples of cancer include those referred to herein (including embodiments and examples).

[0059] The term "pharmaceutical composition" refers to a composition that exists in a form that allows for the biological activity of the active ingredient contained therein, and that does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition. In addition to the active ingredient, such as an ADC, it typically contains ingredients such as pharmaceutically acceptable carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. The pharmaceutical compositions described herein are intended to provide the expected biological activity of the active ingredient and / or achieve the therapeutic effect if permitted for administration.

[0060] The terms "pharmaceutically acceptable carrier" and "physiologically acceptable carrier" refer to a carrier or diluent that does not cause significant irritation to the subject and does not eliminate the bioactivity and properties of the applied active ingredient, such as an ADC compound or composition and / or any other therapeutic agent in the composition. Pharmaceutically acceptable carriers may enhance or stabilize a composition, or may be used to facilitate the preparation of a composition. Pharmaceutically acceptable carriers may include solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and combinations thereof, as known to those skilled in the art (see, for example, Remington). ’ (See *Pharmaceutical Sciences*, 18th edition, Mack Printing Company, 1990, pp. 1289-1329). Unless any conventional carrier is incompatible with the active ingredient to date, its use in therapeutic or pharmaceutical compositions should be considered. The carrier may be selected to minimize adverse side effects in the subject and / or to minimize degradation of the active ingredient. Adjuvants may also be included in any of these formulations.

[0061] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the active ingredient. Formulations intended for parenteral administration may contain, for example, excipients such as sterile water or saline; polyalkylene glycols (such as polyethylene glycol); vegetable oils; or hydrogenated naphthalene. Other exemplary excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, ethylene-vinyl acetate copolymer particles, and surfactants, including, for example, polysorbate 20.

[0062] The term "pharmaceutically acceptable salt" means a salt that does not eliminate the biological activity and properties of the compounds of the present invention and does not cause significant irritation to the subject to which it is administered. Examples of such salts include, but are not limited to: (a) acid addition salts formed from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.; and salts formed from organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, etc.; and (b) salts formed from basic anions, such as chloride ions, bromide ions, and iodide ions.

[0063] The terms “drug combination,” “combination product,” “drug conjugate,” or “combination product” refer to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term “non-fixed combination” means that the active ingredients (e.g., (i) the antibody molecule or ADC molecule of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of two or more active agents. In some embodiments, the antibody molecule or ADC molecule of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when used alone. The term “fixed combination” means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in a separate formulation, and their formulations may be the same or different.

[0064] The terms "combination therapy" or "treatment in combination" refer to the administration of two or more therapeutic agents or modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0065] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0066] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0067] The term "antibody" refers to any form of antibody that has the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, human antibodies, chimeric antibodies, CrossMab antibodies, or camel-derived single-domain antibodies.

[0068] The term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations that may exist in small amounts. The modifier "monoclonal" indicates the characteristic of an antibody derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of the antibody by any particular method.

[0069] The term “isolated antibody or antigen-binding fragment” refers to the purified state of an antibody or antigen-binding fragment. For example, “isolated” can mean that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. However, as those skilled in the art will appreciate, the term “isolated” does not mean the complete absence or non-existence of such substances, unless they are present in amounts that significantly interfere with the experimental or therapeutic application of the antibody described herein. In some embodiments, the isolated antibody or antigen-binding fragment may have a purity greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0070] The terms "full-length antibody" and "intact antibody" are used interchangeably in this document and refer to antibodies with a structure substantially similar to that of natural antibodies. "Natural antibody" refers to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain consists of a variable region (VH) and a constant region. The constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region. The constant region consists of the CL domain. The VH and VL regions can be further divided into hypervariable regions (complementarity-determining regions or CDRs), with more conserved regions (framework regions or FRs) inserted in between. Each VH or VL consists of three CDRs and four FRs, arranged in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in antibody-antigen binding but exhibits various effector functions. The heavy chain of an antibody can be classified into one of five types (called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM)), some of which can be further subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Based on the amino acid sequence of its constant region, the light chain of an antibody can be classified into one of two types, called κ (kappa) and λ (lambda).

[0071] The complementarity-determining region (CDR) or CDR is a region within the antibody variable region that is hypervariable in sequence and forms a structurally defined loop (“hypervariant loop”) and / or contains antigen contact residues (“antigen contact sites”). CDRs are primarily responsible for binding to epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located in the antibody heavy chain variable region are designated HCDR1, HCDR2, and HCDR3, while those located in the antibody light chain variable region are designated LCDR1, LCDR2, and LCDR3. Each VH or VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In a given VH or VL amino acid sequence, the precise amino acid sequence boundary of each CDR can be determined by using any of a variety of well-known schemes or combinations thereof, including, for example: the Chothia scheme (Chothia et al., Canonical structures for the hypervariable regions of immunoglobulins, Journal of Molecular Biology, 196, 901-917 (1987)); the Kabat scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact (University College London); and the North scheme (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406, 228-256 (2011)). The CDR of the antibody in this invention can be determined according to any scheme or combination thereof in the art and human evaluation. In some embodiments, the CDR of the antibody in this invention can be determined according to the Kabat scheme.

[0072] The term "antibody fragment" refers to a molecule distinct from a whole antibody that contains a portion of the whole antibody that binds to the antigen bound to the whole antibody. Examples of antibody fragments include, but are not limited to, Fv, such as disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), Fab, Fab', Fab'-SH, and F(ab')2; biantibodies such as disulfide-stabilized biantibodies (ds diabody), triantibodies, tetraantibodies, and crossFab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv) and scFv dimers (divalent biantibodies); and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). Antibody fragments can be prepared using a variety of techniques, including but not limited to the proteolytic digestion of whole antibodies and production from recombinant host cells (e.g., E. coli or bacteriophages).

[0073] The term "antigen-binding fragment" includes a fragment of an antibody that binds to the antigen. Typically, the antigen-binding fragment includes at least one fragment (e.g., one or more CDRs) of the antigen-binding region or variable region of the antibody and retains at least some of the binding properties of the antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, such as disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), Fab, Fab', Fab'-SH, F(ab')2; biantibodies such as disulfide-stabilized biantibodies (dS diabody), triantibodies, tetraantibodies, crossFab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv) and scFv dimers (divalent biantibodies); single-domain antibodies; nanobodies; and multispecific antibodies. When antigen-binding activity is expressed on a molar concentration basis, the antigen-binding fragment typically retains at least 10% of the antigen-binding activity of the antibody from which it originates. The preferred antigen-binding fragments retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding activity of the antibody from which they originate.

[0074] The term "chimeric antibody" refers to an antibody possessing a variable region of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies originate from different species. Typically, the variable region is derived from antibodies from laboratory animals such as rodents, while the constant domain sequence is derived from human antibodies. This results in chimeric antibodies being less likely to induce adverse immune responses in human subjects compared to antibodies from the aforementioned laboratory animals.

[0075] The term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, a humanized antibody contains at least one, typically two, variable regions, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are the framework regions of a human immunoglobulin. Optionally, a humanized antibody may contain at least a portion of the constant region (Fc) of a human immunoglobulin. In some cases, as those skilled in the art will know, amino acid mutations may be introduced into the humanized antibody (e.g., the variable region, framework region, and / or constant region (if present)) to, for example, improve certain properties of the antibody; such antibody forms still fall within the scope of the "humanized antibody" of this invention.

[0076] As those skilled in the art will appreciate, antibodies can have a glycan form suitable for the cells used to produce the antibody. For example, when produced in mice, in mouse cells, or in hybridomas derived from mouse cells, antibodies may contain mouse glycans. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, antibodies may contain rat glycans.

[0077] The term "Fc region" is used to define a C-terminal region in an immunoglobulin heavy chain that contains at least a portion of a constant region. This term includes native Fc regions and Fc region variants. Native Fc regions encompass the various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subclasses and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the C-terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0078] The terms “Fc variant” or “variant Fc region” are used interchangeably herein to refer to an Fc region containing modifications relative to the native sequence Fc region. Variants obtained by mutating an Fc region for specific properties are known in the art. The Fc variants of the present invention are defined according to the amino acid modifications that constitute them. In one embodiment, the Fc region is modified to possess the properties of an effector function (e.g., complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to the wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from: using a native Fc isotype having a reduced or eliminated effector function, and Fc region modification. In a preferred embodiment, the Fc region has a reduced effector function mediated by the Fc region, such as a reduced or eliminated ADCC or ADCP or CDC effector function, for example, by including mutations that achieve the above functions.

[0079] "Sequence identity" refers to the degree of sequence identity based on comparing nucleotides or amino acids one by one within a comparison window. "(Percentage) sequence identity" can be calculated as follows: Compare two optimally aligned sequences within the comparison window, determine the number of positions in the two sequences that have the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiply the result by 100 to produce the percentage of sequence identity. For the purpose of determining the percentage of sequence identity, optimal alignment can be performed in various ways known in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm that requires maximal alignment across the full length of the sequence being compared or a region of the target sequence. In this invention, for antibody sequences, in one embodiment, the percentage of identity between the two amino acid sequences is determined by performing optimal alignment between a candidate antibody sequence and a reference antibody sequence, followed by optimal alignment according to a numbering rule.

[0080] The term "conservative substitution" or "conservative amino acid substitution" refers to the substitution of one or more amino acids with one or more chemically or functionally similar amino acids. Tables providing conserved substitutions of similar amino acids are well known in the art. Peptide sequences having such substitutions are referred to as "variants with conserved modifications." Such variants with conserved modifications are complementary to, but not excluded from, polymorphic variants, interspecific homologs, and alleles. Conserved substitutions are illustrated in Table A.

[0081] Table A

[0082] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, such as HEK 293 or 293F cells or 293FT cells or Expi293 cells). In another embodiment, the host cell is prokaryotic. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells or 293FT cells or Expi293 cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), and human liver cells (Hep). G2), CHO cells, NSO cells, and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / 0.

[0083] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors." In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector is pCDNA3.4.

[0084] Detailed Implementation Plan

[0085] Implementation Plan I

[0086] The drug conjugate shown in formula (III) of implementation scheme I-1,

[0087] Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof. Here, Ab represents the target portion, such as an antibody or its antigen-binding fragment; p is an integer selected from 1 to 16, for example, an integer selected from 1 to 10, 1 to 9, 2 to 8, 4 to 10, 6 to 8, 3 to 7, 4 to 8, 2 to 6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12; D represents the active pharmaceutical molecule; L a Selected from -C 1-12 alkylene-, -C 1-12 Alkylene -C(O)-, -C 1-12 Alkylene-CONH-(CH2CH2O) n -CH2-C(O)-, -(CH2CH2O) n -CH2-C(O)-, where n is an integer selected from 1 to 18, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; "Cycle A" is an optional substituted 3-8 membered heterocyclic alkyl or 6-10 membered heterocyclic aryl; L1 is selected from -CH2-CHR1-, -O-CH2-, and is absent; R1 is -OH, -NH2, or -C. 1-4 alkylene -OH or -C 1-4 Alkylene-NH2; L2 is a linker unit, selected from short peptide residues composed of 2-10 amino acids, wherein the amino acids are natural or non-natural amino acids; L3 is a spacing unit; and m can be 1, 2, 3 or 4.

[0088] It should be understood that Ab (sulfuric acid) undergoes a partial covalent bond formation with maleimide via the reduction of its disulfide bond to obtain a thiol group. In other words, Ab is partially covalently bonded to maleimide via its sulfur atom.

[0089] Implementation Scheme I-2. The drug conjugate or its pharmaceutically acceptable salt as described in Implementation Scheme I-1, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, wherein, L a Selected from -C 1-6 alkylene-, -C 1-6 Alkylene -C(O)-, -C 1-6 Alkylene-CONH-(CH2CH2O) n -CH2-C(O)-, -(CH2CH2O) n -CH2-C(O)-, where n is an integer selected from 1 to 12, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; "Ring A" is a 5- or 6-membered nitrogen-containing saturated heterocyclic alkyl group or a 5- or 6-membered nitrogen-containing unsaturated heterocyclic alkyl group substituted with oxygen (=O). When ring A is a 5- or 6-membered nitrogen-containing unsaturated heterocyclic alkyl group substituted with oxygen, L1 is absent.

[0090] Implementation Scheme I-3. The drug conjugate or its pharmaceutically acceptable salt described in any one of Implementation Schemes I-1 to I-2, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, wherein, Ring A is selected from and When ring A is When L1 does not exist.

[0091] Implementation Scheme I-4. The drug conjugate or its pharmaceutically acceptable salt as described in any one of Implementation Schemes I-1 to I-3, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, wherein: L1 is selected from , , and -O-CH2-; R1 is -OH, -NH2, -C 1-4 alkylene -OH or -C 1-4 Alkylene-NH2.

[0092] Implementation Scheme I-5. The drug conjugate or its pharmaceutically acceptable salt as described in any one of Implementation Schemes I-1 to I-4, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, wherein: L a Selected from: , , and ,in, # represents the position where it is attached to the maleimide group, and ## represents the position where it is attached to ring A.

[0093] Implementation Scheme I-6. The drug conjugate or its pharmaceutically acceptable salt as described in any one of Implementation Schemes I-1 to I-5, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, wherein: L2 is the linker unit, selected from short peptide residues consisting of 2-10 amino acid residues, wherein the amino acid residues may be independently selected from the L- or D-isomers of the following amino acids: alanine (including β-alanine), arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, valine, glutamine, phenylalanine, lysine, leucine, isoleucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynyl acid, aminoalkyl diacid, heterocyclic carboxylic acid, citrulline, selenocysteine ​​[1-2], pyrrolyl lysine, diaminoalkyl acid and its derivatives. Derivatives; preferably L2 is selected from: -Phe-Lys-, -Val-Lys-, -Phe-Phe-Lys-, -D-Phe-Phe-Lys-, -Gly-Phe-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Iie-Cit-, -Trp-Cit-, -Phe-Ala-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Val-Ala-, -Gly-Gly-Phe-Gly. -Gly-Cit-, -Cit-Val-, -Cit-Ala-, -Lys-Val-, -Val-Lys(Ac)-, -Phe-Lys(Ac-), -Ala-Ala-, -Ala-Ala-Ala-, -Ala-Ala-Asn-, -Ala-Ala-Asp-, -Lys-Ala-Ala-Asn-, -Lys-Ala-Ala-Ala-Asp-, -D-Val-Leu-Lys-, -Gly-Gly-Arg-, -Gly-Gly-Phe-, -Val-Lys-Gly-, -Val-Lys-Gly-Gly- and -Lys-Ala-Asn- or derivatives thereof; more preferably, L2 is -Val-Cit- or derivatives thereof.

[0094] Implementation Scheme I-7. The drug conjugate or its pharmaceutically acceptable salt, or its solvates, racemic mixtures, enantiomers, diastereomers, or tautomers, as described in any one of Implementation Schemes I-1 to I-6, wherein L2 has the structure shown in formula (I-4):

[0095] R3 is selected from: -NHCONH2, -NHCOCH2(OCH2CH2) n1 OCH3、-(CH2) n2 SO2CH3, -(CH2) n2 P(O)(CH3)2; where n1 is an integer selected from 1 to 18, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; n2 is 0, 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, R3 is selected from: -NHCONH2, -NHCOCH2(OCH2CH2) n1 OCH3, -SO2CH3, -P(O)(CH3)2; where n1 is an integer selected from 6-12.

[0096] Implementation Scheme I-8. The drug conjugate or its pharmaceutically acceptable salt as described in any one of Implementation Schemes I-1 to I-7, or their solvates, racemic mixtures, enantiomers, diastereomers, or tautomers. L3 is the structure shown in equation (II-5): ; R4 is selected from -(CH2). n2 SO2CH3, -(CH2) n2 P(O)(CH3)2 or hydrogen; where n2 is 0, 1, 2, 3, 4, 5, 6, 7 or 8; n3 is 1 or 2; $ is the active site linked to L2, $$ is the site linked to the active drug molecule; Preferably, L3 is the structure shown in formula (II-6). The symbols are defined as in equation (II-5).

[0097] Implementation Scheme I-9. The drug conjugate or its pharmaceutically acceptable salt as described in Implementation Scheme I-8, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, wherein the structure of L3 is selected from: and ; Where $ represents the linking site with L2, and $$ represents the linking site with the active pharmaceutical molecule.

[0098] Implementation Scheme I-10. The drug conjugate or its pharmaceutically acceptable salt as described in any one of Implementation Schemes I-1 to I-9, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, Where D represents Dolatatin 10 and its derivatives, preferably selected from: , , , , , , , ; Preferably, D is a Dolastatin 10 derivative selected from: , , , ; More preferably, D is selected from: , ; The wavy line represents the connection point with L3.

[0099] Implementation Scheme I-11. The drug conjugate or its pharmaceutically acceptable salt as described in Implementation Schemes I-1 to I-10, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, wherein p is selected from 2, 3, 4, 5, 6, 7 and 8.

[0100] Implementation Scheme I-12. The drug conjugate or a pharmaceutically acceptable salt thereof as described in any of Implementation Schemes I-1 to I-11, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein Ab is an anti-CD38 antibody or an antigen-binding fragment thereof, an anti-CD20 antibody or an antigen-binding fragment thereof, an anti-CD70 antibody or an antigen-binding fragment thereof, or a PD-L1 antibody or an antigen-binding fragment thereof.

[0101] In some embodiments, the anti-CD20 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of rituximab, teimomab, tosimob, oflamb, oliquizumab, osuzumab, osuzumab, repertuzumab, Ublituximab, or zabetuzumab. In some embodiments, the anti-CD20 antibody comprises the heavy chain variable region and the light chain variable region of rituximab, teimomab, tosimob, oflamb, osuzumab, osuzumab, repertuzumab, Ublituximab, or zabetuzumab. In some embodiments, the anti-CD20 antibody comprises a heavy chain and a light chain of rituximab, teimomab, tosimob, oflamb, oliquizumab, osuzumab, repertuzumab, Ublituximab, or zabetuzumab. In some embodiments, the anti-CD20 antibody comprises two heavy chains and two light chains of rituximab, teimomab, tosimob, oflamb, osuzumab, osuzumab, repertuzumab, Ublituximab, or zabetuzumab.

[0102] In some embodiments, the anti-CD70 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of Vorsetuzumab. In some embodiments, the anti-CD70 antibody comprises a heavy chain variable region and a light chain variable region of Vorsetuzumab. In some embodiments, the anti-CD70 antibody comprises both a heavy chain and a light chain of Vorsetuzumab. In some embodiments, the anti-CD70 antibody comprises two heavy chains and two light chains of Vorsetuzumab.

[0103] In some embodiments, the PD-L1 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of adebelimumab, sugemalimab, envomarab, durvalumab, acitumab, or atezolizumab. In some embodiments, the PD-L1 antibody comprises a heavy chain variable region and a light chain variable region of adebelimumab, sugemalimab, envomarab, durvalumab, acitumab, or atezolizumab. In some embodiments, the PD-L1 antibody comprises both the heavy and light chains of adebelimumab, sugemalimab, envomarab, durvalumab, acitumab, or atezolizumab. In some embodiments, the PD-L1 antibody comprises two heavy chains and two light chains of adebelimumab, sugemalimab, envorimab, durvalumab, acitumab, or atezolizumab.

[0104] Implementation Plan I-13. The anti-CD38 antibody is selected from Daratumumab or an anti-CD38 antibody as defined herein, or an antigen-binding fragment thereof, as described in any of Embodiments I-1 to I-12, or an anti-CD38 antibody or an antigen-binding fragment thereof, as described in any of Embodiments IV-1 to IV-14; the anti-CD20 antibody is selected from Rituximab, Tiimumab, Tosimob, Ofamumab, Otoruzumab, Otoruzumab, Repertuzumab, Ublituximab, and Zabetuzumab; the anti-CD70 antibody is selected from the anti-CD70 antibody, Vorsetuzumab; and the PD-L1 antibody is selected from Adebelimumab, Sugemalimab, Envorimab, Duvalimumab, Acimenumab, and Atezolizumab.

[0105] Implementation Scheme I-14. The drug conjugate or its pharmaceutically acceptable salt as described in Implementation Scheme I-13, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, wherein the Ab is an anti-CD38 antibody or its antigen-binding fragment.

[0106] Implementation Scheme I-15. The drug conjugate or its pharmaceutically acceptable salt as described in Implementation Scheme I-13, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, The Ab is an anti-CD38 antibody or its antigen-binding fragment, comprising a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein... The HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 19; the LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 20, the LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 21, and the LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 22. Optionally, the VH comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 15, or is composed of the amino acid sequence shown in SEQ ID NO: 16; and / or the VL comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16, or is composed of the amino acid sequence shown in SEQ ID NO: 16. Optionally, the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:15, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:16; or the VH comprises the amino acid sequence shown in SEQ ID NO:15, and the VL comprises the amino acid sequence shown in SEQ ID NO:16; or the VH is composed of the amino acid sequence shown in SEQ ID NO:15, and the VL is composed of the amino acid sequence shown in SEQ ID NO:16. Optionally, the anti-CD38 antibody or its antigen-binding fragment is an IgG subtype, such as IgG1, IgG2, IgG3, or IgG4. For example, the anti-CD38 antibody or its antigen-binding fragment may further comprise a heavy chain constant region, such as the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, such as the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. For example, the heavy chain constant region may comprise an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity as shown in SEQ ID NO: 13 or 23, or may be composed of or consist of the amino acid sequence shown in SEQ ID NO: 23. Optionally, the anti-CD38 antibody or its antigen-binding fragment further comprises a light chain constant region, for example, the light chain constant region is the light chain constant region of Kappa or Lambda, such as the light chain constant region of human Kappa or human Lambda, for example, the light chain constant region comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity as shown in SEQ ID NO: 14, or is composed of said amino acid sequence; Optionally, the anti-CD38 antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises or is composed of the amino acid sequence shown in SEQ ID NO:13 or 23, and the light chain constant region comprises or is composed of the amino acid sequence shown in SEQ ID NO:14. Optionally, the anti-CD38 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, or is composed of a heavy chain variable region and a heavy chain constant region; and / or the light chain comprises a light chain variable region and a light chain constant region, or is composed of a light chain variable region and a light chain constant region. For example, the anti-CD38 antibody comprises two heavy chains and two light chains, such as two identical heavy chains and light chains. Optionally, the heavy chain comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11, or is composed of said amino acid sequence; and / or the light chain comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12, or is composed of said amino acid sequence. For example, the heavy chain comprises SEQ ID NO: 11. The amino acid sequence shown in NO:11 or composed of therein, and the light chain contains or is composed of the amino acid sequence shown in SEQ ID NO:12; Optionally, the anti-CD38 antibody is a full-length antibody; Optionally, the anti-CD38 antibody is Daratumumab.

[0107] Implementation Scheme I-16. The drug conjugate as described in Implementation Scheme I-14, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein Ab is an anti-CD38 antibody as defined herein or an antigen-binding fragment thereof, such as an antigen-binding fragment of an anti-CD38 antibody as described in any of Implementation Schemes IV-1 to IV-14, for example Ab1 or huAb1.

[0108] Implementation scheme I-17. Drug conjugate as shown in formula (III-2),

[0109] Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein X is N or CH, and the remaining variables (Ab, L) a L1, L2, L3, D, m and p) are defined as in equation (III).

[0110] Implementation scheme I-18. Drug conjugate as shown in formula (III-3),

[0111] Or their pharmaceutically acceptable salts, or their solvates, racemic mixtures, enantiomers, diastereomers, or tautomers, wherein each variable (Ab, L) a R1, L2, L3, D, m and p) are as defined in equation (III) above.

[0112] Implementation Scheme I-19. The drug conjugate as described in any one of Implementation Schemes I-1 to I-18, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the drug conjugate is selected from: ; ; ; ; ; ; ; ; ; ; ; ; ;

[0113] Ab and p are defined as above.

[0114] Implementation Scheme I-20. The drug conjugate as described in Implementation Scheme I-19, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein the Ab is Daratumumab and p is 2, 3, 4, 5, 6, 7 or 8.

[0115] Implementation Scheme I-21. The drug conjugate as described in Implementation Scheme I-19, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein Ab is an antigen-binding fragment of an anti-CD38 antibody as described in any of Implementation Schemes IV-1 to IV-14, such as Ab1 or huAb1; p is 2, 3, 4, 5, 6, 7 or 8.

[0116] Implementation Scheme I-22. The drug conjugate, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, as described in any of Implementation Schemes I-1 to I-21, having an average DAR of 2-8, for example 3-4, 3.8-4.2, 3-5, 4-5.5, 5-7, 6.5-8 or 6-8.

[0117] Implementation Scheme I-23. A pharmaceutical composition comprising a pharmaceutical conjugate or a pharmaceutically acceptable salt thereof, or a solvate thereof, racemic mixture thereof, enantiomer, diastereomer or tautomer thereof, as described in any one of Implementation Schemes I-1 to I-22, and optionally comprising a pharmaceutically acceptable carrier.

[0118] Implementation Scheme I-24. Use of any of the drug conjugates described in Implementation Schemes I-1 to I-22 or their pharmaceutically acceptable salts or solvates, racemic mixtures, enantiomers, diastereomers or tautomers in the preparation of a medicament for the treatment of cancer.

[0119] Implementation Plan II

[0120] Implementation scheme II-1. The compound represented by formula (I): , Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein: L a Rings A, L1, L2, and m are as defined in Implementation Scheme I above; L 3a For interval units.

[0121] Implementation Scheme II-2. The compound as described in Implementation Scheme II-1 or its pharmaceutically acceptable salt, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, L 3a The structure shown in equation (I-5) is as follows: ; R4 is selected from -(CH2). n2 SO2CH3, -(CH2) n2 P(O)(CH3)2 or hydrogen; where n2 is 0, 1, 2, 3, 4, 5, 6, 7 or 8; n3 is 1 or 2; $ is the active site linked to L2; Preferably, L 3a The structure shown in equation (I-6) The symbols are defined as shown in equation (I-5).

[0122] Implementation Scheme II-3. The compound as described in Implementation Scheme II-2 or its pharmaceutically acceptable salt, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, L 3a The structure is selected from: , ; Here, $ represents the connection site with L2.

[0123] Implementation Scheme II-4. The compound described in any one of Implementation Schemes II-1 to II-3, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the compound is a compound represented by formula (I-2):

[0124] Where X is N or CH; the remaining variables (L a L1, L2, L 3a (and m) as defined in one of the implementation schemes II-1 to II-3.

[0125] Implementation Scheme II-5. The compound described in any one of Implementation Schemes II-1 to II-4, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the compound is a compound represented by formula (I-3):

[0126] R1 is defined as in Implementation Scheme I-4, and the other variables are defined as in one of Implementation Schemes II-1 to II-4.

[0127] Implementation Scheme II-6. The compound as described in Implementation Scheme II-5 or a pharmaceutically acceptable salt thereof, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, wherein: R1 is -OH, -NH2, -CH2-OH, or -CH2-NH2.

[0128] Implementation Scheme II-7. The compound as described in Implementation Scheme II-1 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the compound is selected from: , , , , , , , , , , , , , , , , .

[0129] It should be understood that, unless otherwise specified and without contradiction in the context, the bonds on the left side of the divalent group shown herein are connected to the groups on the left side of the divalent group in its structure, and the bonds on the right side of the divalent group are connected to the groups on the right side of the divalent group in its structure. For example, when ring A is At that time, the bond on the nitrogen atom is with L a The bond on the carbon atom on the right is connected to L1. This also applies to other compounds, including ADC implementations.

[0130] Implementation Plan III

[0131] Implementation Scheme III-1. The compound shown in formula (II)

[0132] Or pharmaceutically acceptable salts thereof, or solvates, racemic mixtures, enantiomers, diastereomers or tautomers thereof, wherein D, L a Rings A, L1, L2, L3, and m are as defined in embodiment I of the present invention (e.g., any one of embodiments I-1 to I-19).

[0133] Implementation Scheme III-2. The compound of Implementation Scheme III-1 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein the compound is a compound represented by formula (II-2):

[0134] The variables are defined as in Embodiment I of the present invention, particularly Embodiment I-17.

[0135] Implementation scheme III-3. The compound represented by formula (II-3)

[0136] Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture thereof, enantiomer, diastereomer or tautomer thereof, wherein each variable is as defined in Embodiment I of the present invention, particularly Embodiment I-18.

[0137] Implementation Scheme III-4. The compound as described in Implementation Scheme III-1 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the compound is selected from: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; .

[0138] Implementation Plan IV

[0139] Implementation Scheme IV-1. An isolated anti-CD38 antibody or its antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises three heavy chain complementarity-determining regions (HCDRs) as shown in SEQ ID NO: 7 or 9; and the light chain variable region comprises three light chain complementarity-determining regions (LCDRs) as shown in SEQ ID NO: 8 or 10. Optionally, the HCDR and LCDR can be determined by any CDR definition scheme known in the art, such as Kabat, Chothia, AbM, or Contact, or a combination thereof. Preferably, the HCDR and LCDR are determined by the Kabat scheme.

[0140] Implementation Scheme IV-2. An isolated anti-CD38 antibody or its antigen-binding fragment, comprising a heavy chain variable region (VH) and a light chain variable region (VL); wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein

[0141] The HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO: 2; and the HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO: 6. Optionally, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6. Optionally, HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 3; LCDR1 consists of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 6.

[0142] Implementation Scheme IV-3. The anti-CD38 antibody or its antigen-binding fragment as described in Implementation Scheme IV-1 or IV-2, wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7 or 9, or is composed of the amino acid sequence shown in SEQ ID NO: 8 or 10, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10, or is composed of the amino acid sequence shown in SEQ ID NO: 8 or 10.

[0143] Implementation Scheme IV-4. The anti-CD38 antibody or its antigen-binding fragment as described in Implementation Scheme IV-3, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:7, and / or the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:8; Optionally, the VH comprises the amino acid sequence shown in SEQ ID NO:7, and the VL comprises the amino acid sequence shown in SEQ ID NO:8; Optionally, the VH consists of an amino acid sequence as shown in SEQ ID NO:7, and the VL consists of an amino acid sequence as shown in SEQ ID NO:8.

[0144] Implementation Scheme IV-5. The anti-CD38 antibody or its antigen-binding fragment as described in Implementation Scheme IV-3, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO: 9, and / or the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO: 10; Optionally, the VH comprises the amino acid sequence shown in SEQ ID NO:9, and the VL comprises the amino acid sequence shown in SEQ ID NO:10; Optionally, the VH consists of an amino acid sequence as shown in SEQ ID NO:9, and the VL consists of an amino acid sequence as shown in SEQ ID NO:10.

[0145] Implementation Scheme IV-6. The anti-CD38 antibody or its antigen-binding fragment as described in any of Implementation Schemes IV-1 to IV-5 is an IgG subtype, such as IgG1, IgG2, IgG3 or IgG4 subtype.

[0146] Implementation Scheme IV-7. The anti-CD38 antibody or its antigen-binding fragment as described in any of Implementation Schemes IV-1 to IV-6 further comprises a heavy chain constant region, for example, the heavy chain constant region is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, such as the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0147] Implementation Scheme IV-8. An anti-CD38 antibody or its antigen-binding fragment as described in Implementation Scheme IV-7, wherein the heavy chain constant region comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 13 or 23, or is composed of said amino acid sequence.

[0148] Implementation Scheme IV-9. The anti-CD38 antibody or its antigen-binding fragment as described in any of Implementation Schemes IV-1 to IV-8 further comprises a light chain constant region, for example, the light chain constant region is the light chain constant region of Kappa or Lambda, such as the light chain constant region of human Kappa or human Lambda.

[0149] Implementation Scheme IV-10. An anti-CD38 antibody or its antigen-binding fragment as described in Implementation Scheme IV-9, wherein the light chain constant region comprises or consists of an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity as shown in SEQ ID NO: 14.

[0150] Implementation Scheme IV-11. An anti-CD38 antibody or its antigen-binding fragment as described in any of Implementation Schemes IV-1 to IV-10, further comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises or is composed of the amino acid sequence shown in SEQ ID NO:13 or 23, and the light chain constant region comprises or is composed of the amino acid sequence shown in SEQ ID NO:14.

[0151] Implementation Scheme IV-12. An anti-CD38 antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes IV-1 to IV-11, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, or is composed of a heavy chain variable region and a heavy chain constant region; and / or the light chain comprises a light chain variable region and a light chain constant region, or is composed of a light chain variable region and a light chain constant region; Optionally, it comprises two heavy chains and two light chains, for example, two identical heavy chains and two identical light chains.

[0152] Implementation scheme IV-13. The anti-CD38 antibody or its antigen-binding fragment as described in any of implementation schemes IV-1 to IV-12 is a murine antibody, a chimeric antibody or a humanized antibody.

[0153] Implementation Scheme IV-14. An anti-CD38 antibody or its antigen-binding fragment as described in any of Implementation Schemes IV-1 to IV-13, which is a full-length antibody, a disulfide-bonded stable Fv fragment (dSFv), a single-domain antibody such as VHH, a Fab antibody (e.g., Fab' antibody, Fab'-SH), a (Fab')2 antibody, a single-chain antibody such as scFv, Fv, dAb (domain antibody), or a multispecific antibody such as a bispecific antibody.

[0154] Embodiment IV-15: An isolated nucleic acid encoding an anti-CD38 antibody or its antigen-binding fragment as described in any of Embodiments IV-1 to IV-14. Further, the nucleic acid encodes the heavy chain and / or light chain of the anti-CD38 antibody or its antigen-binding fragment as described in any of Embodiments IV-14. Further, the nucleic acid encodes the heavy chain variable region and / or light chain variable region of the anti-CD38 antibody or its antigen-binding fragment as described in any of Embodiments IV-1 to IV-14.

[0155] Implementation Scheme IV-16. A recombinant vector or expression vector comprising one or more nucleic acids as described in Implementation Scheme IV-15, further wherein the vector is adapted for recombinant generation of an anti-CD38 antibody or an antigen-binding fragment thereof as described in any of Implementation Schemes IV-1 to IV-13, for example, the vector being a pcDNA series vector, such as pcDNA3.1, pCDNA3.4.

[0156] Implementation Scheme IV-17. A host cell comprising one or more nucleic acids as described in Implementation Scheme IV-15 or recombinant vectors or expression vectors as described in Implementation Scheme IV-16, for example, the host cell being a CHO cell or a 293 cell, such as Expi293.

[0157] Implementation Scheme IV-18. A method for producing an anti-CD38 antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes IV-1 to IV-14, the method comprising: Culture of host cells containing a polypeptide chain encoding the antibody under conditions suitable for expression; optionally further comprising assembling the polypeptide chain to produce the molecule under conditions suitable for assembly of the polypeptide chain into the molecule; or Under conditions suitable for the chain expression of the antibody, host cells containing the nucleic acid molecule of embodiment IV-15 or the recombinant vector or expression vector of embodiment IV-16, or host cells of embodiment IV-17, are cultured, and optionally the antibody is recovered from the host cells (or host cell culture medium).

[0158] Embodiment IV-19. An immunoconjugate or immunofusion comprising an anti-CD38 antibody or an antigen-binding fragment thereof as described in any of Embodiments IV-1 to IV-14. Further, the immunoconjugate or immunofusion comprises other active molecules, such as pharmaceutically active molecules as defined in this invention (e.g., Dolasatin 10 and Dolasatin 10 derivatives as defined in Embodiment I-10).

[0159] In some embodiments, the antibody or its antigen-binding fragment is conjugated to a pharmaceutically active molecule via a linker described herein. In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0160] Implementation Scheme IV-20. A pharmaceutical composition comprising an anti-CD38 antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes IV-1 to IV-14, a nucleic acid as described in Implementation Scheme IV-15, a recombinant vector or expression vector as described in Implementation Scheme IV-16, a host cell as described in Implementation Scheme IV-17, an immunoconjugate or immunofusion as described in Implementation Scheme IV-19, and optionally comprising at least one pharmaceutically acceptable excipient (e.g., a pharmaceutical carrier or pharmaceutical excipient).

[0161] Implementation Scheme IV-21. Use of any anti-CD38 antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes IV-1 to IV-14, nucleic acid as described in Implementation Scheme IV-15, recombinant vector or expression vector as described in Implementation Scheme IV-16, host cell as described in Implementation Scheme IV-17, immunoconjugate or immunofusion compound as described in Implementation Scheme IV-19, or pharmaceutical composition as described in Implementation Scheme IV-20 in the preparation of a medicament for treating cancer.

[0162] Implementation Scheme IV-22: An anti-CD38 antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes IV-1 to IV-14, a nucleic acid as described in Implementation Scheme IV-15, a recombinant vector or expression vector as described in Implementation Scheme IV-16, a host cell as described in Implementation Scheme IV-17, an immunoconjugate or immunofusion as described in Implementation Scheme IV-19, or a pharmaceutical composition as described in Implementation Scheme IV-20, for use in a therapy, such as for the treatment of cancer.

[0163] Implementation Plan IV-23. A method of treating cancer, comprising administering to a subject an effective amount of an anti-CD38 antibody or an antigen-binding fragment thereof as described in any one of Implementation Plans IV-1 to IV-14, a nucleic acid as described in Implementation Plan IV-15, a recombinant vector or expression vector as described in Implementation Plan IV-16, a host cell as described in Implementation Plan IV-17, an immunoconjugate or immunofusion as described in Implementation Plan IV-19, or a pharmaceutical composition as described in Implementation Plan IV-20.

[0164] Implementation Scheme IV-24. Use of the anti-CD38 antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes IV-1 to IV-14, the nucleic acid as described in Implementation Scheme IV-15, the recombinant vector or expression vector as described in Implementation Scheme IV-16, the host cell as described in Implementation Scheme IV-17, the immunoconjugate or immunofusion as described in Implementation Scheme IV-19, or the pharmaceutical composition as described in Implementation Scheme IV-20 for the treatment of cancer.

[0165] Implementation Plan V

[0166] Scheme V-1. Compounds of formula (IV-1), or pharmaceutically acceptable salts thereof, or their solvates, racemic mixtures, enantiomers, diastereomers, or tautomers: ; Among them, L a Rings A, L1, and m are as defined in implementation scheme I or II.

[0167] Implementation Scheme V-2. The compound as described in Implementation Scheme V-1, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the compound is a compound of formula (IV-2):

[0168] Among them, L a X, L1, and m are as defined in implementation scheme I or II.

[0169] Implementation Scheme V-3. A compound as described in Implementation Scheme V-1, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the compound is a compound of formula (IV-3):

[0170] Among them, L a R1, L1, and m are as defined in implementation scheme I or II.

[0171] Implementation Scheme V-4. The compounds described in Implementation Schemes V-1 to V-4, or pharmaceutically acceptable salts thereof, or solvates, racemic mixtures, enantiomers, diastereomers, or tautomers thereof, wherein the compounds are selected from: , .

[0172] Implementation scheme V-5. The compound shown in formula (V-1), or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof:

[0173] R5 is an amino protecting group; rings A, L1, and m are as defined in embodiment I or II.

[0174] Implementation scheme V-6. The compound shown in formula (V-2), or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof,

[0175] R5 is an amino protecting group; X, L1, and m are as defined in embodiment I or II.

[0176] Implementation scheme V-7. The compound shown in formula (V-3), or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof,

[0177] R5 is an amino protecting group; the other variables are as defined in Implementation Scheme I or II.

[0178] Implementation Scheme V-8. The compounds described in Implementation Schemes V-5 to V-7, or pharmaceutically acceptable salts thereof, or solvates, racemic mixtures, enantiomers, diastereomers, or tautomers thereof, wherein the compounds are selected from: ; .

[0179] Scheme V-9. The compound of formula (VI-1), or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, ; Wherein, X, L1, R3, R4 and m are as defined in implementation scheme I or II.

[0180] Implementation scheme V-10. The compound shown in formula (VI-2), or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof: ; R1, R3, R4 and m are as defined in implementation scheme I or II.

[0181] Implementation Scheme V-11. The compound as described in Implementation Scheme V-9 or Implementation Scheme V-10 is selected from: ; ; ; ; ; ;

[0182] Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof.

[0183] The various embodiments of the present invention (including the embodiments below) and the features of the various embodiments should be understood to be arbitrarily combined with each other, and all such combinations are included within the scope of the present invention, just as such combinations are specifically and individually listed herein, unless the context clearly indicates otherwise.

[0184] General Synthesis Method

[0185] The compounds of formulas (I), (II), and (III) and / or their pharmaceutically acceptable salts described in this invention can be synthesized using commercially available starting materials, by methods known in the art, or by the methods disclosed in this patent application. The following examples illustrate general synthetic methods for the compounds of this invention.

[0186] On the one hand, the compound represented by formula (II) provided by the present invention can be synthesized by the following general synthetic method: Method A:

[0187] L a X, L1, L2, R3, R4, R5, D and m are as defined in this document.

[0188] Step A1-1: As shown in Method A, the compound of formula (A) reacts with the compound of formula (B) under basic conditions (e.g., but not limited to DIPEA) and in the presence of a condensing agent (e.g., but not limited to EDCI, HOBT or HATU) to give the compound of formula (F). Alternatively, in step A1-2: the compound of formula (A) reacts with the compound of formula (C) under alkaline conditions (e.g., but not limited to DIPEA) and in the presence of a condensing agent (e.g., but not limited to EDCI, HOBT, or HATU) to obtain the compound of formula (D). R6 is a carboxyl protecting group. When R6 is a different carboxyl protecting group, the compound of formula (D) reacts under the corresponding conditions (e.g., but not limited to, the compound of formula (D) reacts under alkaline conditions (e.g., but not limited to NaOH, etc.) or under catalytic conditions (e.g., but not limited to Pd / C, etc.) or under acidic conditions (e.g., but not limited to TFA, etc.) to obtain the compound of formula (V-2). Compound (V-2) reacts with compound (E) under alkaline conditions (e.g., but not limited to DIPEA) and in the presence of a condensing agent (e.g., but not limited to EDCI, HOBT, or HATU) to give compound (F). Step A2: The compound of formula (F) is reacted under acidic conditions (e.g., but not limited to TFA) or basic conditions (e.g., but not limited to Et2NH) to give the compound of formula (VI-1); Step A3: When L a Selected from -C 1-12 Alkylene -C(O)-, -C 1-12 Alkylene-CONH-(CH2CH2O) n -CH2-C(O)-, -(CH2CH2O) n When -CH2-C(O)- and R7 is OH, the compound of formula (G1) reacts with the compound of formula (VI-1) under basic conditions (e.g., but not limited to DIPEA) and in the presence of a condensing agent (e.g., but not limited to EDCI, HOBT, or HATU) to give the compound of formula (II-2); when L a Selected from -C 1-12 When alkylene oxides are present, the compound of formula (G2) reacts with the compound of formula (VI-1) in the presence of a reducing agent (e.g., but not limited to NaBH(OAc)3, etc.) to give the compound of formula (II-2); when L a Selected from -C 1-12 Alkylene -C(O)-, -C 1-12 Alkylene-CONH-(CH2CH2O) n -CH2-C(O)-, -(CH2CH2O) n When -CH2-C(O)- and R7 is Osu, the compound of formula (G1) reacts with the compound of formula (VI-1) under basic conditions (e.g., but not limited to Et3N or DIPEA) to give the compound of formula (II-2).

[0189] Method B:

[0190] L a Rings A, L1, L2, L3, D, and m are as defined in this paper.

[0191] As shown in Method B, the compound of formula (IV-1) reacts with the H-L2-L3-D compound under basic conditions (e.g., but not limited to DIPEA) and in the presence of a condensing agent (e.g., but not limited to HATU or HBTU) to give the compound of formula (II).

[0192] The substituents of the compounds obtained by the above methods can be further modified to obtain other desired compounds. For synthetic chemical transformation methods, see, for example: R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette (eds.), Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions.

[0193] Before use, the compounds of the present invention can be purified by column chromatography, high performance liquid chromatography, crystallization or other suitable methods.

[0194] Furthermore, the present invention provides a method for preparing the drug conjugate shown in formula (III).

[0195] The variables are as defined in the text. The method includes the following steps: (a) Add Ab (targeting part preferably antibody or its antigen-binding fragment) to a buffer solution, add reducing agent, and then incubate; (b) Couple the compound of formula (II) or its pharmaceutically acceptable salt, or their solvates to the reaction solution in step (a) to obtain the crude product;

[0196] The variables are as defined in the text. and (c) Optionally, the crude product is purified to obtain the drug conjugate shown in formula (III).

[0197] In some embodiments, the drug conjugate represented by formula (III) can be obtained by conjugating the linker moiety of the compound of formula (II) with the active group of an antibody moiety (e.g., Daratumumab or Ab1, HuAb1) in water and / or an organic solvent. Suitable organic solvents may be selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol), and any combination thereof. The conjugation method may employ a reducing agent (e.g., TCEP) to open the disulfide bond of the antibody, yielding a thiol group, which then reacts with the linker moiety of the compound of formula (II) to form a CS bond for conjugation. The resulting conjugated product can be purified by chromatographic methods (e.g., one or more of ion exchange chromatography, hydrophobic chromatography, reversed-phase chromatography, or affinity chromatography).

[0198] In some implementations, the method includes the following two steps: Step 1: Add TCEP solution (10 mmol / L, 2.5-3.0 eq) to a daratumumab solution with a concentration of 4-8 mg / mL prepared with reaction buffer. Incubate the resulting solution at 0℃-22℃ for 1-24 h. The reaction buffer is one of PBS-D (10 mmol / L, pH 7.0), histidine-acetate buffer (20 mmol / L, pH 5.5), or histidine buffer (10 mmol / L, pH 7.0). TCEP is dissolved in water or histidine buffer (10 mmol / L, pH 7.0). Step 2: Add a DMSO (75%) / H2O solution (5 mmol / L, 10-15 eq) of the compound of formula (II) or its pharmaceutically acceptable salt, or their solvates, to the above system, and continue the reaction at 4℃-25℃ for 60-180 min. Add 20 equivalents of L-cysteine, quench at 4℃ for 1 h, and replace the reaction solution with a dialysis card (Slide-A-Lyzer® Dislysis Cassette) or directly with an ultrafiltration centrifuge tube (Millipore Amicon® Ultra). After adding buffer, the product is obtained. The buffer is one of 10 mmol / L PBS (pH 7.0), 10 mmol / L citrate buffer (pH 5.5), or 10 mmol / L His / His·HCl (pH 6.2).

[0199] In some implementations, the steps are performed under the specific reaction conditions disclosed in the embodiments.

[0200] It should be noted that implementation schemes obtained by varying the range or specific values ​​of the specific reaction conditions disclosed in the embodiments by 100%, 80%, 60%, 40%, 20%, or 10% are also under consideration in this invention.

[0201] Pharmaceutical Composition

[0202] The compounds of the present invention (such as those in any of the embodiments herein) can be formulated into pharmaceutical compositions, alone or in combination with one or more other therapeutic agents. A pharmaceutical composition comprises: (a) a compound of the present invention; (b) a pharmaceutically acceptable excipient (e.g., one or more pharmaceutically acceptable carriers); and optionally (c) at least one other therapeutic agent. It should be understood that the active ingredient (the compound of the present invention) in the pharmaceutical composition is present in an effective amount. In some embodiments, the compounds of the present invention comprise compounds as defined in the embodiments above, such as compounds of formula (II), pharmaceutical conjugates of formula (III), or pharmaceutically acceptable salts thereof, or solvates, racemic mixtures thereof, enantiomers, diastereomers, or tautomers, antibodies or antigen-binding fragments thereof as defined in embodiment IV, nucleic acids, recombinant vectors or expression vectors, host cells, immunoconjugates or immunofusions, etc.

[0203] In some implementations, the additional therapeutic agent is selected from, for example, antitumor agents, anti-inflammatory agents, or immunomodulators, including chemotherapeutic agents, immune checkpoint inhibitors or agonists, and targeted therapeutic agents. As used herein, the term "antitumor agent" refers to any agent administered to a subject with cancer for the purpose of treating cancer, including but not limited to chemotherapeutic agents, immune checkpoint inhibitors or agonists, and targeted therapeutic agents.

[0204] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0205] Drug combinations and pillboxes

[0206] In some embodiments, the present invention also provides pharmaceutical combinations or pharmaceutical combination products comprising the compounds of the present invention, and one or more other therapeutic agents, wherein the therapeutic agents are as defined above.

[0207] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0208] In one embodiment, the complete medicine box of the present invention comprises, within the same package: - A first container containing a pharmaceutical composition comprising the compounds of the present invention; - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0209] Other therapeutic agents may be administered separately from the compounds of the present invention, or may be included in the same pharmaceutical composition, such as a fixed-dose combination drug, according to the disclosure of the present invention. Combination therapy can be used to improve efficacy (e.g., by including compounds that enhance the potency or effectiveness of the compounds of the present invention in the combination therapy), reduce one or more side effects, or reduce the required dosage of the compounds of the present invention.

[0210] Uses and methods

[0211] The compounds of the present invention show sufficient preclinical practical use to warrant clinical trials and are expected to demonstrate beneficial therapeutic or preventive effects, for example, in individuals with cancer.

[0212] The compounds of the present invention include compounds as defined in the embodiments above, such as compounds of formula (II), drug conjugates of formula (III), or pharmaceutically acceptable salts thereof, or solvates, racemic mixtures, enantiomers, diastereomers or tautomers thereof, antibodies or antigen-binding fragments thereof as defined in embodiment IV, nucleic acids, recombinant vectors or expression vectors, host cells, immunoconjugates or immunofusions, etc.

[0213] In one aspect, the present invention provides a method for preventing or treating a disease in a subject, including administering to the subject an effective amount of the compound, pharmaceutical composition, pharmaceutical combination, or cassette of the present invention.

[0214] In some embodiments, the disease is a tumor, including solid tumors and non-solid tumors such as hematologic malignancies. In some embodiments, the tumor is cancer. In some embodiments, the cancer includes solid tumors and non-solid tumors such as hematologic malignancies. Non-limiting examples of hematologic malignancies include hematopoietic lymphoid tissue tumors, myeloma, leukemia, etc.

[0215] In some embodiments, hematopoietic lymphoid tissue tumors include, but are not limited to, non-Hodgkin lymphoma; in some embodiments, non-Hodgkin lymphoma is B-cell lymphoma (e.g., but not limited to diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Burkitt lymphoma, etc.); in some embodiments, myeloma includes, but is not limited to, multiple myeloma (MM); in some embodiments, leukemia includes, but is not limited to, chronic myeloid leukemia, chronic lymphocytic leukemia, or acute lymphoblastic leukemia. In one embodiment, the disease is a CD38, CD20, CD70, or PD-L1-related disease. In some embodiments, the disease is associated with abnormal expression or abnormal activity of CD38, CD20, CD70, or PD-L1.

[0216] In some embodiments, the tumor, such as cancer, is CD38, CD20, CD70, or PD-L1 positive, for example, it comprises tumor cells expressing CD38, CD20, CD70, or PD-L1. In some embodiments, the tumor, such as cancer, is CD38, CD20, CD70, or PD-L1 negative, for example, it does not express or expresses low levels of CD38, CD20, CD70, or PD-L1. In some embodiments, the patient's tumor comprises tumor cells expressing CD38, CD20, CD70, or PD-L1. In some embodiments, the patient's tumor cells express CD38, CD20, CD70, or PD-L1, for example, moderately expressing CD38, CD20, CD70, or PD-L1, preferably highly expressing CD38, CD20, CD70, or PD-L1. In some embodiments, the tumor tissue of the patient with the tumor (e.g., cancer) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of CD38, CD20, CD70, or PD-L1, for example, compared to the levels of CD38, CD20, CD70, or PD-L1 in the same tissue of a healthy individual or in healthy tissue adjacent to the patient's tumor tissue. In some embodiments, the tumor cells of the patient have (e.g., elevated levels, such as nucleic acid or protein levels or activity) of CD38, CD20, CD70, or PD-L1, for example, compared to the levels of CD38, CD20, CD70, or PD-L1 in the same cells of a healthy individual or in healthy cells adjacent to the patient's tumor cells.

[0217] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from the diseases described herein). In one embodiment, the subject suffers from or is at risk of suffering from the diseases described herein (e.g., cancer). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0218] In other respects, the present invention provides the use of the above-described compounds or pharmaceutical compositions or assemblies of the present invention in the production or preparation of a medicine for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0219] In other respects, the present invention provides the above-described compounds or pharmaceutical compositions or kits for therapeutic purposes, such as for the uses described herein, for example for the prevention or treatment of the related diseases or conditions mentioned herein.

[0220] In some embodiments, the compounds or pharmaceutical compositions or drug combinations or kits of the present invention can delay the onset of symptoms and / or symptoms associated with the disease.

[0221] In some embodiments, the compounds or pharmaceutical compositions of the present invention may also be administered in combination with one or more other therapies, such as treatments and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0222] In some implementations, the therapeutic agent is as defined above.

[0223] The combination therapy of the present invention covers combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which the administration of the compounds of the present invention may occur before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceuticals.

[0224] The drug composition can be administered via known methods, such as oral, intravenous injection, intraperitoneal, intracerebral (internal parenchyma), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; via a continuous release system or via an implantable device. In some embodiments, the composition can be administered by bolus injection, continuous infusion, or via an implantable device.

[0225] The composition can also be applied topically via an implantable membrane, a sponge, or another suitable material on which the desired molecules are absorbed or encapsulated. In some embodiments, when an implantable device is used, the device can be implanted into any suitable tissue or organ and can deliver the desired molecules via diffusion, timed release of a large pellet, or continuous administration.

[0226] The compounds of the present invention can be used to achieve beneficial therapeutic or preventive effects, for example, in individuals suffering from cancer.

[0227] The drug conjugates of the present invention have the following advantages: (1) It specifically binds to target cells and has a similar affinity to naked antibodies; (2) It has excellent in vitro cell proliferation inhibitory activity and significant in vivo activity; (3) It can enter cells through endocytosis and kill target cells; (4) It has high internalization efficiency; (5) It has a certain bystander lethality effect; (6) It has better product uniformity; (7) It has good physicochemical stability, including plasma stability; and / or (8) It has good drug-like properties. Attached Figure Description

[0228] Figure 1 The binding activity of anti-CD38 antibody to human CD38 protein.

[0229] Figure 2 The binding activity of anti-CD38 antibody to monkey CD38 protein.

[0230] Figure 3 The binding activity of anti-CD38 antibody to CD38 on the surface of Daudi cells.

[0231] Figure 4 The binding activity of anti-CD38 antibody to CD38 on the surface of HCI-H929 cells.

[0232] Figure 5 Anti-CD38 antibody promotes the phagocytosis of Daudi and HCI-H929 tumor cells by human macrophages.

[0233] Figure 6 Anti-CD38 antibody mediates complement Daudi cell killing.

[0234] Figure 7 The binding activity of anti-CD38 antibody to NK cells.

[0235] Figure 8. Internal signal values ​​of the ADC product of the present invention.

[0236] Figure 9 Antitumor activity of Daratumumab and ADC-10 in Daudi subcutaneous xenografts.

[0237] Figure 10 Antitumor activity of Daratumumab and ADC-10 in KMS-11 subcutaneous xenografts. Detailed Implementation

[0238] The following examples are illustrative of the invention and do not limit the invention in any way. The data given (e.g., quantities, temperatures, etc.) are intended to be accurate; however, those skilled in the art will understand that some experimental errors and biases may occur. Unless otherwise stated, all parts are by weight, temperatures are in Celsius, and pressures are at or near atmospheric pressure. All mass spectrometry data were obtained using Agilent 6120 and 1100. All nuclear magnetic resonance data were obtained using a Varian 400MR. Except for synthetic intermediates, all reagents and raw materials used in this invention were commercially available. All compound names, except for reagents, were generated using Chemdraw 20.1.

[0239] In any structural formula of this application, if there is a vacant valence on any atom, the vacant valence is actually for the sake of simplicity and is not specifically described as a hydrogen atom.

[0240] In this application, if both the name and structural formula of a compound are given, and the two are inconsistent, the structure of the compound shall prevail, unless the context indicates that the structure of the compound is incorrect while the name is correct.

[0241] The following list of abbreviations is used in the embodiments:

[0242] Example 1 Preparation of intermediates

[0243] Preparation of Intermediate-1

[0244] A solution of compound A (1.41 g, 10 mmol) in 100 mL of dichloromethane was cooled to 0 °C, and Dysmart reagent (CAS No.: 87413-09-0) (6.36 g, 15 mmol) was added in portions. The resulting reaction mixture was transferred to room temperature and stirred for 3 hours. The solid was removed by filtration, and the filtrate was concentrated to give intermediate-1, a yellow solid, 1.11 g, in 80% yield. MS (m / z): 140.1 [M+H] +

[0245] Preparation of intermediate-2

[0246] 1) Synthesis of compound B

[0247] Compound A (4g, 21.5mmol), ( S Methyl 2,3-epoxypropionate (2.63 g, 25.8 mmol) and DIPEA (11.2 mL, 64.5 mmol) were dissolved in DMF (30 mL). The reaction mixture was stirred at 80 °C for 18 hours. After the reaction mixture cooled to room temperature, water (40 mL) was added. The resulting mixture was extracted with ethyl acetate (25 mL × 3). The organic phases were combined and washed once with water (40 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give 5.26 g of pale yellow solid B, yield 85%, MS (m / z): 289.2 [M+H]. +

[0248] 2) Synthesis of intermediate-2

[0249] Compound B (2.88 g, 10 mmol) and lithium hydroxide monohydrate (2 g, 50 mmol) were dissolved in a methanol / water mixture (15 mL / 6 mL). The reaction mixture was heated to 40 °C and stirred for 18 hours. After cooling to room temperature, it was further cooled in an ice-water bath, and then the pH of the reaction mixture was adjusted to 7.0 with 5 N hydrochloric acid in an ice-water bath. The resulting mixture was concentrated under reduced pressure and separated by ISCO (mobile phase: H₂O (0.1% HCOOH) / CH₃CN = 100:0–0:100) to give intermediate-2, a pale yellow solid, 2.41 g, yield 88%, MS (m / z): 275.2 [M+H]. +

[0250] Preparation of intermediate-3

[0251] 1) Synthesis of compound B

[0252] Compound A (19.2 g, 120 mmol), 2-oxo-1,2-dihydropyridine-3-onitrile (7.2 g, 60 mmol), and triphenylphosphine (23.6 g, 90 mmol) were dissolved in tetrahydrofuran (200 mL). The reaction mixture was cooled to 0 °C, and di-tert-butyl azodicarbonate (20.6 g, 90 mmol) was added in portions. After stirring at room temperature for 1 hour, the mixture was concentrated under reduced pressure and separated by ISCO (mobile phase: ethyl acetate / petroleum ether = 0:100–100:0) to give compound B as a white solid, 6.8 g, yield 43%. MS (m / z): 286.1 [M+Na] +

[0253] 2) Synthesis of compound C

[0254] Compound B (6.8 g, 25.8 mmol) was dissolved in dichloromethane (150 mL), and methanesulfonic acid (16.8 mL, 258 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes, then neutralized with ammonia water to pH 8. The solution was extracted with dichloromethane, and the organic phase was concentrated to dryness under reduced pressure to give compound C, a yellow oil, 4.2 g, yield 99%. MS (m / z): 164.1 [M+H] +

[0255] 3) Synthesis of compound D

[0256] Maleic anhydride (2.8 g, 28.4 mmol) was dissolved in diethyl ether (100 mL), followed by the addition of compound C (4.2 g, 25.8 mmol). The mixture was stirred at room temperature for 30 minutes, filtered, and the white precipitate was collected. After drying, compound D was obtained as a white solid, 6.5 g, with a yield of 97%. MS (m / z): 262.1 [M+H] +

[0257] 4) Synthesis of compound E

[0258] Compound D (3.6 g, 13.8 mmol) was dissolved in acetic anhydride (50 mL), and sodium acetate (1.6 g, 19.6 mmol) was added. The reaction mixture was stirred at 80 °C for 1 hour, followed by the addition of water (50 mL) and extraction with diethyl ether. The organic phase was concentrated under reduced pressure, and the residue was separated by ISCO (mobile phase: acetonitrile / water (0.1% trifluoroacetic acid) = 0:100–100:0) to give compound E as a white solid, 2.1 g, yield 62%. MS (m / z): 244.2 [M+H] +

[0259] 5) Synthesis of compound F

[0260] Compound E (1 g, 4.1 mmol) was dissolved in concentrated hydrochloric acid and heated under reflux for 30 minutes. The reaction solution was concentrated under reduced pressure and separated using ISCO (mobile phase: acetonitrile / water (0.1% trifluoroacetic acid) = 0:100–100:0) to give compound F as a yellow solid, 180 mg, yield 15%, MS (m / z): 299.1 [M+H] +

[0261] 6) Synthesis of intermediate-3

[0262] Compound F (180 mg, 0.6 mmol) and triethylamine (121 mg, 1.2 mmol) were dissolved in dichloromethane (5 mL). After stirring at room temperature for 30 minutes, the solution was concentrated under reduced pressure and then separated using ISCO (mobile phase: acetonitrile / water (0.1% trifluoroacetic acid) = 0:100–100:0) to give intermediate-3 as a yellow solid, 60 mg, yield 38%, MS (m / z): 263.1 [M+H]. +

[0263] Preparation of intermediate-4

[0264] 1) Synthesis of compound B

[0265] Compound A (4g, 21.5mmol), ( R Methyl 2,3-epoxypropionate (2.63 g, 25.8 mmol) and DIPEA (11.2 mL, 64.5 mmol) were dissolved in DMF (30 mL) and stirred at 80 °C for 18 hours. After the reaction mixture cooled to room temperature, water (40 mL) was added, and the mixture was extracted with ethyl acetate (25 mL × 3). The combined organic phases were washed once with water (40 mL), dried over anhydrous sodium sulfate, and the filtrate was filtered and concentrated to give compound B as a pale yellow solid, 5.6 g, yield 90%, MS (m / z): 289.2 [M+H]. +

[0266] 2) Synthesis of intermediate-4

[0267] Compound B (2.88 g, 10 mmol) and lithium hydroxide monohydrate (2 g, 50 mmol) were dissolved in a methanol / water mixture (15 mL / 6 mL). The mixture was heated to 40 °C and stirred for 18 hours, then cooled to room temperature. The pH of the reaction solution was then adjusted to 7.0 with 5 N hydrochloric acid under an ice-water bath. The resulting mixture was concentrated under reduced pressure, and the residue was separated using ISCO (mobile phase: H₂O (0.1% formic acid) / CH₃CN = 100:0–0:100) to give intermediate-4, a pale yellow solid, 2.2 g, yield 80%, MS (m / z): 275.2 [M+H].+

[0268] Preparation of intermediate-5

[0269] 1) Synthesis of compound A

[0270] Sodium hydride (239 mg, 5.96 mmol) was added to a dry THF (20 mL) solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (1 g, 4.96 mmol) at 0 °C. After stirring for 30 minutes, methyl 2-bromoacetate (912 mg, 5.96 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was quenched by adding saturated NH4Cl (100 mL), followed by extraction with ethyl acetate (50 mL × 3). The combined organic phases were dried over Na2SO4, and the solvent was removed by concentration under reduced pressure. The residue was purified by ISCO (methanol:water (0.1% formic acid) = 0:100–100:0) to give compound A as a pale yellow oil, 500 mg, yield 37%. MS (m / z) = 218.1 [M+H] +

[0271] 2) Synthesis of intermediate-5

[0272] Sodium hydroxide (10 mol / L, 3.7 mL) was added to a MeOH / THF / H₂O solution (2 mL / 1 mL / 1 mL) of compound A (500 mg, 1.83 mmol), and the mixture was stirred at room temperature for 4 h. The pH of the reaction solution was adjusted to 7.0 with 3 mol / L hydrochloric acid, and then extracted with ethyl acetate (20 mL × 3). The solvent was removed by concentration under reduced pressure. The residue was purified by ISCO (methanol:water (0.1% formic acid) = 0:100–100:0) to give intermediate-5 as a white solid, 300 mg, yield 63%. MS (m / z) = 204.1 [M+H] +

[0273] Preparation of intermediate-6

[0274] 1) Synthesis of compound B

[0275] Compound A (3.0 g, 25.0 mmol) and DIPEA (7.0 g, 55.0 mmol) were dissolved in dichloromethane (100 mL). Cb2-Cl (4.7 g, 27.5 mmol) was added under ice bath conditions, and the mixture was stirred overnight at room temperature under nitrogen protection. After the reaction was complete, the mixture was washed with water, and the aqueous phase was extracted with dichloromethane. The resulting organic phases were combined, dried, and concentrated. The residue was purified using ISCO (water (0.1% TFA) / acetonitrile = 100:0–0:100) to give compound B as a white solid, 3.5 g, yield 54.7%. MS (m / z): 254.2 [M+H] +

[0276] 2) Synthesis of compound C

[0277] Compound B (3.5 g, 13.8 mmol) and Et3N (1.5 g, 15.2 mmol) were dissolved in dichloromethane (100 mL). MsCl (1.7 g, 15.2 mmol) was added in an ice bath. The mixture was stirred overnight at room temperature under nitrogen protection. After the reaction was complete, the mixture was washed with water, and the aqueous phase was extracted with dichloromethane. The resulting organic phases were combined, dried, and concentrated to give crude compound C as a white solid (3.5 g), yield: 77.8%. The crude product was used directly in the next synthesis. MS (m / z): 354.2 [M+Na] +

[0278] 3) Synthesis of compound D

[0279] Compound C (3.5 g, 10.6 mmol) and Et3N (1.3 g, 12.7 mmol) were dissolved in dichloromethane (100 mL). Piperazine-1-carboxylic acid tert-butyl ester (2.4 g, 12.7 mmol) was added under ice bath conditions, and the mixture was stirred overnight at room temperature under nitrogen protection. After the reaction was complete, the mixture was washed with water, and the aqueous phase was extracted with dichloromethane. The resulting organic phases were combined, dried, and concentrated. The residue was purified by ISCO (PE / EA = 100:0-0:100) to give compound D as a white solid, 2.0 g, yield 45.5%. MS (m / z): 422.6 [M+H] +

[0280] 4) Synthesis of compound E

[0281] Compound D (2.0 g, 4.8 mmol) and LiOH·H₂O (1.0 g, 24.0 mmol) were added to methanol / water (3:1, 50 mL), and the mixture was purged with nitrogen and stirred at 40 °C for 3 hours. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was purified by ISCO (water (0.1% TFA) / acetonitrile = 100:0–0:100) to give compound E as 1.0 g of white solid, yield 52.6%. MS (m / z): 408.6 [M+H] +

[0282] 5) Synthesis of compound F

[0283] Compound E (1.0 g, 2.5 mmol) and Pd / C (100 mg) were added to methanol (20 mL). After displacing hydrogen at atmospheric pressure, the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, yielding compound F as a light-colored solid, 0.65 g, in 97.0% yield. MS (m / z): 274.2 [M+H] +

[0284] 6) Synthesis of intermediate-6

[0285] Compound F (0.65 g, 2.4 mmol), Fmoc-OSu (0.88 g, 2.6 mmol), and sodium bicarbonate (0.61 g, 7.2 mmol) were added to dioxane / H₂O (1:1, 20 mL), and stirred at room temperature for 5 hours under nitrogen protection. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was purified by ISCO (water (0.1% TFA): acetonitrile = 100:0–0:100 gradient elution) to give intermediate -6 as 0.7 g of white solid, yield 59.8%. MS (m / z): 496.4 [M+H] +

[0286] Preparation of intermediate-7

[0287] 1) Synthesis of compound B

[0288] Compound A (305 mg, 1.75 mmol), DIPEA (285 mg, 2.19 mmol), and HATU (832 mg, 2.19 mmol) were added to a DMF solution of intermediate-4 (400 mg, 1.46 mmol) under ice-water bath and nitrogen protection. After addition, the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, dried, concentrated, and purified by ISCO (mobile phase: CH3CN / H2O = 0 / 100-100 / 0) to give compound B as a white solid, 470 mg, yield 75%. MS (m / z): 430.2 [M+H] +

[0289] 2) Synthesis of intermediate-7

[0290] Compound B (400 mg, 1.46 mmol) was added to a mixed solvent of TFA / DCM (1:1, 4 mL) under ice-water bath conditions. After the addition was complete, the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated, and the resulting product was dissolved in a mixed solution of dioxane / H₂O (3:2), followed by the addition of sodium bicarbonate (368 mg, 4.38 mmol) and Fmoc-OSu (492 mg, 1.46 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 hours. The resulting reaction solution was concentrated under reduced pressure and purified by ISCO (mobile phase: CH₃CN:H₂O = 0:100-100:0) to give intermediate-7 as a white solid, 420 mg, yield 78%. MS (m / z): 496.4 [M+H] +

[0291] Preparation of intermediate-8

[0292] Intermediate-4 (500 mg, 1.82 mmol) was added to a TFA / DCM solution (1:1, 3 mL), and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was dissolved in THF, followed by the addition of DIPEA (712 mg, 5.47 mmol) and compound A (674 mg, 2.19 mmol). After the addition was complete, the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure and purified by ISCO (mobile phase: CH3CN:H2O = 0:100-100:0) to give intermediate-8 as a light gray solid, 260 mg, yield 38%. MS (m / z): 367.4 [M+H] +

[0293] Preparation of intermediate-9

[0294] 1) Synthesis of compound B

[0295] Under ice-water bath and nitrogen protection, sodium hydride (155 mg, 3.87 mmol) was added in a single dose to a dry DMF solution of compound A (1.3 g, 2.58 mmol). After the addition was complete, the reaction solution was stirred in an ice-water bath for 15 min. Iodomethane (440 mg, 3.10 mmol) was then added dropwise to the reaction solution. After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was poured into water and extracted with ethyl acetate. The extract was washed with saturated potassium hydrogen sulfate solution, then with saturated brine. After drying with sodium sulfate, the solution was concentrated to obtain compound B, which was an oily crude product. No purification was required before proceeding to the next reaction. MS (m / z): 519.2 [M+H] +

[0296] 2) Synthesis of intermediate-9

[0297] Pd / C was added to a THF solution of compound B, and the resulting reaction solution was stirred at 60°C for 3 hours under hydrogen protection. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated. The residue was dissolved in dichloromethane (30 mL), followed by the addition of silver oxide (155 mg, 3.87 mmol), compound C (155 mg, 3.87 mmol), and potassium iodide (155 mg, 3.87 mmol). The reaction solution was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated. The residue was dissolved in TFA / DCM (3 mL, 1:1), and stirred at room temperature for 1 hour. The residue obtained after concentration was purified by ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give intermediate-9, a colorless oil, 770 mg, yield 61%. MS (m / z): 487.3 [M+H] +

[0298] Preparation of intermediate-10

[0299] 1) Synthesis of compound B

[0300] MMAE (1 g, 1.4 mmol), compound A (1.34 g, 1.75 mmol), HOBT (117 mg, 0.87 mmol), and DIPEA (0.61 mL, 3.5 mmol) were dissolved in DMF (12 mL). The reaction mixture was stirred at room temperature for 18 hours and then concentrated under reduced pressure. The residue was purified by ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give compound B as a yellow solid, 1.12 g, yield 60%. MS (m / z): 1346.8 [M+H] +

[0301] 2) Synthesis of intermediate-10

[0302] Compound B (1.12 g, 0.83 mmol) and triethylamine (2.3 mL, 16.6 mmol) were dissolved in DMF (10 mL) and stirred at room temperature for 18 hours. The reaction solution was concentrated under reduced pressure and purified by ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give intermediate-10 as a yellow solid, 700 mg, yield 75%. MS (m / z): 1123.6 [M+H] +

[0303] Preparation of intermediate-11

[0304] 1) Synthesis of compound A

[0305] Intermediate-10 (500 mg, 0.45 mmol), intermediate-4 (367 mg, 1.33 mmol), EDCI (171 mg, 0.89 mmol), HOBT (120 mg, 0.89 mmol), and DIPEA (0.78 mL, 4.45 mmol) were dissolved in DMF (10 mL). The resulting reaction solution was stirred at room temperature for 18 hours and then concentrated under reduced pressure. The residue was purified by ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give compound A as a yellow solid, 420 mg, yield 68%. MS (m / z): 1379.6 [M+H] +

[0306] 2) Synthesis of intermediate-11

[0307] Compound A (420 mg, 0.304 mmol) was dissolved in a mixture of dichloromethane and trifluoroacetic acid (3 mL / 3 mL) and stirred at room temperature for 1 hour. The resulting reaction solution was concentrated under reduced pressure and purified by ISCO (mobile phase: water (0.1% TFA) / acetonitrile = 100:0–0:100) to give intermediate-11 as a yellow solid, 390 mg, yield 98%. MS (m / z): 1279.6 [M+H] +

[0308] The following intermediates were prepared using the same raw materials and reagents as intermediate-11, under conditions recognized by those skilled in the art.

[0309]

[0310] Preparation of Intermediate-15

[0311] 1) Synthesis of compound B

[0312] Intermediate-4 (274 mg, 1.0 mmol), compound A (249 mg, 1.5 mmol), and HATU (570 mg, 1.5 mmol) were added to DMA (5 mL). DIPEA (695 µL, 4.0 mmol) was slowly added dropwise at room temperature. After the addition was complete, the reaction was continued at room temperature for 2 hours. The reaction was then concentrated under reduced pressure. The residue was purified by reversed-phase silica gel rapid column chromatography (methanol:water (0.1% FA) = 0:100-100:0) to give compound B as a yellow oil, 284 mg, yield 61.3%. MS (m / z) = 464.3 [M+H] +

[0313] 2) Synthesis of intermediate-15

[0314] Compound B (284 mg, 0.6 mmol) and 10% Pd / C (30 mg) were added to methanol (10 mL), and the mixture was reacted at room temperature for 4 hours under hydrogen protection. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give intermediate-15 as a white solid, 211 mg, in a yield of 92.2%. MS (m / z) = 374.2 [M+H] +

[0315] Preparation of intermediate-16

[0316] 1) Synthesis of compound C

[0317] Compound A (350 mg, 0.88 mmol) and compound B (344 mg, 4.41 mmol) were added to n-butanol (10 mL) and reacted at 120 °C for 4 hours under nitrogen protection. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was purified by reversed-phase silica gel rapid column chromatography (methanol:water (0.1% TFA) = 0:100–100:0 gradient elution) to give compound C as a white solid, 175 mg, yield 67.6%. MS (m / z) = 294.4 [M+H] +

[0318] 2) Synthesis of compound D

[0319] Compound C (175 mg, 0.60 mmol), (4-aminophenyl)methanol (96 mg, 0.78 mmol), and HATU (342 mg, 0.90 mmol) were added to DMA (5 mL). DIPEA (313 µL, 1.80 mmol) was slowly added dropwise at room temperature. After the addition was complete, the reaction was allowed to continue overnight at room temperature. The mixture was concentrated under reduced pressure after the reaction was complete. The residue was purified by ISCO (methanol:water (0.1% FA) = 0:100-100:0) to give compound D as a yellow solid, 120 mg, yield 50.2%. MS (m / z) = 399.2 [M+H] +

[0320] 3) Synthesis of compound E

[0321] Compound D (120 mg, 0.30 mmol) and bis(4-nitrophenyl) carbonate (183 mg, 0.60 mmol) were added to DMA (2 mL). DIPEA (43 µL, 0.45 mmol) was then slowly added dropwise at room temperature. After the addition was complete, the reaction was allowed to continue overnight at room temperature. The mixture was concentrated under reduced pressure after the reaction was complete, and the residue was purified by ISCO (methanol:dichloromethane = 0:100-100:0) to give compound E, 157 mg, as a yellow solid, in 92.9% yield. MS (m / z) = 564.6 [M+H] +

[0322] 4) Synthesis of compound F

[0323] Compound E (157 mg, 0.28 mmol), MMAE (300 mg, 0.42 mmol), and HOBT (38 mg, 0.28 mmol) were added to a mixed solution of DMF (1.5 mL) and pyridine (0.5 mL). DIPEA (127 µL, 0.73 mmol) was slowly added dropwise at room temperature. After the addition was complete, the reaction was continued at room temperature for 24 hours. After the reaction was complete, the solution was concentrated under reduced pressure. The residue was purified by ISCO (acetonitrile:water (0.1% TFA) = 0:100-100:0) to give compound F as a white solid, 248 mg, yield 77.5%. MS (m / z) = 1142.2 [M+H] +

[0324] 5) Synthesis of compound G

[0325] Compound F (248 mg, 0.22 mmol) was added to dichloromethane (2 mL), cooled to 0 °C in an ice bath, and trifluoroacetic acid (1 mL) was slowly added dropwise. After the addition was complete, the mixture was brought to room temperature and reacted for 15 minutes. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified by ISCO (acetonitrile:water (0.1% TFA) = 0:100-100:0 gradient elution) to give compound G as a white solid, 228 mg, yield 100.8%. MS (m / z) = 1042.4 [M+H] +

[0326] 6) Synthesis of compound H

[0327] Compound G (115 mg, 0.11 mmol), intermediate-15 (54 mg, 0.14 mmol), EDCI (63 mg, 0.33 mmol), and HOBT (22 mg, 0.17 mmol) were added to DMA (2 mL). DIPEA (77 µL, 0.44 mmol) was slowly added dropwise at room temperature. After the addition was complete, the reaction was allowed to continue overnight at room temperature. The reaction was concentrated under reduced pressure after the reaction was complete. The residue was purified by ISCO (acetonitrile:water (0.1% TFA) = 0:100-100:0 gradient elution) to give compound H as a white solid, 110 mg, yield 71.5%. MS (m / z) = 1398.4 [M+H] +

[0328] 7) Synthesis of intermediate-16

[0329] Compound H (110 mg, 0.08 mmol) was added to dichloromethane (2 mL), cooled to 0 °C in an ice bath, and trifluoroacetic acid (1 mL) was slowly added dropwise. After the addition was complete, the mixture was brought to room temperature and reacted for 15 minutes. The reaction was then concentrated under reduced pressure, and the residue was purified by ISCO (acetonitrile:water (0.1% TFA) = 0:100-100:0) to give intermediate -16 as a white solid, 73.8 mg, yield 72.3%. MS (m / z) = 1297.2 [M+H] +

[0330] Preparation of intermediate-17

[0331] 1) Synthesis of compound C

[0332] Under ice-water bath and nitrogen protection, pyridine (1 mL) and HATU (1260 mg, 3.32 mmol) were added to DMF solutions of compound A (330 mg, 1.66 mmol) and compound B (790 mg, 1.99 mmol). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the solution was concentrated under reduced pressure, and the residue was purified by ISCO (mobile phase: MeOH / H2O = 0 / 100-100 / 0) to give compound C as a pale yellow solid, 360 mg, yield 37%. MS (m / z) = 579.2 [M+H] +

[0333] 2) Synthesis of compound E

[0334] Under ice-water bath and nitrogen protection, pyridine (0.5 mL) and compound D (379 mg, 1.25 mmol) were added separately to a THF solution of compound C (360 mg, 0.62 mmol). The reaction was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified by ISCO (mobile phase: DCM:MeOH = 100:0-50:50) to give compound E as a white solid, 420 mg, yield 90%. MS (m / z) = 744.2 [M+H] +

[0335] 3) Synthesis of compound F

[0336] Under ice-water bath and nitrogen protection, pyridine (0.5 mL), HOBT (84 mg, 0.62 mmol), and MMAE (460 mg, 0.62 mmol) were added to a DMF solution of compound E (420 mg, 0.57 mmol). The reaction was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified by ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give compound F as a white solid, 380 mg, yield 50%. MS (m / z) = 1322.2 [M+H] +

[0337] 4) Synthesis of compound G

[0338] Et₂N (1 mL) was added to a THF solution of compound F (380 mg) under ice-water bath conditions. The reaction was stirred at room temperature for 1 hour. After the reaction was complete, the solution was concentrated under reduced pressure, and the residue was purified by ISCO (mobile phase MeOH:H₂O = 0:100-100:0) to give compound G as a white solid, 290 mg, yield 90%. MS (m / z) = 1100.2 [M+H] +

[0339] 5) Synthesis of compound H

[0340] Under ice-water bath and nitrogen protection, pyridine (35 mg, 0.44 mmol) and HATU (111 mg, 0.29 mmol) were added to a DMF solution of compound G (160 mg, 0.15 mmol) and intermediate-7 (70 mg, 0.15 mmol). After the addition was complete, the reaction was stirred at room temperature for 3 hours. After the reaction was complete, the solution was poured into ice water, the solid was collected by filtration, the filter cake was dissolved in ethyl acetate and dried, and then concentrated again to give compound H as a white solid, 165 mg, yield 70%. MS (m / z) = 1577.6 [M+H] +

[0341] 6) Synthesis of intermediate-17

[0342] Et₂N (1 mL) was added to a THF solution of compound H (165 mg) under ice-water bath conditions. After the addition was complete, the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solution was concentrated under reduced pressure, and the residue was purified using ISCO (mobile phase: MeOH / H₂O = 0:100-100:0) to give intermediate-17 as a white solid, 128 mg, in 90% yield. MS (m / z) = 1355.6 [M+H] +

[0343] Preparation of intermediate-18

[0344] 1) Synthesis of compound B

[0345] Under ice-water bath and nitrogen protection, MsCl (522 mg, 4.25 mmol) and DIPEA (552 mg, 4.25 mmol) were added to a dry dichloromethane solution of compound A (700 mg, 2.83 mmol). After addition, the reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the concentrated reaction mixture was poured into water and extracted with dichloromethane. The extract was dried, concentrated, and dissolved in CH3SNa / THF (10 mL). The mixture was stirred at room temperature for 3 hours, and then the reaction system was heated to reflux and stirred for 1 hour. The reaction mixture was extracted with ethyl acetate. The extract was dried, concentrated, and then MeOH / H2O and OXONE (2.6 g, 4.25 mmol) were added to the residue. The resulting reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was removed by concentration, and the residue was purified by ISCO (mobile phase: CH3CN:H2O = 0:100-100:0) to give compound B, a colorless oil, 530 mg, yield 63%. MS(m / z) = 240.0 [M-56] +

[0346] 2) Synthesis of compound D

[0347] Under ice-water bath and nitrogen protection, pyridine (0.5 mL) and HATU (820 mg, 2.15 mmol) were added to DMF solutions of compounds B (530 mg, 1.79 mmol) and C (265 mg, 2.15 mmol). After the addition was complete, the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was then poured into ice water, filtered, and the solid was collected and dried to give compound D as a white solid, 500 mg, yield 70%. MS (m / z) = 401.2 [M+H] +

[0348] 3) Synthesis of compound F

[0349] Under ice-water bath and nitrogen protection, DIPEA and compound E (1.1 mg, 3.59 mmol) were added separately to a DMF solution of compound D (500 mg, 1.25 mmol). The reaction mixture was stirred at room temperature for 24 hours. After the reaction was complete, the reaction mixture was concentrated under reduced pressure, and the residue was purified using ISCO (mobile phase: EA:PE = 0:100-100:0, DCM:CH3CN = 1:1) to give compound F as a white solid, 360 mg, yield 49%. MS (m / z) = 588.2 [M+Na] +

[0350] 4) Synthesis of compound G

[0351] Under ice-water bath and nitrogen protection, pyridine (151 mg, 1.91 mmol), HOBT (87 mg, 0.64 mmol), and MMAE (456 mg, 0.64 mmol) were added to a DMF solution of compound F (360 mg, 0.64 mmol). The reaction was stirred at room temperature for 16 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give compound G as a white solid, 390 mg, yield 53%. MS (m / z) = 1144.4 [M+H] +

[0352] 5) Synthesis of compound H

[0353] Compound G (390 mg) was added to a TFA / DCM mixture (1:2, 3 mL) under ice-water bath and nitrogen protection, and stirred for 20 min. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give compound H as a white solid, 260 mg, yield 58%. MS (m / z) = 1044.6 [M+H] +

[0354] 6) Synthesis of Compound I

[0355] Under ice-water bath and nitrogen protection, pyridine (47 mg, 0.59 mmol) and HATU (168 mg, 0.44 mmol) were added to a DMF solution of compound H (260 mg, 0.29 mmol) and intermediate-7 (124 mg, 0.29 mmol). After the addition was complete, the mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give compound I as a white solid, 296 mg, yield 66%. MS (m / z) = 1521.6 [M+H] +

[0356] 7) Synthesis of intermediate-18

[0357] Et₂N (1 mL) was added to a THF solution of compound I (296 mg) under ice-water bath conditions. The reaction mixture was stirred at room temperature for 2 h. After concentration, the residue was washed with petroleum ether to give intermediate-18, a white solid, 240 mg, in 90% yield. MS (m / z) = 1299.6 [M+H] +

[0358] Preparation of intermediate-19

[0359] 1) Synthesis of compound C

[0360] Compound A (1.95 g, 8.5 mmol) and compound B (1.73 g, 9.4 mmol) were dissolved in anhydrous ethanol (30 mL) and the mixture was heated under reflux for 12 hours. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was purified by ISCO (mobile phase: PE:EA = 100:0-0:100) to give compound C as a white solid, 200 mg, yield 5.7%, MS (m / z) = 417.4 (M+1). + .

[0361] 2) Synthesis of compound D

[0362] Compound C (200 mg, 0.5 mmol) and LiOH·H₂O (100 mg, 2.5 mmol) were added to methanol / water (3:1, 100 mL) and incubated under nitrogen protection at 40 °C. o After stirring at C for 3 hours, the reaction was completed. The solvent was removed by concentration under reduced pressure. The residue was purified using ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give compound D, a white solid, 73 mg, in 37.8% yield. MS (m / z) = 403.4 [M+H] +

[0363] 3) Synthesis of compound E

[0364] Compound D (73 mg, 0.18 mmol), intermediate-10 (168 mg, 0.15 mmol), DIPEA (93 mg, 0.72 mmol), EDCI (68 mg, 0.6 mmol), and HOBT (49 mg, 0.36 mmol) were added to DMF (10 mL). The mixture was stirred at room temperature for 5 h under nitrogen protection. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was purified by ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give compound E as a white solid, 80 mg, yield 29.3%. MS (m / z) = 1508.0 [M+H] +

[0365] 4) Synthesis of compound F

[0366] Compound E (80 mg, 0.05 mmol) was added to 1 mL of THF under ice bath conditions, followed by 0.2 mL of TBFA (1 min THF). The mixture was stirred at room temperature for 15 min under nitrogen protection. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was purified using ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give compound F as an oily substance, 70 mg, yield 94.6%. MS (m / z) = 697.6 [M / 2+H] +

[0367] 5) Synthesis of intermediate-19

[0368] Compound F (70 mg, 0.05 mmol) was added to a mixed solution of trifluoroacetic acid / dichloromethane (TFA:DCM = 1:1, 10 mL), and stirred at room temperature for 0.5 h under nitrogen protection. After the reaction was complete, the solvent was removed by concentration under reduced pressure. The residue was purified by ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give compound G as an oily substance, 50 mg, yield 76.9%. MS (m / z) = 1293.8 [M+H] +

[0369] Preparation of Intermediate-20

[0370] 1) Synthesis of compound C

[0371] Under ice-water bath and nitrogen protection, Ag₂O (4.8 g, 20.83 mmol), KI (1.4 g, 8.33 mmol), and compound B (3.22 g, 16.67 mmol) were added to a dichloromethane solution of compound A (7.0 g, 13.88 mmol), and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the reaction solution was filtered, the filtrate was washed with saturated sodium bisulfate aqueous solution, the organic phase was separated, dried, and concentrated, and the residue was dissolved in THF, with Pd / C added. The resulting reaction solution was stirred at 60 °C for 3 h under hydrogen protection. After the reaction was complete, the reaction solution was filtered and concentrated. The resulting residue was then dissolved in toluene (60 mL), with DPPA (5.7 g, 20.83 mmol) and DBU (4.2 g, 27.78 mmol) added. After the addition was complete, the reaction was stirred at 105 °C for 20 h. After cooling to room temperature, the extract was poured into water and extracted with ethyl acetate. The extract was washed with saturated sodium bicarbonate solution, dried, and concentrated. The residue was purified using ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give compound C as a yellow oil, 4.1 g, yield 53%. MS (m / z) = 554.2 [M+H] +

[0372] 2) Synthesis of compound D

[0373] Pd / C was added to a THF / H₂O (60 mL) solution of compound C (4.1 g), and the mixture was stirred at room temperature for 3 hours under hydrogen protection. The reaction solution was filtered and concentrated. The residue was purified using ISCO (mobile phase: MeOH:H₂O = 0:100-100:0) to give compound D, a yellow oil, 3.6 g, in 90% yield. MS (m / z) = 528.2 [M+H] +

[0374] 3) Synthesis of intermediate-20

[0375] Under ice-water bath and nitrogen protection, DIPEA (185 mg, 1.42 mmol) was added to a THF solution of compound D (500 mg, 0.95 mmol) and compound E (350 mg, 1.13 mmol). After the addition, the reaction mixture was stirred overnight at room temperature. The reaction solution was then filtered, and the filtrate was concentrated and dissolved in a TFA / DCM (1:1, v / v) mixture (2 mL), and stirred at room temperature for 2 hours. The solvent was removed by concentrating the reaction solution, and the residue was purified by ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give intermediate -20, a colorless oil, 560 mg, yield 88%. MS (m / z) = 665.4 [M+H] +

[0376] Example 2 Preparation of Compound 1-14 (Linker-payload)

[0377] Preparation of compound-1

[0378] Intermediate-11 (50 mg, 0.039 mmol), NaBH(OAc)3 (82 mg, 0.39 mmol), and intermediate-1 (27 mg, 0.195 mmol) were dissolved in methanol (4 mL) and stirred overnight at room temperature. The reaction mixture was then concentrated under reduced pressure, and the residue was passed through ISCO (mobile phase: acetonitrile:water (0.1% TFA) = 0:100-100:0) to give compound-1 as a yellow solid, 24 mg, yield 44%, MS (m / z) = 1402.6 [M+H]. +

[0379] 1H NMR (400 MHz, CDCl3) δ 9.09 (s, 1H), 8.20 (s, 2H), 7.80-7.70 (m,1H), 7.56-7.51 (m, 2H), 7.37 – 7.26 (m, 8H), 7.23-7.20 (m, 1H), 6.68 (s, 2H),6.45-6.35 (m, 1H), 5.80-5.50 (m, 1H), 5.22-4.99 (m, 3H), 4.94-4.80 (m, 2H),4.69-4.63 (m, 2H), 4.44-4.37 (m, 1H), 4.32-4.17 (m, 4H), 4.16-3.97 (m, 5H), 3.86-3.82 (m, 1H), 3.63-3.60 (m, 3H), 3.37 (s, 3H), 3.35-3.33 (s, 1H), 3.32-3.28 (m, 5H), 3.15-3.13 (m, 1H), 3.00 (s, 3H), 2.90-2.85 (m, 6H), 2.71-2.66(m, 3H), 2.63-2.54 (m, 3H), 2.45 – 2.35 (m, 3H), 2.27 – 2.14 (m, 3H), 2.07-2.01 (m, 2H), 1.95-1.88 (m, 2H), 1.85-1.79 (m, 3H), 1.74 – 1.66 (m, 2H),1.57-1.51 (m, 2H), 1.23-1.20 (m, 3H), 1.02-1.00 (m, 3H), 0.97-0.94 (m, 8H),0.88-0.84 (m, 9H), 0.80-0.77 (m, 3H), 0.75-0.71 (m, 3H).

[0380] Preparation of compound-2

[0381] Intermediate-12 (56 mg, 0.039 mmol), compound A (16 mg, 0.051 mmol), and triethylamine (22 µl, 0.156 mmol) were dissolved in DMF (5 mL). After stirring at room temperature for 18 hours, the reaction solution was concentrated under reduced pressure and separated by ISCO (mobile phase: water (0.1% formic acid): acetonitrile = 100:0–0:100) to give compound-2 as a yellow solid, 44 mg, yield 28%, MS (m / z) = 1628.8 [M+H]. +

[0382] 1 H NMR (400 MHz, CD3OD) δ 8.18 (s, 1H), 7.99-7.88 (m, 1H), 7.86-7.72(m, 1H), 7.61-7.54 (m, 2H), 7.41-7.35 (m, 3H), 7.34 – 7.27 (m, 3H), 7.23-7.17(m, 1H), 6.79 (s, 2H), 5.36-5.30 (m, 1H), 5.23 – 5.11 (m, 2H), 5.10-5.03 (m,1H), 4.74-4.58 (m, 3H), 4.54-4.46 (m, 2H), 4.30 – 4.26 (m, 2H), 4.24 – 4.17(m, 2H), 3.73-3.59 (m, 9H), 3.50-3.46 (m, 2H), 3.35 – 3.33 (m, 5H), 3.27-3.25(m, 1H), 3.22-3.14 (m, 2H), 3.12-3.07 (m, 2H), 2.96-2.90 (m, 3H), 2.82-2.77(m, 5H), 2.75-2.67 (m, 5H), 2.64-2.58 (m, 1H), 2.52 – 2.43 (m, 2H), 2.40-2.34(m, 2H), 2.22 – 2.15 (m, 2H), 2.13 – 2.09 (m, 1H), 2.05 – 1.99 (m, 2H), 1.91 – 1.82 (m, 3H), 1.78 – 1.70 (m, 2H), 1.65-1.50 (m, 10H), 1.20-1.09 (m, 8H),1.02 – 0.93 (m, 13H), 0.93 – 0.82 (m, 18H).

[0383] The following compounds were prepared using the same preparation process as compound-2, with appropriate intermediates and reagents, under conditions deemed suitable by those skilled in the art.

[0384]

[0385] Preparation of compound-5

[0386] Intermediate-10 (31 mg, 0.12 mmol), intermediate-3 (67 mg, 0.06 mmol), HBTU (46 mg, 0.12 mmol), and DIPEA (77 mg, 0.6 mmol) were dissolved in DMF (4 mL). After stirring at room temperature for 3 hours, the reaction solution was concentrated under reduced pressure. The residue was purified by ISCO (mobile phase: acetonitrile / water (0.1% TFA) = 0:100-100:0) to give compound-5 as a yellow solid, 70 mg, yield 85%, MS (m / z) = 1368.6 [M+H]. +

[0387] 1H NMR (400 MHz, CD3OD) δ 8.53 – 8.48 (m, 1H), 8.44 – 8.38 (m, 1H), 8.35 – 8.32 (m, 1H), 8.29 – 8.26 (m,1H), 8.00 – 7.86 (m, 1H), 7.81 – 7.71(m, 1H), 7.66 – 7.60 (m, 2H), 7.44 – 7.39 (m, 3H), 7.38 – 7.32 (m, 3H),7.26 – 7.21 (m, 1H), 7.16 – 7.11 (m, 1H), 6.78 – 6.73 (m, 2H), 5.27 – 5.16(m, 2H), 5.15 – 5.06 (m, 1H), 4.83 – 4.74 (m, 2H), 4.73 – 4.67 (m, 1H), 4.67– 4.61 (m, 1H), 4.62 – 4.47 (m, 4H), 4.29 – 4.17 (m, 3H), 4.14 – 4.05 (m,1H), 4.04 – 3.85 (m, 3H), 3.80 – 3.66 (m, 1H), 3.61 – 3.53 (m, 1H), 3.48 –3.42 (m, 1H), 3.30 (s, 2H), 3.27 – 3.17 (m, 2H), 3.17 – 3.11 (m, 2H), 3.01– 2.94 (m, 3H), 2.56 – 2.46 (m, 2H), 2.34 – 2.17 (m, 3H), 2.11 – 2.01 (m,1H), 1.99 – 1.76 (m, 6H), 1.68 – 1.52 (m, 4H), 1.50 – 1.30 (m, 3H), 1.23 –1.13 (m, 7H), 1.09 – 0.99 (m, 12H), 0.98 – 0.77 (m, 18H).

[0388] Preparation of compound-8

[0389] 1) Synthesis of compound A

[0390] Intermediate-6 (150 mg, 0.3 mmol), intermediate-10 (270 mg, 0.24 mmol), DIPEA (150 mg, 1.2 mmol), EDCI (115 mg, 0.6 mmol), and HOBT (81 mg, 0.6 mmol) were added to 10 mL of DMF and stirred at room temperature for 5 h under nitrogen protection. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give compound A as 200 mg of white solid. Yield: 41.2%. MS (m / z) = 801.6 [M / 2+H] +

[0391] 2) Synthesis of compound B

[0392] Compound A (200 mg, 0.13 mmol) was added to a mixed solution of trifluoroacetic acid and dichloromethane (1:1, 10 mL), and stirred at room temperature for 0.5 h under nitrogen protection. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give compound B as an oil, 90 mg. Yield: 48.1%. MS (m / z) = 760.1 [M / 2+H] +

[0393] 3) Synthesis of compound D

[0394] Compound B (90 mg, 0.06 mmol), compound C (28 mg, 0.09 mmol), and DIPEA (31 mg, 0.24 mmol) were added to DMF (3 mL). The mixture was stirred at room temperature for 5 h under nitrogen protection. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The residue was purified using ISCO (mobile phase: water (0.1% TFA): acetonitrile = 100:0–0:100) to give compound D as a white solid, 60 mg. Yield: 59.4%. MS (m / z) = 847.8 [M / 2+H] +

[0395] 4) Synthesis of Compound-8

[0396] Compound D (60 mg, 0.03 mmol) and TEA (0.3 mL) were dissolved in DMF (2 mL), and the mixture was stirred at room temperature for 2 hours. The reaction solution was then concentrated under reduced pressure, and the residue was purified by ISCO (mobile phase: water (0.1% formic acid): acetonitrile = 100:0–0:100) to give compound-8, a yellow solid, 17 mg, yield 32.7%, MS (m / z) = 1471.8 [M+H]. +

[0397] 1 H NMR (400 MHz, CD3OD) δ 9.91 (s, 1H), 8.46 – 8.34 (m, 1H), 8.00 –7.88 (m, 1H), 7.81 – 7.70 (m, 1H), 7.54 (s, 2H), 7.43 – 7.25 (m, 7H), 7.19(s, 1H), 6.78 (s, 2H), 5.19 – 5.07 (m, 1H), 4.92 (s, 4H), 4.82 (s, 2H), 4.67– 4.45 (m, 1H), 4.32 – 4.07 (m, 2H), 3.74 – 3.54 (m, 5H), 3.52 – 3.43 (m,3H), 3.39 – 3.32 (m, 11H), 3.21 – 3.02 (m, 3H), 2.98 – 2.88 (m, 3H), 2.74 –2.42 (m, 4H), 2.40 – 2.32 (m, 1H), 2.27 – 1.99 (m, 1H), 1.95 – 1.67 (m, 3H), 1.66 – 1.52 (m, 8H), 1.37 – 1.25 (m, 4H), 1.20 – 1.07 (m, 7H), 1.05 – 0.90(m, 19H), 0.89 – 0.76 (m, 13H).

[0398] Preparation of compound-13

[0399] 1) Synthesis of compound C

[0400] Under ice-water bath and nitrogen protection, DIPEA (180 mg, 1.39 mmol) and compound B (425 mg, 1.39 mmol) were added to a DMF solution of compound A (460 mg, 0.69 mmol). After stirring at room temperature for 16 h, the reaction solution was concentrated, and the residue was purified using ISCO (mobile phase: EA:PE = 0:100-100:0, DCM:CH3CN = 1:1) to give compound C as a white solid, 560 mg, yield 90%. MS (m / z) = 845.8 [M+Na] +

[0401] 2) Synthesis of compound D

[0402] Under ice-water bath and nitrogen protection, pyridine (0.5 mL), HOBT (95 mg, 0.69 mmol), and MMAE (501 mg, 0.69 mmol) were added to a DMF solution of compound C (560 mg, 0.68 mmol). The reaction mixture was stirred at room temperature for 16 h. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified using ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give compound D, a white solid, 370 mg, yield 37%. MS (m / z) = 1402.4 [M+H] +

[0403] 3) Synthesis of compound E

[0404] Compound D (370 mg) was added to a mixed solution of CF3COOH / DCM (1:2, 3 mL) and stirred for 20 min under ice-water bath and nitrogen protection. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give compound E, a white solid, 170 mg, yield 50%. MS (m / z) = 1303.4 [M+H] +

[0405] 4) Synthesis of compound F

[0406] Under ice-water bath and nitrogen protection, DIPEA (105 mg, 0.80 mmol) and HATU (122 mg, 0.32 mmol) were added to a DMF solution of compound E (170 mg, 0.13 mmol) and intermediate-9 (130 mg, 0.26 mmol), and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give compound F, a white solid, 57 mg, yield 25%. MS (m / z) = 1771.6 [M+H] +

[0407] 5) Synthesis of compound G

[0408] Et₂NH (59 mg, 0.80 mmol) was added to a THF solution of compound F (57 mg) in an ice-water bath. After the addition was complete, the mixture was stirred at room temperature for 2 h. After the reaction was complete, the reaction solution was concentrated, the residue was washed with petroleum ether, and dried to give compound G, a white solid, 46 mg, in 90% yield. MS (m / z) = 1549.6 [M+H] +

[0409] 6) Synthesis of Compound-13

[0410] Under ice-water bath and nitrogen protection, DIPEA (5 mg, 0.035 mmol) and HATU (14 mg, 0.035 mmol) were added to a DMF solution of compound G (46 mg, 0.029 mmol) and intermediate-8 (14 mg, 0.035 mmol). After addition, the mixture was stirred at room temperature for 2 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by ISCO (mobile phase: MeOH:H2O = 0:100-100:0) to give compound-13 as a white solid, 29 mg, yield 51%. MS (m / z) = 1897.8 [M+H] +

[0411] 1 H NMR (400 MHz, DMSO) δ 10.04 (s, 1H), 8.41 – 8.28 (m, 2H), 8.06 (d, J = 8.6 Hz, 0.5H), 7.88 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 8.9 Hz, 1H), 7.73(t, J = 5.7 Hz, 1H), 7.62 (d, J = 8.2 Hz, 0.5H), 7.54 (d, J = 7.2 Hz, 2H),7.33 – 7.21 (m, 7H), 7.16 (d, J= 7.2 Hz, 1H), 6.98 (s, 2H), 5.47 – 5.31 (m,2H), 5.09-4.93 (m, 2H), 4.76 – 4.56 (m, 1H), 4.48-4.34 (m, 3H), 4.29 – 4.21(m, 2H), 4.10 – 3.93 (m, 3H), 3.83 (s, 2H), 3.53-3.45 (m, 36H), 3.41-3.37 (m,5H), 3.22-3.19 (m, 7H), 3.18-3.14 (m, 3H), 3.11-3.07 (m, 3H), 2.95 (s, 1H),2.88 – 2.79 (m, 3H), 2.65 – 2.52 (m, 3H), 2.43 – 2.29 (m, 6H), 2.28-2.18 (m,3H), 2.16 – 2.03 (m, 2H), 2.02 – 1.89 (m, 2H), 1.80 – 1.64 (m, 3H), 1.53-1.40 (m, 8H), 1.24-1.13 (m, 4H), 1.04-0.91 (m, 8H), 0.87-0.72 (m, 25H).

[0412] Preparation of compound-14

[0413] Under nitrogen protection, DIPEA (14 mg, 0.10 mmol) and HATU (40 mg, 0.10 mmol) were added to a DMF (1.5 mL) solution of intermediate-11 (48 mg, 0.037 mmol) and intermediate-20 (51 mg, 0.076 mmol). The mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated to remove the solvent, and the residue was purified by ISCO (mobile phase: MeOH:H₂O (0.1% HCOOH) = 0:100-100:0) to give compound-14 as a colorless viscous substance, 20 mg, yield 27.7%. MS (m / z) = 1925.6 [M+H] +

[0414] 1 H NMR (400 MHz, DMSO) δ 8.60 – 8.52 (m, 1H), 8.44 (d, J = 8.3 Hz, 0.5H), 8.30 (t, J = 5.0 Hz, 1H), 8.12 (t, J= 6.6 Hz, 1H), 8.06 (t, J = 5.6Hz, 1H), 7.99 (t, J = 5.2 Hz, 1H), 7.95-7.85 (m, 1H), 7.61 (d, J = 9.0 Hz,0.5H), 7.30 – 7.21 (m, 8H), 7.18-7.13 (m, 2H), 6.97 (s, 2H), 4.76 – 4.38 (m,6H), 4.33 – 3.87 (m, 3H), 3.80 – 3.51 (m, 8H), 3.31-3.26 (m, 3H), 3.22 – 2.94 (m, 12H), 2.88-2.74 (m, 4H), 2.59-2.51 (m, 1H), 2.41 – 2.18 (m, 2H), 2.08 (t, J = 7.4 Hz, 3H), 2.03-1.88 (m, 1H), 1.82 – 1.61 (m, 2H), 1.54 – 1.39 (m, 6H), 1.32 – 1.11 (m, 4H), 1.06 – 0.92 (m, 7H), 0.91-0.70(m, 20H).

[0415] Example 3: Preparation of ADC

[0416] Preparation of ADC-4

[0417] A 6 mg / mL daratumumab solution (0.84 mL, 5 mg) prepared with PBS-D buffer (10 mmol / L, pH 7.0) was added to a TCEP solution (20 μL, 10 mmol / L ddH2O, 6.0 eq), and the resulting solution was reacted at 37 °C for 120 min. A DMSO:H2O solution (3:1, v / v) of compound 3 (100 μL, 5 mmol / L, 15 eq) was added to the system, and the reaction was continued overnight at 4 °C. 20 equivalents of L-Cysteine ​​were added, and the reaction was quenched at 4 °C for 1 h. The reaction solution was then transferred to a dialysis card (Slide-A-Lyzer® Dislysis Cassette), 10 mmol / L PBS (pH 7.0) was added, and the product was obtained after further solution exchange. The drug / antibody ratio (DAR value) was calculated to be 7.59 by HICI-HPLC.

[0418] The following control compounds were prepared using the same procedure as compound ADC-4, with the corresponding Linker-Payload (see Example 2, for example), under conditions deemed suitable by those skilled in the art.

[0419] Table 1

[0420] ADC-10 preparation

[0421] To a daratumumab solution (0.75 mL, 6 mg) at a concentration of 8 mg / mL prepared with His buffer (10 mmol / L, pH 7.0), His buffer (725 μL), TCEP solution (10 μL, 10 mmol / L in His buffer, 2.5 eq), and DTPA solution (15 μL, 10 mmol / L in His buffer) were added sequentially. The mixture was reacted at 22°C for 60 min. Then, a DMSO:H₂O solution (3:1, v / v) of compound 3 (80 μL, 5 mmol / L, 10 eq) was added, and the reaction was continued at 22°C for another 60 min. The reaction solution was then transferred to an ultrafiltration centrifuge tube (Millipore Amicon® Ultra), and His / His·HCl buffer (10 mmol / L, pH 6.2) was added. After changing the buffer, the target product was obtained. The drug / antibody ratio (DAR) calculated by HICI-HPLC was 3.80.

[0422] The following antibody-drug conjugates were prepared using the same linker-payload (see Example 2 for example) as ADC-10, under conditions deemed suitable by those skilled in the art.

[0423]

[0424] Preparation of control ADC

[0425] The Linker-payload structure used by the ADC is as follows:

[0426] The following control ADC-1 is prepared according to the same preparation process as ADC-4 described above, and the following control ADC-2 is prepared according to the same preparation process as ADC-10 described above, using the corresponding Linker-Payload (MC-VC-PAB-MMAE) and under suitable conditions recognized by those skilled in the art.

[0427] Table 3

[0428] Example 4: Production of antibody HuAb1

[0429] In short, mice were immunized with the recombinant protein CD38-His (ACROBiosystems, Cat: CD8-H5224), which contains the His-tagged extracellular domain of human CD38. Positive mouse plasma cells (binding CD38 and expressing IgG) were isolated and screened using a beacon single-cell photodynamic therapy system. Furthermore, single-cell sequencing was used to obtain the sequence of the variable region encoding the anti-CD38 antibody in the positive mouse plasma cells. Suitable candidate antibodies were selected by analyzing the binding positivity of the antibody secreted by each cell to CD38 and the gene sequence of the antibody encoding it.

[0430] The chimeric antibody variable region sequence was compared with the PDB_Antibody database using BLAST, and the human backbone sequence with the highest similarity was selected for humanization: the heavy chain variable region showed high sequence homology with human germline IGHV1-2, and the light chain variable region showed high sequence homology with human germline IGKV1-33. The amino acid sequence and precise boundaries of the variable region CDR were then defined using the Kabat numbering system. Subsequently, the mouse antibody variable region CDR segment was grafted into the human backbone sequence to achieve antibody humanization. Furthermore, to maintain antibody activity, computer simulation technology was used to analyze the variable region and its surrounding framework amino acid sequences to examine their spatial stereobinding mode. By calculating electrostatic forces, van der Waals forces, hydrophilicity / hydrophobicity, and entropy, key amino acid individuals in each candidate antibody gene sequence that can interact with CD38 and maintain the spatial structure were analyzed. These were then grafted back into the selected human antibody gene framework, and the amino acid sites in the framework region that must be retained were marked, leading to the synthesis of humanized antibodies. Some key sites in the antibody framework region needed to be reverted to the antibody framework region sequence mutated into the chimeric antibody.

[0431] The amino acid sequence of the heavy chain variable region of the example candidate chimeric antibody Ab1 is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. The example humanized antibody is HuAb1, whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10. The amino acid sequences (defined by the Kabat numbering system) of the variable regions CDR of Ab1 and HuAb1 are as follows: HCDR1 as shown in SEQ ID NO: 1; HCDR2 as shown in SEQ ID NO: 2; HCDR3 as shown in SEQ ID NO: 3; LCDR1 as shown in SEQ ID NO: 4; LCDR2 as shown in SEQ ID NO: 5; and LCDR3 as shown in SEQ ID NO: 6.

[0432] Candidate antibody Ab1 and humanized antibody HuAb1 were prepared as follows: The gene encoding the variable region sequence of the candidate antibody or humanized antibody was cloned into an expression vector (PCDNA 3.4, purchased from Life Technologics, ThermoFisher) containing the gene encoding the human IgG1 heavy chain constant region (amino acid sequence as shown in SEQ ID NO: 13) and the kappa constant region (amino acid sequence as shown in SEQ ID NO: 14). Antibody expression was performed using the Expi293 cell transient transfection method. The antibody in the expression supernatant was purified by protein A affinity purification to obtain chimeric antibodies (such as Ab1) or humanized antibodies (such as HuAb1).

[0433] Table 4 Sequence List

[0434] Example 5: ELISA detection of the binding activity of CD38 antibody to CD38

[0435] Recombinant human CD38-His protein (ACRO Biosystems, Cat: CD8-H5224) or monkey CD38-His protein (ACRO Biosystems, Cat: CD8-C5223) was coated onto 96-well plates and incubated overnight at 4°C. After washing the plates three times with PBST (PBS containing 0.5% Tween-20), the plates were blocked with PBST containing 2% BSA for 2 hours. The plate was then washed three times with PBST, and serially diluted (starting concentration 33.3 nM, 4-fold dilution) of the test antibodies (daratumumab, Ab1, HuAb1, and hIgG1) were added and incubated at 37°C for 1 hour. After washing three times with PBST, HRP-labeled anti-human IgG-Fc secondary antibody (Sigma, Cat: A0170) was added and incubated at 37°C for 30 minutes. After washing three times with PBST, the plate was developed using TMD (Surmodics, Cat: TMBW-1000-01) chromogenic buffer and incubated in the dark for 15 minutes. The reaction was then terminated by adding 2N H2SO4, and the OD450 was read using a microplate reader. hIgG1, mentioned here and later in this article, was purchased from Biolegend (Cat: 403502).

[0436] The results are as follows Figure 1 and Figure 2 As shown, Ab1 and HuAb1 exhibit high binding activity to human CD38, and their EC50 values ​​are high. 50 The concentrations were 0.091 nM and 0.099 nM, respectively. In the same experiment, the EC50 of daratumumab was... 50 The value was 0.099 nM, comparable to Ab1 and HuAb1. Meanwhile, Ab1 and HuAb1 were positive for binding to monkey CD38 protein, and their EC50 values ​​were... 50 The concentrations were 0.16 nM and 0.15 nM, respectively, while daratumumab did not bind to monkey CD38.

[0437] Example 6: Biacore detection of the binding activity of anti-CD38 antibody to human CD38

[0438] Biacore determines binding kinetic parameters by measuring surface plasmon resonance (SPR). This technique detects the microscopic rate constants of antibody-antigen binding (ka) and dissociation (kd), and calculates the affinity between the antibody and antigen. The Biacore instrument (Biacore T200) and reagents were purchased from GE Healthcare.

[0439] Purified antibodies (daratumumab, Ab1, HuAb1) were diluted in 1X sample dilution buffer (Cytiva, BR-1006-69) (antibody concentration 1 μg / ml) and flowed through a CM5 chip (Cytiva 29-1275-56) pre-immobilized with protein A. Then, serially diluted (starting concentration 100 nM, 2-fold dilution) human CD38-His (ACROBiosystems, Cat: CD8-H5224) fusion protein was flowed through the same detection chip to measure antigen-antibody binding. Subsequently, 1X mobile phase buffer (Cytiva, BR-1006-69) was flowed through the chip to detect antigen-antibody dissociation. Binding and dissociation signal data of antigen and antibody at different concentrations were collected, and the affinity between antigen and antibody was calculated using a 1:1 Langmuir model.

[0440] The results are shown in Table 5. Anti-CD38 antibodies Ab1 and HuAb1 showed high affinity for human CD38, with KD values ​​of 1.068E-8 (M) and 9.535E-9 (M), respectively, which were significantly higher than 1.543E-7 (M) of daratumumab.

[0441] Table 5. Kinetic constants of anti-CD38 antibody binding to human CD38 as detected by Biacore.

[0442] Example 7: FACS detection of the binding activity of anti-CD38 antibody to CD38 on cell surface

[0443] Human Burkitt lymphoma cell line Daudi (ATCC, Cat: CCL-213) and human myeloma cell line HCI-H929 (ATCC, Cat: CRL-3580) both endogenously express human CD38 on their surface. Antibodies (Ab1, HuAb1, and hIgG1 (Biolegend, Cat: 403502)) were serially diluted (starting at 100 nM, 3-fold dilution) into PBS containing 2% fetal bovine serum (FBS, Gibco, Cat: 10100147). The diluted antibodies were then mixed with the test cells (5 cells per well). 10 5After mixing (number of cells), the mixture was incubated at 4°C for 1 hour. The cells were then washed three times with PBS containing 2% fetal bovine serum (FBS), and PE-labeled mouse anti-human IgG Fc antibody (Biolegend, Cat: 409304) was added. The mixture was then incubated at 4°C in the dark for 1 hour. After washing the cells three times with PBS containing 2% fetal bovine serum (FBS), fluorescence signals were detected using a CantoII (BD Biosciences) flow cytometer. A concentration-dependent curve was fitted using GraphPad based on the mean fluorescence intensity (MFI), and EC50 was calculated. 50 .

[0444] like Figure 3 and Figure 4 As shown, anti-CD38 antibodies Ab1 and HuAb1 bind to CD38 on Daudi cells and HCI-H929 cells. EC2 cells bound to Daudi... 50 The concentrations of EC bound to HCI-H929 cells were 0.87 nM and 0.82 nM, respectively. 50 The values ​​are 1.09 nM and 0.77 nM, respectively.

[0445] Example 8: Effect of anti-CD38 antibody on the phagocytosis of tumor cells by human macrophages

[0446] The ability of the anti-CD38 antibody of this invention to promote the phagocytosis of tumor cells by macrophages was detected using a flow cytometry-based assay. Fresh blood was collected from donors and peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Cat: 17-1440-02). Mononuclear cells were further isolated using a human total mononuclear cell isolation kit (Miltenyi Biotec, Cat: 130-096-537), and cultured with macrophage colony-stimulating factor (M-CSF, R&D Systems, Cat: 216-MC) for 7 consecutive days to induce differentiation into macrophages. On the day of the phagocytosis assay, the differentiated macrophages were starved in serum-free culture medium for 2 hours. Simultaneously, target tumor cells (Daudi) were fluorescently labeled according to the labeling procedure recommended in the CFSE (eBioscience, Cat: 65-0850-85) instructions. Labeled tumor cells and macrophages were mixed at a 4:1 ratio, and experimental antibodies (Ab1, HuAb1, and hIgG1 (Biolegend, Cat: 403502)) at the target concentration (initial concentration 10 ug / ml, 10-fold dilution) were added and incubated at 37°C for 2 hours. The cells were then washed twice with PBS, digested with trypsin (Gibco, Cat: 25200072), and incubated with APC-labeled anti-CD14 antibody (Biolegend, Cat: 325608) on ice in PBS containing 2% fetal bovine serum for 30 minutes in the dark. The cells were washed twice and analyzed by flow cytometry.

[0447] Calculate the percentage of CFSE-positive cells within the CD14-positive macrophage population. For example... Figure 5 As shown, anti-CD38 antibodies Ab1 and HuAb1 can effectively promote the phagocytosis of tumor cells by macrophages.

[0448] Example 9: Anti-CD38 antibody-mediated complement killing effect on Daudi cells

[0449] Complement-mediated cell killing was once considered an important component of antibody-mediated cell killing. C1q molecules in the complement system bind to and activate antigen-antibody complexes on the cell surface. This activation, through a series of cascade enzymatic reactions via the classical complement activation pathway, leads to the formation of a membrane attack complex on the target cell surface, resulting in cell lysis and death. However, recent studies have shown that complement activation induced during antibody-mediated complement killing of target cells is closely related to infusion reactions observed in patients during treatment. See Antibodies (Basel). 2018 Mar; 7(1): 14.Infusion reactions are a common treatment-related side effect during daratumumab treatment, significantly impacting its application and closely related to the development of drug resistance mechanisms. The in vitro complement killing assay was used to evaluate the complement-mediated killing effect of the anti-CD38 antibody of this invention on CD38-expressing tumor cells (Daudi).

[0450] Specifically, Daudi cells were washed twice with FBS-free cell culture medium and added to the test plate at a density of 1E4 cells / well. Then, 100 nM of the test antibody (daratumumab, Ab1, HuAb1, and hIgG1 (Biolegend, Cat: 403502)) was added, and the plate was incubated in a cell culture incubator for 30 minutes. Complement-containing serum (Quidel Corporation, Cat: A113) was added to bring the working concentration to 10%. The test plate was incubated in a cell culture incubator for 5 hours, and then 50 µl of Cell Titer-Glo was added to each well. The plate was mixed in a shaker for 2 minutes to induce cell lysis and incubated at room temperature for 10 minutes. Chemiluminescence was detected using Envision readings.

[0451] Using the formula: "Cytotoxicity% = 100" Cytotoxicity (%) was calculated using the formula: [Maximum Kill - Sample] / [Maximum Kill - Background]. Experimental results are shown below. Figure 6 As shown, daratumumab exhibits a strong mediating effect of complement killing CD38-expressing cells, while Ab1 and HuAb1 have very weak complement killing effects. This suggests a lower risk of infusion reactions in patients during clinical application.

[0452] Example 10: Binding of anti-CD38 antibody to NK cells

[0453] In the clinical application of the anti-CD38 antibody daratumumab, a decrease in NK cell counts is frequently observed in patients, and this decrease is considered closely related to daratumumab treatment failure. Since NK cells also express CD38 on their surface, the ADCC, CDC, and ADCP effects generated after the anti-CD38 antibody binds to it may be one of the reasons for the decrease in NK cell counts. The binding of anti-CD38 antibody to NK cells in human peripheral blood was detected using the FACS method.

[0454] Fresh blood was collected from donors, and peripheral blood mononuclear cells (PBMCs) were isolated. The experimental antibody was serially diluted (starting concentration 100 nM, 10-fold dilution) with PBS containing 2% fetal bovine serum (FBS, Gibco, Cat: 10100147). The diluted antibody was then mixed with PBMCs (8 cells per well). 10 5 After mixing (number of cells), the mixture was incubated at 4°C for 30 minutes. The cells were then washed three times with PBS containing 2% fetal bovine serum (FBS), and PE-labeled anti-human IgG Fc antibody (Biolegend, Cat:366904) and Pacific Blue-labeled anti-human CD56 antibody (Biolegend, Cat:304629) for labeling NK cells were added. The mixture was incubated at 4°C in the dark for 30 minutes. The cells were washed three times with PBS containing 2% fetal bovine serum (FBS), and fluorescence signals were detected using a CantoII (BD Biosciences) flow cytometer. Figure 7 As shown, all the tested anti-CD38 antibodies bound to human NK cells, but the binding of antibodies Ab1 and HuAb1 to NK cells was significantly weaker than that of daratumumab, and therefore their toxic effects on NK cells were also weaker.

[0455] Example 11 SEC-HPLC Detection

[0456] The ADC sample prepared in Example 3 was analyzed by filtering with a Millex® 0.22 µm Hydrophilic Durapore (PVDF) Membrane.

[0457] Instrument: Agilent 1260 Infinity II

[0458] Column: Waters XBridge® BEH200Å SEC 3.5µm, 7.8×300mm

[0459] Mobile phase: A: 10 mmol / L PBS + 15% IPA

[0460] Conditions: 0.7 ml / min, 100% A, 20 min, detection wavelength: 280 nm

[0461] The aggregation degree of each ADC can be determined by SEC-HPLC detection, and the data are summarized in Table 6.

[0462] Table 6

[0463] Conclusion: ADC products incorporating the connectors described in this invention exhibit lower aggregation rates.

[0464] Example 12: Testing the binding of the antibody or antibody-drug conjugate of the present invention to CD38 positive cells.

[0465] Experimental methods

[0466] First, Ramos cells (CRL-1596), H929 cells (CRL-9068), or RPMI-8226 cells (CCL-155) (all purchased from the American Center for Standard Biological Collections, ATCC) were cultured in RPMI-1640 medium (Gibco, A1049101) and 10% heat-inactivated fetal bovine serum (FBS) (Gibco, C20012500BT) at 5% CO2 in a cell culture incubator at 37°C. The suspended Ramos cells were collected, centrifuged, and resuspended in staining buffer (PBS (Gibco, C20012500BT) + 2% FBS) at 4°C. Cells were then cultured at 1.0 × 10⁻⁶ cells / cm². 5 Add 50 μL of cells / well to each well of a 96-well V-bottom plate (BD Falcon). Serially dilute the antibody or antibody-drug conjugate 4-fold with staining buffer to the appropriate concentrations. Then, add 50 µL / well of the diluted antibody or antibody-drug conjugate at different concentrations (final concentrations: 150, 37.5, 9.38, 2.34, 0.586, 0.146, 0.037, 0.0092 nM) or 50 µL / well of human IgG1 isotype control antibody (Biolegend, 403502) to the corresponding wells. Incubate at 4°C for 1.0 h. Centrifuge at 300 g for 5 minutes, aspirate the supernatant, resuspend in 200 µL / well of staining buffer, and wash twice. Add 100 µL / well of PE-labeled anti-human IgG Fc antibody (Biolegend, 410708) diluted in staining buffer, and incubate at 4°C for 45 minutes in the dark. Centrifuge at 300g for 5 minutes, aspirate the supernatant, and resuspend in 200 µL / well staining buffer, repeating the washing twice. Then, resuspend the cells in 200 µL / well FACS fixation buffer (PBS + 2% FBS + 1% tissue cell fixative (Beijing Dingguo, AR-0211)) and fix at room temperature for 15 minutes or overnight at 4°C.

[0467] Detection and data analysis

[0468] Fluorescence signals were detected on a BD FACSCanto™ II flow cytometer (BD Biosciences) to obtain the mean fluorescence intensity (MFI) of the antibody or antibody-drug conjugate binding to cells. EC50 was then calculated using the Log (agonist) vs. response equation in GraphPad Prism software. 50 Values. See Tables 7-1 to 7-3.

[0469] Table 7-1

[0470] Table 7-2

[0471] Table 7-3

[0472] Conclusion: As shown in Tables 7-1, 7-2 and 7-3, the ADC product of the present invention exhibits cell binding activity similar to that of daratum antibody in Ramos (CD38+) cell line, H929 (CD38+) cell line and RPMI-8226 cell line.

[0473] Example 13: Internalization experiment of the antibody or antibody-drug conjugate of the present invention on CD38-positive Ramos cells.

[0474] Experimental methods

[0475] Ramos cells (ATCC, CRL-1596) were first cultured in RPMI-1640 medium (Gibco, A1049101) and 10% heat-inactivated fetal bovine serum (FBS) (Gibco, 10100147) at 37°C with 5% CO2. The suspended Ramos cells were collected, centrifuged, and resuspended in staining buffer (PBS (Gibco, C2001200CP) + 2% FBS) at 4°C. Cells were then cultured at 1.0 x 10⁻⁶ cells / mL. 5Add 50 μL of cells / well to each well of a 96-well V-bottom plate (BD Falcon). Dilute the antibody or antibody-drug conjugate to the appropriate concentration (20 µg / mL) with staining buffer, then add 50 µL / well of the diluted antibody or antibody-drug conjugate or human IgG1 isotype control antibody (Biolegend, 403502) to the corresponding cell wells. Incubate at 4°C for 1.0 h. Centrifuge at 300 g for 5 min, aspirate the supernatant, resuspend in 200 µL / well of staining buffer, and wash twice. Add 100 µL / well of PE-labeled anti-human IgG Fc antibody (Biolegend, 410708) diluted in staining buffer, incubate in the dark at 4°C for 45 min. Centrifuge at 300 g for 5 min, aspirate the supernatant, resuspend in 200 µL / well of staining buffer, and wash twice. After centrifugation, resuspend cells in 100 µL / well of complete culture medium and incubate at 37°C (5% CO2) or 4°C for 4 hours. Centrifuge at 300g for 5 minutes, aspirate the supernatant, and resuspend in 200 µL / well of hydrochloric acid-acidified staining buffer (pH=2.8), incubate at 4°C for 5 minutes. Centrifuge at 300g for 5 minutes, aspirate the supernatant, and resuspend in 200 µL / well of staining buffer, repeating the washing process twice. Then, resuspend cells in 200 µL / well of FACS fixation buffer (PBS + 2% FBS + 1% tissue cell fixative (Beijing Dingguo, AR-0211)) and fix at room temperature for 15 minutes or at 4°C overnight.

[0476] Detection and data analysis

[0477] Fluorescence signals were detected using a BD FACSCanto™ II flow cytometer (BD Biosciences) to obtain the mean fluorescence intensity (MFI) of the antibody or antibody-drug conjugate (ADC) binding to cells. The internalization signal value of the antibody or ADC is expressed as the MFI obtained after labeling cells were incubated at 37°C and washed with acid; the control internalization signal value (no internalization signal value) is expressed as the MFI obtained after labeling cells were incubated at 4°C and washed with acid. Internalization signal values ​​for each ADC product are listed below. Figure 8 .

[0478] Conclusion: From Figure 8 The data shows that the ADC product of this invention exhibits good internalization.

[0479] Example 14: Cell proliferation inhibition experiment

[0480] Experimental methods

[0481] Ramos (CD38 positive) (CLORD CRL-1596), RPMI-8226 (CD38 positive) (CLORD CCL-155), and SU-DHL-2 (CD38 negative) (CLORD CRL-2956) cells (all purchased from the American Center for Standard Biological Collections, ATCC) were cultured in RPMI-1640 medium (Gibco, A1049101) and 10% heat-inactivated fetal bovine serum (FBS) (Gibco, 10100147) at 37°C with 5% CO2. Cells were cultured at 1.0 x 10⁻⁶ cells / mL. 4 100 μL of cells / well was added to each well of a 96-well plate and cultured in a cell culture incubator at 37°C with 5% CO2. The test antibody (e.g., antibody or ADC) was serially diluted 4-fold with PBS (Gibco, catalog number C20012500BT) to the appropriate concentration. Then, 10 µL / well of the diluted test antibody at different concentrations (final concentrations of 200, 50.0, 12.5, 3.13, 0.781, 0.195, 0.049, 0.012 nM) or 10 µL / well of control solution (PBS) was added to each 100 µL / well cell culture system. The cells were incubated in a cell culture incubator at 37°C with 5% CO2 for 72 hours.

[0482] After 72 hours of incubation, add 50 µL / well of CellTiter-Glo® reagent (Promega, catalog number G9243) to the wells, shake to mix, and incubate for 10 minutes.

[0483] Detection and data analysis

[0484] The luminescence signal was read on an EnVision™ multi-sensor microplate reader (PerkinElmer) to calculate the inhibition rate (%) of the compound, and then the half-maximal inhibitory concentration (IC50) was obtained using XLfit5 software (ID Business Solutions Limited). 50 ) value. All samples were taken in repeat wells.

[0485] Inhibition rate % = (1 - (Lum) 抗体类药物 -Lum 背景 ) / (Lum 细胞 -Lum 背景 ))×100%

[0486] in, Lum 抗体类药物 This indicates the luminescent signal in the well of the antibody drug to be tested; Lum 细胞 This indicates the luminescent signal in the cell control wells; Lum 背景 This represents the luminescence signal in the cell-free well control.

[0487] Other cell sources and catalog numbers are as follows: OPM-2 cells (German Microbial Culture Collection DSMZ, catalog number ACC 50 / G), SU-DHL-6 (ATCC, catalog number CRL-2959), SU-DHL-4 (ATCC, catalog number CRL-2957), Daudi (ATCC, catalog number CCL-213), Mino (ATCC, catalog number CRL-3000), MAVER-1 (ATCC, catalog number CRL-3008), H1975 (ATCC, catalog number CRL-5908), HCC4006 (ATCC, catalog number CRL-2871), MFE-280 (European Certified Cell Culture Collection ECACC, catalog number 98050131), and Caki-1 (CD70+) (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, SIBS).

[0488] The obtained data are shown in Tables 8-1 to 8-5 below.

[0489] Table 8-1

[0490] Table 8-2

[0491] Table 8-3

[0492] Table 8-4

[0493] Table 8-5

[0494] Conclusion: The ADC product of this invention exhibits in vitro proliferation inhibitory activity dependent on cellular CD38 expression (Table 8-1). According to the above method, the inhibitory activity of ADC-10 and control ADC-2 was measured in parallel in the OPM-2 proliferation inhibition assay; the former had an IC50 of 0.69 nM. 50 The latter has an IC size of 1.2 nM. 50ADC-10 showed significantly increased activity compared to the control ADC-2 (p = 0.00033), but its activity was weaker on CD38-negative SU-DHL-2 cells (Table 8-1), suggesting that ADC-10 has lower cytotoxicity to CD38-negative cells. In addition to its activity against multiple myeloma (MM) cells, it also exhibited strong proliferative inhibitory activity against other CD38-positive B-cell lymphomas (such as diffuse large B-cell lymphoma, Burkitt lymphoma, and mantle cell lymphoma) (Table 8-2).

[0495] The data in Tables 8-3 to 8-5 show that the linker of the present invention is still compatible with other antibodies and exhibits excellent activity in positive cells and lower cytotoxicity in negative cell lines.

[0496] Example 15 Plasma stability of the ADC of the present invention

[0497] Experimental methods: Control ADC-1 and control ADC-2 were added to mouse, rat, and human plasma at a final concentration of 100 ug / ml; ADC-1 and ADC-4 at a final concentration of 10 ug / ml; and ADC-7, ADC-9, ADC-10, ADC-12, ADC-16, and ADC-17 at a final concentration of 100 nM. After thorough mixing, the specified volume of sample was immediately removed and frozen at -60°C. The -80℃ freezer was used as the 0 h sample, and the remaining sample was incubated in a 37℃, 5% CO2 cell culture incubator. After 96 h, 50 μL of the sample was transferred, and the free toxin was detected by LC-MS / MS.

[0498] Add 200 μL of acetonitrile solution containing internal standard working solution to 50 μL of sample to precipitate, vortex mix for 3 minutes, centrifuge at 4400 rpm for 10 minutes, take 50 μL of supernatant, dilute appropriately, and perform LC / MS / MS (AB SCIEX).

[0499] Free toxin release rate % = (Free toxin concentration / Theoretical incubation concentration of antibody-conjugated toxin) × 100%

[0500] Experimental results: After incubation in mouse plasma for 96 hours (4 days), the toxin release rate of all ADCs was less than 25%, with the highest release rate observed in control ADC-2 (21.6%). No free toxin release was detected in ADC-16 after 96 hours of incubation in mouse plasma (below the limit of detection). The toxin release rate of all ADCs after 96 hours of incubation in rat plasma was significantly lower than that in mouse plasma, with no detectable release of free toxins in ADC-1 and ADC-16 after 96 hours of incubation in rat plasma. Except for control ADC-1, control ADC-2 and ADC-7 showed detectable toxin release after 96 hours of incubation in human plasma. The release rate of free toxin was 0.2%, while no free toxin was detected in the other ADCs after incubation in human plasma for 96 hours. Specific results are shown in Table 9.

[0501] Table 9

[0502] ND: Undetectable.

[0503] Example 16: In vivo antitumor activity of the antibody-drug conjugate (ADC-10) of the present invention

[0504] Objective: To evaluate the antitumor activity of the antibody-drug conjugate (ADC-10) of the present invention against the Daudi subcutaneous tumor model in BALB / c nude mice.

[0505] Methods: Human B-cell lymphoma cells Daudi (ATCC) ® CRL-213™ was cultured and proliferated in RPMI 1640 medium containing 10% fetal bovine serum. Tumor cells in the exponential growth phase were collected and suspended in RPMI 1640 medium containing matrix gel, at a concentration of 5 × 10⁶ cells per mouse. 6 Cells were implanted subcutaneously in the right flank of female BALB / c nude mice (Shanghai Lingchang Biotechnology Co., Ltd.).

[0506] After tumor cell inoculation, mice were randomly divided into 4 groups according to tumor volume. Daratumumab and antibody-drug conjugate ADC-10 were diluted with physiological saline and administered according to the regimen shown in Table 10. Daratumumab was purchased from Johnson & Johnson Pharmaceuticals, and antibody-drug conjugate ADC-10 was prepared according to the method in Example 3.

[0507] Table 10. Dosing regimens for Daratumumab and ADC-10

[0508] Note: Day 0 is the day of grouping, and Day 1 is the day after grouping when medication is administered.

[0509] Regularly measure tumor volume (tumor volume = 0.5 × major diameter × minor diameter). 2 Mouse body weight. The tumor growth inhibition rate and tumor shrinkage rate of each treatment group were statistically analyzed after drug administration.

[0510] The formula for calculating the tumor growth inhibition rate in each treatment group is: 1-[ (D t Tumor volume - D0 tumor volume) 治疗组 / (D t Tumor volume - D0 tumor volume) 溶媒组 ]×100%.

[0511] If the tumor volume after treatment is smaller than the tumor volume at the time of initial grouping, the tumor shrinkage rate is calculated. The calculation formula is: [(D0 tumor volume - D...]] t [(Tumor volume) / D0 tumor volume] × 100%.

[0512] D t The tumor volume is the tumor volume on day t, and the tumor volume D0 is the tumor volume on day 0.

[0513] Pairwise comparisons between each drug-treated group and the solvent control group, as well as among the drug-treated groups, were performed using t-tests. The p-value was calculated, with p < 0.05 considered statistically significant. Mouse body weight was measured periodically, and relative body weight was calculated using the formula: (mouse body weight on the day of measurement / mouse body weight at the time of grouping) × 100%.

[0514] result: On day 26, daratumumab (10 mg / kg, administered on days 1, 5, 8, and 12) significantly inhibited the growth of subcutaneous tumors in Daudi, with a tumor growth inhibition rate of 96.8%. ADC-10 (2.5 mg / kg, administered on days 1, 5, 8, and 12) showed similar antitumor effects to the daratumumab group, with a tumor growth inhibition rate of 95.9%. In the ADC-10 (10 mg / kg, administered on days 1 and 5) group, tumors completely disappeared in all animals by day 16, with a tumor shrinkage rate of 100%, and no tumor reappearance or growth was observed until day 26. (See attached image). Figure 9 As shown in the figure. Tumor-bearing mice tolerated all of the test products well. On day 26, the mean relative body weight of mice in the ADC-10 administration groups (2.5 mg / kg and 10 mg / kg) was in the range of 105%-111%.

[0515] Based on the above data, ADC-10 showed a strong and dose-dependent antitumor effect on the Daudi nude mouse subcutaneous xenograft model, and the efficacy of 10 mg / kg administration for one week was significantly stronger than that of daratumumab administration for two weeks at the same dose (p<0.01).

[0516] Example 17: In vivo antitumor activity of the antibody-drug conjugate (ADC-10) of the present invention.

[0517] Objective: To evaluate the antitumor activity of the antibody-drug conjugate (ADC-10) of the present invention against the KMS-11 subcutaneous tumor model in BALB / c nude mice.

[0518] Methods: Human multiple myeloma cells KMS-11 (HSRRB) ® JCRB1179 was cultured and proliferated in RPMI 1640 medium containing 10% fetal bovine serum. Tumor cells in the exponential growth phase were collected and cultured at a concentration of 1 × 10⁻⁶ cells per mouse. 7 Cells were implanted subcutaneously in the right flank of female BALB / c nude mice (Shanghai Lingchang Biotechnology Co., Ltd.).

[0519] After tumor cell inoculation, mice were randomly divided into 5 groups according to tumor volume. Daratumumab and antibody-drug conjugate ADC-10 were diluted with physiological saline and administered according to the regimens shown in Table 12. Daratumumab was purchased from Johnson & Johnson Pharmaceuticals, and antibody-drug conjugate ADC-10 was prepared according to the method in Example 3.

[0520] Table 12. Dosing regimens for Daratumumab and ADC-10

[0521] Note: Day 0 is the day of grouping, and Day 1 is the day after grouping when medication is administered.

[0522] Regularly measure tumor volume (tumor volume = 0.5 × major diameter × minor diameter). 2 Mouse body weight. The tumor growth inhibition rate and tumor shrinkage rate of each treatment group were statistically analyzed after drug administration.

[0523] The formula for calculating the tumor growth inhibition rate in each treatment group is: 1-[ (D t Tumor volume - D0 tumor volume) 治疗组 / (D t Tumor volume - D0 tumor volume) 溶媒组 ]×100%.

[0524] If the tumor volume after treatment is smaller than the tumor volume at the time of initial grouping, the tumor shrinkage rate is calculated. The calculation formula is: [(D0 tumor volume - D...]] t[(Tumor volume) / D0 tumor volume] × 100%.

[0525] D t The tumor volume is the tumor volume on day t, and the tumor volume D0 is the tumor volume on day 0.

[0526] Pairwise comparisons between each drug-treated group and the solvent control group, as well as among the drug-treated groups, were performed using t-tests. The p-value was calculated, with p < 0.05 considered statistically significant. Mouse body weight was measured periodically, and relative body weight was calculated using the formula: (mouse body weight on the day of measurement / mouse body weight at the time of grouping) × 100%.

[0527] result: On day 25, daratumumab (10 mg / kg, administered on days 1, 5, 8, and 12) significantly inhibited the growth of KMS-11 subcutaneous tumors, achieving a tumor growth inhibition rate of 89.8%. In the ADC-10 (2.5 mg / kg, 5.0 mg / kg, administered on days 1, 5, 8, and 12) and ADC-10 (10 mg / kg, administered on day 1) groups, all tumors completely disappeared on days 13, 11, and 11, respectively, with a tumor shrinkage rate of 100%. No tumor reappearance or growth was observed until day 25. (See attached image). Figure 10 As shown in the figure. Tumor-bearing mice tolerated all of the test products well. On day 25, the mean relative body weight of mice in the ADC-10 administration groups (2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg) was in the range of 105%-111%.

[0528] Based on the above data, ADC-10 showed a strong anti-tumor effect on the KMS-11 mouse subcutaneous xenograft model, and the efficacy of a single dose of 10 mg / kg was significantly stronger than that of Daratumumab administered at the same dose for two weeks (p<0.01).

[0529] This invention has been illustrated through various specific embodiments. However, those skilled in the art will understand that this invention is not limited to the specific embodiments, and various modifications or variations can be made within the scope of this invention. Furthermore, the various technical features mentioned throughout this specification can be combined with each other without departing from the spirit and scope of this invention. Such modifications and variations are all within the scope of this invention.

Claims

1. The drug conjugate shown in formula (III), Or their pharmaceutically acceptable salts, or their solvates, racemic mixtures, enantiomers, diastereomers, or tautomers. in, Ab refers to the target region, such as an antibody or its antigen-binding fragment. p is an integer selected from 1 to 16, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12; D represents the active pharmaceutical molecule; L a Selected from -C 1-12 alkylene-, -C 1-12 Alkylene -C(O)-, -C 1-12 Alkylene-CONH-(CH2CH2O) n -CH2-C(O)-, -(CH2CH2O) n -CH2-C(O)-, where n is an integer selected from 1 to 18, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; "Cycle A" is an optional substituted 3-8 membered heterocyclic alkyl or 6-10 membered heterocyclic aryl; L1 is selected from -CH2-CHR1-, -O-CH2-, and is absent; R1 is -OH, -NH2, or -C. 1-4 alkylene -OH or -C 1-4 Alkylene-NH2; L2 is a linker unit, selected from short peptide residues composed of 2-10 amino acids, wherein the amino acids are natural or non-natural amino acids; L3 is a spacing unit; and m can be 1, 2, 3 or 4.

2. The drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein, L a Selected from -C 1-6 alkylene-, -C 1-6 Alkylene -C(O)-, -C 1-6 Alkylene-CONH-(CH2CH2O) n -CH2-C(O)-, -(CH2CH2O) n -CH2-C(O)-, where n is an integer selected from 1 to 12; "Ring A" is a 5- or 6-membered nitrogen-containing saturated heterocyclic alkyl group or a 5- or 6-membered nitrogen-containing unsaturated heterocyclic alkyl group substituted with oxygen (=O). When ring A is a 5- or 6-membered nitrogen-containing unsaturated heterocyclic alkyl group substituted with oxygen, L1 is absent.

3. The drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 2, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein, Ring A is selected from and When ring A is When L1 does not exist.

4. The drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein: L1 is selected from , , and -O-CH2-; R1 is -OH, -NH2, -C 1-4 alkylene -OH or -C 1-4 Alkylene-NH2.

5. The drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 4, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein: L a Selected from: , , and ,in, # represents the position where it is attached to the maleimide group, and ## represents the position where it is attached to ring A.

6. The drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein: L2 is the linker unit, selected from short peptide residues consisting of 2-10 amino acid residues, wherein the amino acid residues may be independently selected from the L- or D-isomers of the following amino acids: alanine (including β-alanine), arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, valine, glutamine, phenylalanine, lysine, leucine, isoleucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynyl acid, aminoalkyl diacid, heterocyclic carboxylic acid, citrulline, selenocysteine ​​[1-2], pyrrolyl lysine, diaminoalkyl acid and its derivatives. Derivatives; preferably L2 is selected from: -Phe-Lys-, -Val-Lys-, -Phe-Phe-Lys-, -D-Phe-Phe-Lys-, -Gly-Phe-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Iie-Cit-, -Trp-Cit-, -Phe-Ala-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Val-Ala-, -Gly-Gly-Phe-Gly. -Gly-Cit-, -Cit-Val-, -Cit-Ala-, -Lys-Val-, -Val-Lys(Ac)-, -Phe-Lys(Ac-), -Ala-Ala-, -Ala-Ala-Ala-, -Ala-Ala-Asn-, -Ala-Ala-Asp-, -Lys-Ala-Ala-Asn-, -Lys-Ala-Ala-Ala-Asp-, -D-Val-Leu-Lys-, -Gly-Gly-Arg-, -Gly-Gly-Phe-, -Val-Lys-Gly-, -Val-Lys-Gly-Gly- and -Lys-Ala-Asn- or derivatives thereof; more preferably, L2 is -Val-Cit- or derivatives thereof.

7. The drug conjugate or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, as described in any one of claims 1 to 6, wherein L2 has the structure shown in formula (I-4): in, R3 is selected from: -NHCONH2, -NHCOCH2(OCH2CH2) n1 OCH3、-(CH2) n2 SO2CH3, -(CH2) n2 P(O)(CH3)2; where n1 is an integer selected from 1 to 18, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; n2 is 0, 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, R3 is selected from: -NHCONH2, -NHCOCH2(OCH2CH2) n1 OCH3, -SO2CH3, -P(O)(CH3)2; where n1 is an integer selected from 6-12.

8. The drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof. L3 is the structure shown in equation (II-5): ； in, R4 is selected from -(CH2). n2 SO2CH3, -(CH2) n2 P(O)(CH3)2 or hydrogen; where n2 is 0, 1, 2, 3, 4, 5, 6, 7 or 8; n3 is 1 or 2; $ is the active site linked to L2, $$ is the site linked to the active drug molecule; Preferably, L3 is the structure shown in formula (II-6). The symbols are defined as in equation (II-5).

9. The drug conjugate of claim 8 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the structure of L3 is selected from: and ; in, $ represents the binding site with L2, and $$ represents the binding site with the active drug molecule.

10. The drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 9, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof. Where D represents Dolatatin 10 and its derivatives, preferably selected from: ， ， ， ， ， ， ， ； Preferably, D is a Dolastatin 10 derivative selected from: ， ， ， ； More preferably, D is selected from: ， ； in, The wavy line represents the connection point with L3.

11. The drug conjugate or a pharmaceutically acceptable salt thereof as claimed in claims 1 to 10, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein, p is selected from 2, 3, 4, 5, 6, 7, and 8.

12. The drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 11, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein, Ab is an anti-CD38 antibody or its antigen-binding fragment, an anti-CD20 antibody or its antigen-binding fragment, an anti-CD70 antibody or its antigen-binding fragment, or a PD-L1 antibody or its antigen-binding fragment.

13. The drug conjugate or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, as described in any one of claims 1 to 12, wherein the anti-CD20 antibody is selected from rituximab, teimomab, tosimob, ofamumab, ozoglucomannab, ozoglucomannab, repertuzumab, ubluituximab, and zabetuzumab; wherein the anti-CD70 antibody is selected from the anti-CD70 antibody, vorsetuzumab; and wherein the PD-L1 antibody is selected from adebelimumab, sugemalimab, envorimab, durvalumab, acimenumab, and atezolizumab.

14. The drug conjugate of claim 12 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein the Ab is an anti-CD38 antibody or an antigen-binding fragment thereof.

15. The drug conjugate of claim 14, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the Ab is an anti-CD38 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises three heavy chain complementarity-determining regions (HCDRs) as in SEQ ID NO: 7 or 9; and the light chain variable region comprises three light chain complementarity-determining regions (LCDRs) as in SEQ ID NO: 8 or 10. or, The Ab is an anti-CD38 antibody or its antigen-binding fragment, comprising a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein... The HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO: 5, and the LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO: 6; or The HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO: 19; the LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO: 20, the LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO: 21, and the LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

22.

16. The pharmaceutical conjugate of claim 14 or 15, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the VH comprises or is composed of an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7 or 9; and / or the VL comprises or is composed of an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 8 or 10; Alternatively, the VH may contain an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 15, or may be composed of the amino acid sequence shown in SEQ ID NO: 16; and / or the VL may contain an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16, or may be composed of the amino acid sequence shown in SEQ ID NO:

16. Preferably, the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:7, and / or the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:8; Or the VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 9, and / or the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 10; or The VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 15, and / or the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:

16.

17. The drug conjugate of any one of claims 14 to 16, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, wherein the anti-CD38 antibody or its antigen-binding fragment is an IgG subtype, such as IgG1, IgG2, IgG3 or IgG4.

18. The drug conjugate of any one of claims 14 to 17, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the anti-CD38 antibody or its antigen-binding fragment further comprises a heavy chain constant region, for example, the heavy chain constant region being the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, such as the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, preferably the heavy chain constant region comprising an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity as shown in SEQ ID NO: 13 or SEQ ID NO: 23, or composed of said amino acid sequence; and / or The anti-CD38 antibody or its antigen-binding fragment further comprises a light chain constant region, such as the light chain constant region of Kappa or Lambda, for example, the light chain constant region of human Kappa or human Lambda; preferably, the light chain constant region comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity as shown in SEQ ID NO: 14, or is composed of the amino acid sequence shown in SEQ ID NO:

14. Preferably, the anti-CD38 antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:23, and the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:

14.

19. The drug conjugate of any one of claims 14 to 18, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the anti-CD38 antibody or its antigen-binding fragment comprises a heavy chain and a light chain. The heavy chain comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11, or is composed of the amino acid sequence shown in SEQ ID NO: 12; and / or the light chain comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12, or is composed of the amino acid sequence shown in SEQ ID NO:

12. Optionally, the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:11, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:

12. Optionally, the Ab is Daratumumab.

20. The drug conjugate of any one of claims 14 to 19, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the anti-CD38 antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody; and / or The anti-CD38 antibody or its antigen-binding fragment is a full-length antibody, a disulfide-bonded stable Fv fragment (dSFv), a single-domain antibody such as VHH, a Fab antibody (e.g., Fab' antibody, Fab'-SH), a (Fab')2 antibody, a single-chain antibody such as scFv, Fv, dAb (domain antibody), or a multispecific antibody (e.g., a bispecific antibody).

21. The drug conjugate shown in formula (III-2), Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein, X is N or CH, Ab, L a L1, L2, L3, D, m and p are defined in formula (III) of any one of claims 1-20.

22. The drug conjugate shown in formula (III-3), Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein, Ab、L a R1, L2, L3, D, m and p are defined in formula (III) of any one of claims 1-20.

23. The drug conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the drug conjugate is selected from: ； ； ； ； ； ； ； ； ； ； ； ； ； Wherein Ab and p are as defined in any one of claims 1 to 22.

24. The drug conjugate of claim 23, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, Where Ab is Daratumumab, and p is 2, 3, 4, 5, 6, 7, or 8; or Ab is Ab1 or huAb1; p is 2, 3, 4, 5, 6, 7 or 8.

25. The drug conjugate of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof, having an average DAR of 2-8, for example 3-5 or 6-8.

26. The compound represented by formula (I): ， Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein: L a Rings A, L1, L2, and m are as defined in any one of claims 1-23 above; L 3a For interval units.

27. The compound of claim 26 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, L 3a The structure shown in equation (I-5) is as follows: ； in, R4 is selected from -(CH2). n2 SO2CH3, -(CH2) n2 P(O)(CH3)2 or hydrogen; where n2 is 0, 1, 2, 3, 4, 5, 6, 7 or 8; n3 is 1 or 2; $ is the active site linked to L2; Preferably, L 3a The structure shown in equation (I-6) The symbols are defined as shown in equation (I-5).

28. The compound of claim 27 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, L 3a The structure is selected from: ， ； in, $ represents the connection site with L2.

29. The compound of any one of claims 26 to 28, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein, The compound is the compound represented by formula (I-2): Where X is N or CH; L a L1, L2, L 3a 、 and m are as defined in any one of claims 26 to 28.

30. The compound of any one of claims 26 to 29, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein, The compound is the compound represented by formula (I-3): Wherein R1 is defined as in claim 4, and the other variables are defined as in any one of claims 26 to 29; Preferably, R1 is -OH, -NH2, -CH2-OH, or -CH2-NH2.

31. The compound of claim 26 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the compound is selected from: ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ,or .

32. The compound shown in formula (II) Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein D L a Rings A, L1, L2, L3, and m are as defined in any one of claims 1 to 24.

33. The compound of claim 32 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein, The compound is the compound represented by formula (II-2): The variables are defined in claim 21. Preferably, the compound is the compound represented by formula (II-3). Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture thereof, enantiomer, diastereomer or tautomer thereof, wherein each variable is as defined in claim 22.

34. The compound of claim 32 or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, wherein the compound is selected from: ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； 。 35. An isolated anti-CD38 antibody or an antigen-binding fragment thereof, wherein the anti-CD38 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises three heavy chain complementarity-determining regions (HCDRs) as shown in SEQ ID NO: 7 or 9; and the light chain variable region comprises three light chain complementarity-determining regions (LCDRs) as shown in SEQ ID NO: 8 or 10; Optionally, the anti-CD38 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, wherein... The HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 3; the LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 5, and the LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

6. Optionally, the anti-CD38 antibody or its antigen-binding fragment comprises VH and VL, wherein the VH comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7 or 9, or is composed of the amino acid sequence shown in SEQ ID NO: 8 or 10, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10, or is composed of the amino acid sequence shown in SEQ ID NO: 8 or 10. Optionally, the anti-CD38 antibody or its antigen-binding fragment comprises VH and VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:7, and / or the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:8; or the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:9, and / or the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:

10. Optionally, the anti-CD38 antibody or its antigen-binding fragment is an IgG subtype, such as IgG1, IgG2, IgG3 or IgG4 subtype; Optionally, the anti-CD38 antibody or its antigen-binding fragment further comprises a heavy chain constant region, for example, the heavy chain constant region is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, such as the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4. Preferably, the heavy chain constant region comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity as shown in SEQ ID NO:13 or SEQ ID NO:23, or is composed of said amino acid sequence. Optionally, the anti-CD38 antibody or its antigen-binding fragment further comprises a light chain constant region, such as the light chain constant region of Kappa or Lambda, for example, the light chain constant region of human Kappa or human Lambda; preferably, the light chain constant region comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity as shown in SEQ ID NO: 14, or is composed of said amino acid sequence; Optionally, the anti-CD38 antibody or its antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:23, and The light chain constant region contains or is composed of the amino acid sequence shown in SEQ ID NO:14; Optionally, the anti-CD38 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, or is composed of a heavy chain variable region and a heavy chain constant region; and / or the light chain comprises a light chain variable region and a light chain constant region, or is composed of a light chain variable region and a light chain constant region. Optionally, the anti-CD38 antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody; Optionally, the anti-CD38 antibody or its antigen-binding fragment is a full-length antibody, a disulfide-stable Fv fragment (dSFv), a single-domain antibody such as VHH, a Fab antibody (e.g., Fab' antibody, Fab'-SH), a (Fab')2 antibody, a single-chain antibody such as scFv, Fv, dAb (domain antibody), or a bispecific (multispecific) antibody.

36. An isolated nucleic acid encoding the anti-CD38 antibody or its antigen-binding fragment as described in claim 35; further, the nucleic acid encoding the heavy chain and / or light chain of the anti-CD38 antibody or its antigen-binding fragment as described in claim 35; further, the nucleic acid encoding the heavy chain variable region and / or light chain variable region of the heavy chain variable region of the anti-CD38 antibody or its antigen-binding fragment as described in claim 35.

37. A recombinant vector or expression vector comprising one or more nucleic acids as described in claim 36, further wherein the vector is adapted for recombinant generation of the anti-CD38 antibody as described in claim 35 or an antigen-binding fragment thereof, for example, the vector being a pcDNA series vector, such as pCDNA3.1, pCDNA3.

4.

38. A host cell comprising one or more nucleic acids as described in claim 36 or a recombinant vector or expression vector as described in claim 37, for example, the host cell being a CHO cell or a 293 cell, such as Expi293.

39. A method for producing the anti-CD38 antibody as described in claim 35 or an antigen-binding fragment thereof, the method comprising: Culture of host cells containing a polypeptide chain encoding the antibody under conditions suitable for expression; optionally further comprising assembling the polypeptide chain to produce the molecule under conditions suitable for assembly of the polypeptide chain into the molecule; or Under conditions suitable for the expression of the antibody, host cells comprising the nucleic acid molecule of claim 36 or the recombinant vector or expression vector of claim 37, or host cells of claim 38, are cultured, and optionally the antibody is recovered from the host cells (or host cell culture medium).

40. An immunoconjugate or immunofusion comprising the anti-CD38 antibody as described in claim 35 or an antigen-binding fragment thereof; further, the immunoconjugate or immunofusion comprising other active molecules, such as Dolasatin 10 as defined in claim 10 and Dolasatin 10 derivatives.

41. Compounds of formula (IV-1), or pharmaceutically acceptable salts thereof, or their solvates, racemic mixtures, enantiomers, diastereomers, or tautomers: ； in, L a Rings A, L1, and m are as defined in any one of claims 1-5; Preferably, the compound is the compound shown in formula (IV-2): Wherein, X is N or CH, and La, L1 and m are as defined in any one of claims 1-5; Preferably, the compound is the compound shown in formula (IV-3): Among them, L a R1, L1 and m are as defined in claim 22; Preferably, the compound is selected from: ， 。 42. The compound represented by formula (V-1), or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof: in, R5 is an amino protecting group; rings A, L1, and m are as defined in any one of claims 1-5; or Compounds of formula (V-2), or pharmaceutically acceptable salts thereof, or their solvates, racemic mixtures, enantiomers, diastereomers or tautomers, Wherein, R5 is an amino protecting group; X, L1, and m are as defined in claim 41; or Compounds of formula (V-3), or pharmaceutically acceptable salts thereof, or their solvates, racemic mixtures, enantiomers, diastereomers, or tautomers, Wherein, R5 is an amino protecting group; the remaining variables are as defined in claim 41; Preferably, the compound is selected from: ； 。 43. The compound represented by formula (VI-1), or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof, ； in, X is N or CH, and L1, R3, R4 and m are as defined in claim 32; or Compounds of formula (VI-2), or pharmaceutically acceptable salts thereof, or their solvates, racemic mixtures, enantiomers, diastereomers, or tautomers: ； Wherein, R1 is as defined in claim 30 or 31, and R3, R4 and m are as defined in claim 32.

44. The compound of claim 43, selected from: ； ； ； ； ； ； Or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer or tautomer thereof.

45. A pharmaceutical composition comprising a pharmaceutical conjugate or a pharmaceutically acceptable salt thereof or a solvate thereof, a racemic mixture thereof, an enantiomer, a diastereomer, or a tautomer thereof, as described in any one of claims 1 to 25; a pharmaceutical conjugate or a pharmaceutically acceptable salt thereof or a solvate thereof, a racemic mixture thereof, an enantiomer, a diastereomer, or a tautomer thereof, as described in any one of claims 32 to 34; an anti-CD38 antibody or an antigen-binding fragment thereof, as described in any one of claims 35; a nucleic acid, as described in claim 36; a recombinant vector or expression vector, as described in claim 37; a host cell, as described in claim 38; or an immunoconjugate or immunofusion, as described in claim 40; and optionally comprising a pharmaceutically acceptable excipient, such as a pharmaceutical carrier or pharmaceutical excipient.

46. ​​A pharmaceutical combination comprising, as described in any one of claims 1 to 25, a pharmaceutically acceptable salt thereof or a solvate thereof, a racemic mixture, an enantiomer, a diastereomer, or a tautomer; a pharmaceutically acceptable salt thereof or a solvate thereof, a racemic mixture, an enantiomer, a diastereomer, or a tautomer; an anti-CD38 antibody thereof as described in any one of claims 35, an antigen-binding fragment thereof; a nucleic acid as described in claim 36; a recombinant vector or expression vector as described in claim 37; a host cell as described in claim 38; or an immunoconjugate or immunofusion as described in claim 40, and one or more other therapeutic agents.

47. Use of the drug conjugate of any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof; the drug conjugate of any one of claims 32 to 34, or a pharmaceutically acceptable salt thereof, or a solvate, racemic mixture, enantiomer, diastereomer, or tautomer thereof; the anti-CD38 antibody of any one of claims 35, or an antigen-binding fragment thereof; the nucleic acid of claim 36; the recombinant vector or expression vector of claim 37; the host cell of claim 38; the immunoconjugate or immunofusion of claim 40; the pharmaceutical composition of claim 45; or the pharmaceutical composition of claim 46 in the preparation of a medicament for treating tumors, such as cancer.

48. The use as claimed in claim 47, wherein the tumor is selected from hematopoietic lymphoid tissue tumors such as non-Hodgkin's lymphoma, myeloma, and leukemia; preferably selected from B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Burkitt lymphoma, multiple myeloma (MM), chronic myeloid leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.

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