Antibody-drug conjugates and uses thereof

Abstract

The invention provides an antibody drug conjugate and application thereof, in particular to an antibody drug conjugate targeting B7H3. Meanwhile, the invention further provides an antibody targeting B7H3 and application of the antibody.

Description

Antibody-drug conjugates and their uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to two prior applications, Patent Application No. 2023107806802, filed with the State Intellectual Property Office of China on June 29, 2023, and Patent Application No. 2024101255800, filed with the State Intellectual Property Office of China on January 30, 2024, both entitled “Antibody Drug Conjugate.” The entire contents of both prior applications are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to an antibody-drug conjugate, a preparation method thereof, and applications thereof. Background Art

[0004] Antibody-drug conjugates (ADCs) consist of three distinct components: an antibody, a linker, and a bioactive molecule (drug). ADC technology uses a linker to couple the antibody and drug molecules together, leveraging the antibody's specificity to deliver the drug to the target tissue, thereby reducing the drug's systemic side effects, increasing the drug's therapeutic window, and broadening the therapeutic potential of antibodies.

[0005] Bioactive molecules (drugs) are essential for ADCs to function. Camptothecin compounds have attracted widespread attention due to their excellent anti-tumor effects. The marketed ADCs Trodelvy and Enhertu, respectively, utilize camptothecin compounds SN38 and Dxd as cytotoxic agents. Dxd is 10 times more active than the common chemotherapy drug irinotecan. Its strong ability to penetrate cell membranes allows it to kill nearby cancer cells after ingesting the ADC, creating a "bystander effect." Its significantly shortened half-life in the bloodstream helps minimize toxic side effects.

[0006] However, the ADCs ultimately prepared using camptothecin compounds have greatly altered the properties of monoclonal antibodies, with their stability and half-life significantly reduced. Although Enhertu achieved a confirmed objective response rate (ORR) of 79.7% in the DESTINY-Breast 03 clinical study, the DS8201-A-J101 clinical study showed that for HER2-low expressing breast cancer, Enhertu's confirmed objective response rate (ORR) was 37.0%, a significantly lower therapeutic efficacy than that for high or medium expressing breast cancer. In addition, new side effects have emerged with ADC drugs, such as pneumonia and interstitial lung disease with Enhertu.

[0007] WO2020233174Al discloses a class of camptothecin compounds containing a valine-citrulline (Val-Cit)-PAB linker. However, this molecule cannot be coupled with an antibody to obtain an ADC molecule of qualified quality (the polymer content in a qualified ADC product needs to be less than 5%). Therefore, there is an urgent need in this field to provide more suitable antibody-drug conjugates based on camptothecin drugs (such as exatecan, belotecan, etc.) to achieve efficient, simple, and practical chemical preparation and coupling, and to improve the pharmaceutical properties, metabolic properties, efficacy, and safety of existing antibody-drug conjugates, such as improving the stability of ADC molecules and increasing the therapeutic window.

[0008] B7H3, short for B7 homolog 3, also known as CD276, is a newly identified B7 family ligand belonging to the type I transmembrane glycoprotein. Its extracellular domain is composed of two immunoglobulin constant (IgC) and variable (IgV) domains. It exists in two main splice forms: 2IgB7H3, in which the extracellular region consists of IgV-IgC, and 4IgB7H3, which consists of IgV-IgC-IgV-IgC. Both mouse and human B7H3 include 2IgB7-H3, but humans and other primates also possess the four immunoglobulin-like domains of 4IgB7H3.

[0009] The receptor for B7H3 has not yet been identified, and its mechanism of action is not fully understood. Current research suggests that B7H3 possesses both immunosuppressive and immunostimulatory functions. The mainstream view is that B7H3 primarily plays an immunosuppressive role. Some studies suggest that B7H3 is a negative regulator of T cell function, primarily affecting Th1 cells. However, other studies have shown that it primarily inhibits CD8+ T cells and NK cells, directly inhibiting NK activation, with no significant effect on CD4+ T cells. B7H3 exerts its inhibitory effect on T cells by inhibiting IL-2 secretion. Exogenous addition of IL-2 reverses this effect, suggesting that B7H3 is an upstream regulatory molecule of IL-2. When DCs are co-cultured with Tregs, DCs upregulate B7H3 and reduce pMHC expression, impairing their T cell-stimulating function. B7H3 expression is negatively correlated with response to PD-1 therapy in NSCLC. B7H3 expression is also negatively correlated with CD8+ T cell infiltration. Patients with high CD8+ T cell infiltration have a better response to IO therapy.

[0010] In tumor cells, B7H3 may be involved in tumor growth and immune escape mechanisms. B7H3 can regulate the migration, invasion, and adhesion of various cancer cells to fibronectin through the Jak2 / Stat3 / MMP-9 signaling pathway. Furthermore, overexpression of B7H3 in colorectal and breast cancer cells can enhance resistance to apoptosis by activating the Jak2 / STAT3 / survivin signaling pathway, thereby weakening the sensitivity of tumor cells to the chemotherapy drug paclitaxel. B7H3 expression can promote cancer cell glycolysis. Finally, B7H3 can promote VEGF expression and thus tumor angiogenesis. Clinical data from patients with multiple cancer types show that overexpression of B7H3 is associated with poor prognosis, including tumor progression and metastasis.

[0011] B7H3 mRNA is widely present in many human tissues, such as the heart, prostate, pancreas, liver, colon, and thymus. However, immunohistochemical (IHC) analysis of a large number of normal tissues reveals low or undetectable expression of B7H3 in only a few tissues. Tissue microarrays from Biomax (USA) using a B7H3-specific monoclonal antibody revealed only weak cytoplasmic staining in salivary gland acinar cells, gastric epithelial cells, and adrenal cells. Other studies have also observed only weak staining in the basal membrane of the stomach, gallbladder, prostate, cervix, and endometrium. B7H3 is widely expressed in various solid tumors, such as head and neck cancer, bladder cancer, lung cancer, kidney cancer, glioma, prostate cancer, breast cancer, and ovarian cancer, making it a promising target for ADC drugs.

[0012] Summary of the Invention

[0013] The present invention provides an antibody drug conjugate having a structure represented by formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: A-(LD)d (I)

[0014] wherein A is selected from an antibody (such as a monoclonal antibody) or an antigen-binding fragment thereof;

[0015] L is a linker moiety, one end of which is connected to A and the other end is connected to the bioactive molecule D;

[0016] D is a bioactive molecule;

[0017] d represents the molar ratio of the bioactive molecule to A (also known as DAR, i.e., drug-antibody conjugation ratio), which is an integer or decimal selected from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12);

[0018] When d is a decimal, it refers to the average number of linker-bioactive molecules (LD) conjugated per A.

[0019] In some embodiments, A is selected from a monoclonal antibody or an antigen-binding fragment thereof.

[0020] In some embodiments, A is selected from an antibody or antigen-binding fragment thereof that targets HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4), EGFR, dll3, TROP2, B7H3, B7H4, TF (Tissue factor), c-Met, CD20, CD22, CD30, CD33, CD44, CD47, CD56, CD70, CD73, CD79b, CD105, CEA, A33, Cripto, EphA2, G250, MUCl, Lewis Y, EDB-FN, VEGFR, VEDF, PD-1, PD-L1, MET, RET, GPNMB, Integrin, PSMA, Tenascin-C, SLC44A4, Claudin18.2, or Mesothelin.

[0021] In some embodiments, A is selected from antibodies or antigen-binding fragments thereof targeting B7H3, HER2, TROP2, B7H4, TF, EDB-FN, Claudin18.2.

[0022] In some embodiments, A is selected from antibodies or antigen-binding fragments thereof targeting B7H3, HER2.

[0023] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is shown in SEQ ID NO: 3; and the light chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is shown in SEQ ID NO: 6, wherein CDRs are determined according to the Kabat numbering convention.

[0024] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the anti-B7H3 antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the variable region (HV) sequence of the heavy chain is shown in SEQ ID NO:7, and the variable region (LV) sequence of the light chain is shown in SEQ ID NO:8.

[0025] In some embodiments, A is an antibody targeting B7H3, and the anti-B7H3 antibody further comprises a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

[0026] In some embodiments, A is an antigen-binding fragment targeting B7H3, and the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2.

[0027] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting B7H3, and the anti-B7H3 antibody or antigen-binding fragment thereof is a humanized or fully human antibody or antigen-binding fragment thereof.

[0028] In some embodiments, A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the anti-B7H3 antibody comprises a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:9 or SEQ ID NO:10, and the amino acid sequence of the light chain is shown in SEQ ID NO:11.

[0029] In some embodiments, A is selected from 43A or 43B targeting B7H3, DP001 targeting HER2, sacituzumab targeting TROP2, Alsevalimab targeting B7H4, Tisotumab targeting TF, L19A targeting EDB-FN, Zolbetuximab targeting Claudin18.2, or biosimilars thereof.

[0030] 43A includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO:9, and the amino acid sequence of the light chain is shown in SEQ ID NO:11.

[0031] Among them, the amino acid sequences marked in bold are the heavy chain / light chain CDR sequences, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are named SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively:

[0032] HCDR1: DYFMN

[0033] HCDR2: DVNPKTGSPSYNQKFKG

[0034] HCDR3:RYGFLYSMDY

[0035] LCDR1:RASQDISNYLN

[0036] LCDR2:YTSRLHS

[0037] LCDR3:QQGNTHPFT

[0038] The underlined amino acid sequences are the heavy chain / light chain variable region sequences, HV and LV are designated as SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0039] 43B includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain is shown in SEQ ID NO: 11.

[0040] Among them, the amino acid sequences marked in bold are the heavy chain / light chain CDR sequences, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are named SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively:

[0041] HCDR1: DYFMN

[0042] HCDR2: DVNPKTGS PSYNQKFKG

[0043] HCDR3:RYGFLYSMDY

[0044] LCDR1:RASQDISNYLN

[0045] LCDR2:YTSRLHS

[0046] LCDR3:QQGNTHPFT

[0047] The underlined amino acid sequences are the heavy chain / light chain variable region sequences, HV and LV are designated as SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0048] DP001 includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain is shown in SEQ ID NO: 13.

[0049] Sacituzumab includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain is shown in SEQ ID NO: 15.

[0050] Alsevalimab includes two identical heavy chains and light chains, the heavy chain amino acid sequence is shown in SEQ ID NO: 16, and the light chain amino acid sequence is shown in SEQ ID NO: 17.

[0051] Tisotumab includes two identical heavy chains and light chains, the heavy chain amino acid sequence is shown in SEQ ID NO: 18, and the light chain amino acid sequence is shown in SEQ ID NO: 19.

[0052] L19A includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain is shown in SEQ ID NO: 21.

[0053] Zolbetuximab includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is shown in SEQ ID NO: 23.

[0054] The amino acid variable region sequences of the present invention are all determined according to the Kabat numbering convention.

[0055] In some embodiments, -L- is selected from -L1-L2-L3-L4-L5-, wherein L1 is a covalent linking unit that is covalently linked to A, L2 is an extension unit, L3 is selected from a bond or an amino acid residue optionally substituted with a polar hydrophilic group, L4 is selected from a peptide residue consisting of 2-8 amino acids, and L5 is a bond or a self-cleavage fragment or a self-cleavage fragment substituted with a polar hydrophilic group.

[0056] In some embodiments, -L- is selected from -L1-L2-L3-L4-L5-, wherein L1 is a covalent linking unit that is covalently linked to A, L2 is an extension unit, L3 is selected from an amino acid residue optionally substituted with a polar hydrophilic group, L4 is selected from a peptide residue consisting of 2-8 amino acids, and L5 is a bond or a self-cleavable fragment.

[0057] In some embodiments, L1 is selected from the following groups:

[0058] Where * indicates connection with A.

[0059] In some embodiments, L2 is selected from -L 2a -、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2bEach independently selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -alkynylene-C1-C6 alkylene-, -phenylene-, -phenylene-C1-3 alkylene-, 1-50 (preferably 9-30, more preferably 9-26, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, wherein the alkylene group is a straight-chain or branched heteroalkylene group having 45, 46, 47, 48, 49, 50) atoms, and the alkylene group, cycloalkylene group, phenylene group and heteroalkylene group are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 1-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, and the heteroalkylene group contains 1-12 (preferably 1-8, more preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene group are selected from one or more (two or three) of N, O or S (preferably O).

[0060] In some embodiments, L2 is selected from -L 2a -、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2bEach independently selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -alkynylene-C1-C6 alkylene-, 1-50 (preferably 9-30, more preferably 9-26, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50) atoms, each of the alkylene, cycloalkylene and heteroalkylene is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 1-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene contains 1-12 (preferably 1-8, more preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, the heteroatoms of the heteroalkylene are selected from one or more (two or three) of N, O or S (preferably O).

[0061] In some embodiments, L3 is selected from amino acid residues substituted with polar hydrophilic groups, wherein the polar hydrophilic groups include sugar residues and their derivatives, polyethylene glycol residues and their derivatives, polysarcosine residues and their derivatives, or combinations thereof.

[0062] In some embodiments, L4 is selected from a peptide residue consisting of 2-8 amino acids selected from phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine.

[0063] In some embodiments, L5 is selected from a bond, wherein R3 is selected from hydrogen, m is an integer selected from 3-50, preferably an integer of 8-24, more preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. * indicates connection with D.

[0064] In some embodiments, L5 is selected from a bond, Among them, * indicates connection with D.

[0065] In some embodiments, L5 is selected from:

[0066] In some embodiments, L2 is selected from -L 2a-、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from -C1-C6 alkylene-, -C1-C3 alkylene-C3-C6 cycloalkylene-, -ethynylene-C1-C6 alkylene-, -(CH2CH2O) s -、-(CH2CH2O) s C 1-3 Alkylene-, -C 1-3 Alkylene (CH2CH2O) s C 1-3 Alkylene-, wherein s is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0067] In some embodiments, L2 is selected from -L 2a -、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from methylene, ethylene, n-propylene, isopropylene, n-pentylene, methylenecyclohexylene, ethynylenemethylene, ethynyleneethylene, ethynylene-n-propylene, ethynylene-n-butylene, ethynylene-n-pentylene, ethynylene-n-hexylene, phenylene, phenylenemethylene, -(CH2CH2O) s CH2-, -(CH2CH2O) s CH2CH2-、-CH2(OCH2CH2) s -、-CH2CH2(OCH2CH2) s -、-CH2CH2(OCH2CH2) s CH2-, -CH2CH2(OCH2CH2) s CH2CH2-、-CH2(OCH2CH2) s CH2-, wherein s is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0068] In some embodiments, L2 is selected from -L2a -、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from methylene, ethylene, n-propylene, isopropylene, n-pentylene, methylenecyclohexylene, ethynylenemethylene, ethynyleneethylene, ethynylene n-propylene, ethynylene n-butylene, ethynylene n-pentylene, ethynylene n-hexylene, -(CH2CH2O) s CH2-, -(CH2CH2O) s CH2CH2-、-CH2(OCH2CH2) s -、-CH2CH2(OCH2CH2) s -、-CH2CH2(OCH2CH2) s CH2-, -CH2CH2(OCH2CH2) s CH2CH2-、-CH2(OCH2CH2) s CH2-, wherein s is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0069] In some embodiments, L2 is selected from: -(CH2CH2O) s CH2C(O)-、-(CH2CH2O) s CH2CH2-, Where s is defined as above.

[0070] In some embodiments, L2 is selected from: -(CH2CH2O) s CH2C(O)-、-(CH2CH2O) s CH2CH2-, Where s is defined as above.

[0071] In some embodiments, L2 is selected from:

[0072] * indicates connection to L1.

[0073] In some embodiments, L3 is selected from a bond, glutamic acid, glutamic acid substituted with a polar hydrophilic group comprising a sugar residue and its derivatives or a polyethylene glycol residue and its derivatives or a polysarcosine residue and its derivatives or a combination thereof.

[0074] In some embodiments, L3 is selected from glutamic acid substituted with a polar hydrophilic group, wherein the polar hydrophilic group comprises a sugar residue and its derivatives, a polyethylene glycol residue and its derivatives, a polysarcosine residue and its derivatives, or a combination thereof.

[0075] In some embodiments, L3 is selected from: Wherein HG is a polar hydrophilic group; preferably, it is selected from a group comprising sugar residues and derivatives thereof, a group comprising polyethylene glycol residues and derivatives thereof, a group comprising polysarcosine residues and derivatives thereof, or a combination thereof, wherein * indicates connection with L2.

[0076] In some embodiments, HG is selected from: -NH(CH2CH2O)nCH3, wherein n and q are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), preferably 1-24, and more preferably 3-12.

[0077] In some embodiments, L3 is selected from:

[0078] wherein n and q are each independently selected from an integer of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), preferably 1-24, and more preferably 3-12.

[0079] In some embodiments, L4 is selected from:

[0080] * indicates connection to L3.

[0081] In some embodiments, -L1-L2- is selected from:

[0082] Among them, * indicates connection with A.

[0083] In some embodiments, -L1-L2- is selected from:

[0084] Among them, * indicates connection with A.

[0085] In some embodiments, -L3-L4- is selected from:

[0086] The definition of HG is the same as above, and * indicates connection with L2.

[0087] In some embodiments, -L3-L4- is selected from:

[0088] Wherein, * indicates connection with L2, and the definitions of n and q are the same as above.

[0089] In some embodiments, -L- is selected from:

[0090] Wherein, HG and s are as defined above, and * indicates connection with A.

[0091] In some embodiments, -L- is selected from:

[0092] In some embodiments, -L- is selected from:

[0093] In some embodiments of the present invention, -L- is selected from:

[0094] In some embodiments of the invention, -L- is selected from:

[0095] Wherein, n and s are defined as above.

[0096] In some embodiments, -L- is selected from -L1-L2-L4-L5-, wherein L1, L2, L4, and L5 are as defined above.

[0097] In some embodiments, -L- is selected from:

[0098] In some embodiments, -L- is selected from:

[0099] wherein m is selected from an integer of 3-50, preferably an integer of 8-24, and more preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0100] In some embodiments, D is selected from a topoisomerase I inhibitor, a tubulin inhibitor, a DNA damaging agent.

[0101] In some embodiments, D is selected from camptothecin derivatives.

[0102] In some embodiments, D is selected from Wherein R1 and R2 are independently selected from H, C 1-3 Alkyl (preferably methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), or R1 and R2 together with the carbon atom to which they are connected form a 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutane, cyclopentyl, cyclohexyl).

[0103] In some embodiments, D is selected from MMAE, MMAF, eribulin, pyrrolobenzodiazepine,

[0104] In some embodiments, D is selected from:

[0105] In some embodiments, the antibody drug conjugate of formula (I) has the following structure:

[0106] In some embodiments, the antibody drug conjugate of formula (I) has the following structure:

[0107] Wherein, d and s are defined as above.

[0108] In some embodiments, the antibody drug conjugate of formula (I) has the following structure:

[0109] Wherein, n, d, and s have the same definitions as above.

[0110] In some embodiments, the antibody drug conjugate of formula (I) has the following structure:

[0111] Wherein, HG, R1, R2, d, s, and m are as defined above.

[0112] In some embodiments, the antibody drug conjugate of formula (I) has the following structure:

[0113] In some embodiments, the antibody drug conjugate of formula (I) has the following structure:

[0114] In some embodiments, the antibody drug conjugate of formula (I) has the following structure:

[0115] Wherein, n, d, and s have the same definitions as above.

[0116] In some embodiments, in the antibody drug conjugate of formula (I), A is 43A, 43B or DP001.

[0117] In some embodiments, the antibody drug conjugate of formula (I) is selected from:

[0118] wherein d is selected from an integer or decimal of 1-8, preferably an integer or decimal of 2-8, more preferably an integer or decimal of 4-8, and most preferably 2, 4, 6 or 8.

[0119] The present invention also provides a linker-drug compound of the structure shown in formula (II) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: L1'-L2-L3-L4-L5-D (II)

[0120] wherein L1' is selected from L2, L3, L4, L5, and D are defined the same as in formula (I).

[0121] In some embodiments, the linker-drug compound of the structure represented by formula (II) is selected from:

[0122] In some embodiments, the linker-drug compound of the structure represented by formula (II) is selected from:

[0123] In some embodiments, the linker-drug compound of the structure represented by formula (II) is selected from:

[0124] Wherein, n and s are defined as above.

[0125] In some embodiments, the linker-drug compound of the structure represented by formula (II) is selected from:

[0126] Wherein, s is defined as above.

[0127] The partial structure of the antibody-drug conjugate consisting of a linker and a drug is referred to as a "linker-drug compound." The linker-drug compound reacts with one or more of the interchain disulfide bond sites (two sites between heavy chains and two sites between heavy and light chains) in the antibody to form a thiol group (sulfur atom of a cysteine ​​residue) after reduction, or is coupled to the antibody via transglutaminase (mTGase).

[0128] The present invention provides a pharmaceutical composition comprising the antibody-drug conjugate of the present invention and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates, or isotope-labeled forms. The pharmaceutical composition further comprises pharmaceutically acceptable excipients and carriers.

[0129] The present invention provides use of the antibody-drug conjugate of the present invention and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products in the preparation of drugs for treating proliferative diseases. The proliferative disease is preferably a disease related to abnormal expression of HER2 and B7H3, including cancer, and the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (such as small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia) bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma (such as Hodgkin lymphoma, non-Hodgkin lymphoma or relapsed anaplastic large cell lymphoma).

[0130] The present invention provides a method for treating or preventing a proliferative disease, comprising administering to a patient in need thereof a therapeutically effective dose of an antibody-drug conjugate of the present invention, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, or pharmaceutical compositions comprising the same; the proliferative disease is preferably a disease associated with abnormal expression of HER2 or B7H3, including cancer. The cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, or relapsed anaplastic large cell lymphoma).

[0131] In some embodiments, the cancer is a tumor in which tumor cells express HER2 (HER2+) and B7H3 (B7H3+).

[0132] The present invention provides use of the linker-drug compound of the present invention in preparing an antibody-drug conjugate.

[0133] The antibody-drug conjugate of the present invention can be used alone or in combination with other anti-tumor agents.

[0134] The present invention provides a pharmaceutical composition comprising the antibody-drug conjugate of the present invention and other anti-tumor agents.

[0135] The present invention provides a method for preparing the antibody-drug conjugate of the present invention, comprising the following steps:

[0136] S1: Antibody reduction: Replace the antibody medium with PBS 7.0 / EDTA to a concentration of 10 mg / mL. Add appropriate amounts of 10 mM TCEP aqueous solution and 1 M potassium phosphate dibasic aqueous solution, ensuring the solution has the correct pH. Incubate at 37°C for several hours to reduce the disulfide bonds within the antibody.

[0137] S2: Conjugation of the antibody to the linker-drug compound;

[0138] Specifically, a 10 mM solution of the compound dissolved in dimethyl sulfoxide (DMSO) was added to the above solution at room temperature, mixed, and allowed to react at room temperature for 0.5-4 hours to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide was added, followed by stirring at room temperature to terminate the linker-drug reaction.

[0139] Purification of S3 antibody-drug conjugates;

[0140] Specifically, a 1 mL Mabselect SuRe prepacked chromatography column was equilibrated with PBS7.0 / EDTA solution (10 mM PB, 137 mM NaCl, 5 mM EDTA, pH 7.0) on an AKTA system, and the antibody-drug conjugate was loaded using a sample loop. After equilibration with PBS7.0 / EDTA for another 10 to 20 mL, the column was eluted with acetate buffer at pH 3.5 and neutralized by adding 1 / 10 volume of sodium citrate neutralizing solution to obtain the purified antibody-drug conjugate.

[0141] The present invention provides a method for preparing the antibody-drug conjugate of the present invention, comprising the following steps:

[0142] The linker-drug compound and the antibody are reacted in the presence of mTgase (preferably Streptoverticillium mobaraense).

[0143] Specifically, a predetermined volume of linker-drug compound, reaction buffer, antibody, mTgase (preferably Streptoverticillium mobaraense), and HO were added to an EP tube. The ADC was catalyzed by mTgase. The tube was sealed and mixed thoroughly, then incubated at room temperature for no more than four days. When the heavy chain coupling efficiency exceeded 95%, the conjugation reaction was considered complete and immediately purified. ADC purification was performed in the same manner as for antibodies. The reaction was monitored using RP-HPLC.

[0144] Use disinfectant Ready system, load the completed reaction into the sterilized On the Protein A column, the minimum residence time is 5 minutes to ensure complete binding of the ADC product. A depth filter is used to remove any particles from the reactants and / or buffer during purification and to maintain sterility. The column is then washed with excess binding and wash buffer to remove mTGase, unreacted toxin molecules, and any unwanted buffer components before the low pH elution of the desired product. The eluted fraction is collected in a sterile collection bag pre-filled with neutralization buffer.

[0145] The present invention also provides an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is shown in SEQ ID NO: 3; and the light chain comprises three complementarity determining regions (CDRs), wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is shown in SEQ ID NO: 6, wherein the CDRs are determined according to the Kabat numbering convention.

[0146] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the variable region (HV) sequence of the heavy chain is shown in SEQ ID NO:7, and the variable region (LV) sequence of the light chain is shown in SEQ ID NO:8.

[0147] In some embodiments, the anti-B7H3 antibody further comprises a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

[0148] In some embodiments, the antigen binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2.

[0149] In some embodiments, the anti-B7H3 antibody or antigen-binding fragment thereof is a humanized or fully human antibody or antigen-binding fragment thereof.

[0150] In some embodiments, the anti-B7H3 antibody comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO:9 or SEQ ID NO:10, and the amino acid sequence of the light chain (LV) is shown in SEQ ID NO:11.

[0151] The present invention provides a pharmaceutical composition comprising the above-mentioned anti-B7H3 antibody or an antigen-binding fragment thereof.

[0152] The present invention provides a nucleotide sequence encoding the above-mentioned anti-B7H3 antibody or an antigen-binding fragment thereof.

[0153] The present invention provides an expression vector comprising the above-mentioned nucleotide sequence encoding the anti-B7H3 antibody or the antigen-binding fragment thereof.

[0154] The present invention provides a host cell comprising the above expression vector.

[0155] The present invention provides bispecific antibodies or polyspecific antibodies comprising the above-mentioned anti-B7H3 antibody or antigen-binding fragment thereof.

[0156] The present invention provides an antibody-drug conjugate coupled to the above-mentioned anti-B7H3 antibody or an antigen-binding fragment thereof.

[0157] The present invention provides an antibody-drug conjugate comprising the above-mentioned anti-B7H3 antibody or antigen-binding fragment thereof, and a cytotoxic agent.

[0158] Compared with antibody-drug conjugates known in the prior art, such as DS-8201, or experimental samples with good effects at different targets currently used as controls, the antibody-drug conjugates of the present invention exhibit better anti-cell proliferation effects, significant tumor inhibition effects, better stability, including plasma stability and buffer stability, lower toxicity, better bystander effects (i.e., after the death of target gene-positive tumor cells, they can also cause the death of adjacent tumor cells), and a wider therapeutic window. The toxin molecules currently discovered exhibit higher topoisomerase inhibitory activity when highly concentrated.

[0159] Compared with the anti-B7H3 antibodies known in the prior art, such as M30 (CN103687945B), the anti-B7H3 antibodies of the present invention exhibit higher cell binding activity and faster endocytosis rate.

[0160] The sequence of the M30 antibody is as follows:

[0161] definition

[0162] Unless otherwise specified, the term "antibody-drug conjugate" refers to an antibody (eg, monoclonal antibody) or antibody fragment linked to a biologically active cytotoxic drug via a stable chemical linker compound.

[0163] Unless otherwise specified, the term "linker-drug compound" refers to the partial structure of an "antibody-drug conjugate" consisting of a linker compound and a drug compound.

[0164] The linker-drug compound of the present invention is connected to the antibody by conventional coupling methods in the art, including: lysine coupling, inter-light and heavy chain reductive disulfide bond coupling, enzymatic coupling, and directional coupling (Beck A, Reichert JM. Antibody-drug conjugates: Present and future; MAbs, 2014, 6: 15-17; McCombs JR, Owen S C. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. The AAPS journal, 2015, 17: 339-351). The present invention preferably couples the linker-drug compound to the antibody by reducing the inter-light and heavy chain disulfide bond, i.e., one or more of the inter-light and heavy chain disulfide bond sites (two sites between the heavy chain and two sites between the heavy and light chains) are reduced to form a thiol group (sulfur atom of a cysteine ​​residue) for reaction. Also preferred is enzymatic conjugation, whereby the linker-drug compound is coupled to the antibody via transglutaminase (mTGase).

[0165] Unless otherwise specified, the term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with mammalian tissues, particularly human tissues, without excessive toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. For example, pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or separately by reacting the free base or free acid with a suitable reagent.

[0166] Unless otherwise specified, the term "isotopically labeled" means that the compounds of the present invention may exist in an isotopically traced or enriched form containing one or more atoms whose atomic mass or mass number is different from the atomic mass or mass number of the largest atom found in nature. Isotopes may be radioactive or non-radioactive. Isotopes commonly used as isotopic labels are: hydrogen isotopes, 2 H and 3 H; Carbon isotope: 13 C and 14 C; Chlorine isotope: 35 Cl and 37 Cl; Fluorine isotope: 18 F; Iodine isotope: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O,17 O and 18 O and sulfur isotopes 35 These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. 2 H and 13 C, due to their ease of labeling and convenient detection, are more widely used. Certain heavy isotopes, such as deuterium (2H), can enhance metabolic stability and prolong half-life, thereby reducing dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from pre-labeled starting materials using known synthetic techniques similar to those used for non-isotope-labeled compounds.

[0167] Unless otherwise specified, the terms "solvate" and "solvate" mean a physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain circumstances, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be capable of isolation. The solvent molecules in the solvate may exist in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses solution-phase and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0168] Unless otherwise specified, the term "stereoisomer" refers to compounds that have the same chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc. Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on the differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0169] Unless otherwise specified, the term "heteroatom" refers to nitrogen, oxygen, sulfur, and halogen atoms.

[0170] Unless otherwise specified, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 10 carbon atoms (i.e., C 1-10 Alkyl), further preferably containing 1 to 8 carbon atoms (C 1-8 Alkyl), more preferably containing 1-6 carbon atoms (ie C 1-6 Alkyl). For example, "C 1-6The term "alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5 or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl and the like.

[0171] Unless otherwise specified, the term "-alkyl-" or "alkylene" refers to a saturated straight or branched divalent hydrocarbon group. For example, C1-C8 alkylene refers to a straight or branched alkylene group having 1 to 8 carbon atoms.

[0172] Unless otherwise specified, the term "heteroalkyl" means that one or more carbon atoms in an alkyl group is replaced by a heteroatom.

[0173] Unless otherwise specified, the term "heteroalkylene" refers to a divalent "heteroalkyl" group connected to other groups at both ends.

[0174] Unless otherwise specified, the term "phenylene" refers to a divalent "phenyl" group connected to other groups at both ends.

[0175] Unless otherwise specified, the term "cycloalkyl" refers to a fully saturated carbocyclic ring that can exist as a monocycle, a bridged ring, or a spirocycle. Preferably, it contains 3-12 carbon atoms (i.e., C3-12 cycloalkyl), more preferably 3-10 carbon atoms (C3-10 cycloalkyl), further preferably 3-7 carbon atoms (C3-7 cycloalkyl), 4-6 carbon atoms (C4-6 cycloalkyl), 5-6 carbon atoms (C5-6 cycloalkyl). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, etc.

[0176] Unless otherwise specified, the term "cycloalkylene" refers to a divalent "cycloalkyl" group connected to other groups at both ends.

[0177] Unless otherwise specified, the term "amino acid residue" refers to the corresponding residue when one hydrogen atom is removed from the amine and / or one hydroxyl group is removed from the carboxyl terminus of an amino acid.

[0178] Unless otherwise specified, the term "peptide" refers to a short chain of amino acid monomers connected by peptide (amide) bonds. BRIEF DESCRIPTION OF THE DRAWINGS

[0179] Figure 1 shows the DAR value test results of DP001-042.

[0180] Figure 2 shows the DAR value test results of DP001-043.

[0181] Figure 3 shows the DAR value test results of 43A-DXd.

[0182] Figure 4 shows the DAR value test results of 43A-042.

[0183] Figure 5 shows the DAR value test results of 43B-042.

[0184] Figure 6 shows the 43A-043DAR value detection results.

[0185] Figure 7 shows the DAR value detection results of 43B-043.

[0186] FIG8 shows the hydrophobicity test results of antibody-drug conjugates.

[0187] FIG9 shows the affinity test results of 43A antibody.

[0188] FIG10 shows the affinity test results of 43B antibody.

[0189] FIG11 shows the flow cytometry affinity results of antibodies 43A and 43B.

[0190] Figures 12 and 13 show the results of the antibody-drug conjugate inhibitory activity test on SK-OV-3 and SK-BR-3 cells.

[0191] Figures 14 and 15 show the results of the antibody-drug conjugate inhibitory activity test on human melanoma A375 cells.

[0192] FIG16 shows the effect of antibody-drug conjugates on the tumor volume of human lung cancer Calu-6 cell transplants.

[0193] FIG17 is a single crystal diffraction pattern of compound 12.

[0194] FIG18 shows the effect of antibody-drug conjugates on the volume of human melanoma cell A375 transplanted tumors in mice.

[0195] FIG19 shows the effect of antibody-drug conjugates on the volume of human lung cancer cell Calu-6 transplanted tumors in mice.

[0196] FIG20 shows the effect of antibody-drug conjugates on the volume of human melanoma cell A375 transplanted tumors in mice.

[0197] FIG21 shows the effect of antibody-drug conjugates on the volume of human esophageal squamous cell carcinoma KYSE-150 mouse transplanted tumors.

[0198] FIG22 shows the effect of antibody-drug conjugates on the volume of human non-small cell lung cancer SK-MES-1 transplanted tumors in mice.

[0199] FIG23 shows the effect of antibody-drug conjugates on the volume of human liver cancer Hep3B transplanted tumors in mice.

[0200] FIG24 shows the effect of antibody-drug conjugates on the volume of human lung cancer cell Calu-6 transplanted tumors in mice. DETAILED DESCRIPTION

[0201] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to professionals in the field. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials shown herein are for demonstration purposes only.

[0202] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS) and / or liquid chromatography (HPLC). NMR measurements were performed using a Bruker Avance III 400 MHz nuclear magnetic resonance spectrometer; LC-MS measurements were performed using a SHIMADZU LC-20AD-PDA-LCMS-2020 instrument; and HPLC measurements were performed using a SHIMADZU LC-20AD-PDA high-performance liquid chromatograph.

[0203] The starting materials in the examples of the present invention are known and can be purchased commercially, or can be synthesized using or according to methods known in the art.

[0204] Abbreviation Description

[0205] Preparation Example

[0206] 1. Preparation of Linker-Drug Compounds

[0207] Example 1

[0208] 4-((30S,33S,36S)-30-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-33-isopropyl-36-methyl-27,31,34-trioxy-2,5,8,11,14,17,20,23-octaoxa-26,32,35-triazaheptamethylenetetrainoic acid-37-amino)benzyl(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indol[1,2-b]quinolin-1-yl)carbamate (A_10)

[0209] Step 1: Preparation of compound A_2:

[0210] (1) Dissolve Q_6 (10 g, 32.5 mmol) and A_1 (6.59 g, 32.5 mmol) in 120 ml of DMF, add DIEA (12.56 g, 97.4 mmol), and react at room temperature for 2 h.

[0211] (2) After the reaction was completed, the reaction solution was added to 1 mol / L HCl and extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 30 g of a pale yellow oily crude product. The crude product was mixed and subjected to column chromatography (DCM:MeOH = 50:1) to obtain 12 g of a pale yellow oily product A_2. LC-MS [M+H] + :m / z 397.1.

[0212] Step 2: Preparation of compound A_4:

[0213] (1) Dissolve A_2 (1.2 g, 3 mmol) and A_3 (1 g, 3 mmol) in 15 ml of DMF, add DIEA (1.16 g, 9 mmol) and HATU (1.14 g, 3 mmol), and react at room temperature for 2 h.

[0214] (2) LC-MS monitoring of the reaction completion, the product was obtained by reverse phase column (water: acetonitrile = 65%: 35%), and lyophilized to obtain 1 g of light yellow oil A_4. LC-MS [M+H] + :m / z 762.4.

[0215] Step 3: Preparation of compound A_5:

[0216] (1) A_4 (1 g, 3 mmol) was dissolved in 10 ml (DCM:TFA=1:1) and reacted at rt for 17 h.

[0217] (2) The reaction was completed by LCMS monitoring and concentrated to obtain 1.2 g of crude yellow oil. LC-MS [M+H] + :m / z 706.4.

[0218] Step 4: Preparation of compound A_12:

[0219] (1) Dissolve B_4 (1 g, 3.5 mmol) and A_11 (427 mg, 3.5 mmol) in 20 ml of DMF, add 2,4,6-trimethylpyridine (1.1 g, 10.4 mmol) and (2-oxime-ethyl cyanoacetate)-N,N-dimethyl-morpholinourea hexafluorophosphate (COMU) (1.49 g, 3.5 mmol), and react at room temperature for 2 h.

[0220] (2) After the reaction was completed by LC-MS monitoring, the product was obtained by reverse phase column chromatography (water: acetonitrile = 65%: 35%) and freeze-dried to obtain 1.2 g of white solid powder.

[0221] Step 5: Preparation of compound A_14:

[0222] (1) A_12 (500 mg, 1.27 mmol) and A_13 (774 mg, 2.55 mmol) were dissolved in 120 ml of DMF, DIEA (492 mg, 3.81 mmol) was added, and the mixture was reacted at room temperature for 3 h.

[0223] (2) After the reaction was completed by LC-MS monitoring, the reaction solution was added to 1 mol / L HCl and extracted twice with ethyl acetate. The organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, concentrated, and mixed. The product was then subjected to column chromatography (PE:EA=1:1) to obtain 590 mg of a white solid.

[0224] Step 6: Preparation of compound A_15:

[0225] (1) Dissolve isotecan mesylate (169869-90-3) (300 mg, 0.56 mmol), A_14 (315 mg, 0.56 mmol), and HOBt (153 mg, 1.13 mmol) in 9 ml of DMF, add DIEA (219 mg, 1.69 mmol), and react at room temperature for 2 h.

[0226] (2) After the reaction was completed, the reaction solution was purified by reverse phase column chromatography (water:acetonitrile = 40%:60%) to obtain the product, which was lyophilized to obtain 510 mg of a yellow solid. LC-MS [M+H] + :m / z 855.4.

[0227] Step 7: Preparation of compound A-16:

[0228] (1) A_15 (300 mg, 0.56 mmol) was dissolved in 6 ml (DCM:TFA=1:1) and reacted at room temperature for 2 h.

[0229] (2) After the reaction was completed by LC-MS monitoring, the reaction solution was poured into ether, and a yellow solid precipitated. Centrifugation was performed to obtain 150 mg of a crude yellow solid. LC-MS [M+H] + :m / z 755.2.

[0230] Step 8: Preparation of compound A_10:

[0231] (1) A_16 (100 mg, 0.14 mmol) and A_5 (220 mg, 0.28 mmol) were dissolved in 4 ml of DMF, and 2,4,6-trimethylpyridine (54 mg, 0.5 mmol) and HATU (85 mg, 0.27 mmol) were added and reacted at room temperature for 2 h.

[0232] (2) After the reaction was complete, the product was sent for preparation and lyophilized to obtain 22 mg of yellow solid A_10. LC-MS [M+H] + : m / z 1443.6.

[0233] Example 2

[0234] (S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexylamino)-N1-((S)-1-(((S)-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-N5-(2,5,8,11,14,17,20,23-octaoxopentacontan-25-yl)pentanediamine (B_3)

[0235] Step 1: Preparation of compound B_5: (1) Dissolve isotecan mesylate (169869-90-3) (300 mg, 0.56 mmol) and B_4 (163 mg, 0.56 mmol) in 3 ml of DMF, add 2,4,6-trimethylpyridine (205 mg, 1.69 mmol) and COMU (242 mg, 0.56 mmol), and react at room temperature for 0.5 h.

[0236] (2) After the reaction was complete, the reaction solution was added to 40 ml of water. A bluish-gray solid precipitated and was filtered. The filter cake was washed with water and lyophilized to obtain 330 mg of a crude bluish-gray solid. LC-MS [M+H]+: m / z 706.2.

[0237] Step 2: Preparation of compound B_6:

[0238] (1) Dissolve B_5 (330 mg, 0.47 mmol) in 10 ml (DCM:TFA=1:1) and react in an ice bath for 20 min.

[0239] (2) After the reaction was complete, the reaction solution was poured into ether. A bluish-gray solid precipitated and centrifuged. The solid was sent for preparation. The prepared solution was lyophilized to obtain 150 mg of a yellow solid. LC-MS [M+H] + : m / z 606.2.

[0240] Step 3: Preparation of compound B_3:

[0241] (1) Dissolve B_6 (50 mg, 0.0.08 mmol) and A_5 (115 mg, 0.16 mmol) in 3 ml of DMF, add 2,4,6-trimethylpyridine (26.5 mg, 0.25 mmol) and HATU (47 mg, 0.125 mmol), and react at room temperature for 3 h.

[0242] (2) The reaction was completed after LC-MS monitoring and the product was sent to the preparation. The preparation solution was lyophilized to obtain 25 mg of yellow solid powder.

[0243] Example 3: Preparation of Compound 045

[0244] 1. Preparation of intermediate compound 8

[0245] Compound 1 (47.5 g, 189 mmol) and methanol (250 mL) were added to a reaction flask. 80% hydrazine hydrate (35.4 g, 567 mmol) was slowly added at room temperature. The temperature was raised to 70°C and the reaction was refluxed for 6 hours. After cooling, white crystals precipitated. The remaining solid was filtered and washed with methanol (20 mL × 3) to obtain a white solid 2 (47.47 g, 100% yield). MS (ESI): (M+H) + Calculated value 252.1, experimental value 252.2.

[0246] Compound 2 (47.4 g, 189 mmol), potassium hydroxide (12.7 g, 227 mmol) and ethanol (400 mL) were added to a reaction flask and stirred at room temperature to dissolve. After slowly adding carbon disulfide (17 g, 283 mmol), the temperature was raised to 100°C and the reaction was refluxed for 5 hours to complete. After removing the solvent under reduced pressure, water (50 mL) was added and the pH was adjusted to 6 with dilute hydrochloric acid. The mixture was extracted with dichloromethane (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 3 (49.9 g, yield 90%). MS (ESI): (M+H) + Calculated value 294.1, experimental value 294.3.

[0247] Compound 3 (5.86 g, 20 mmol), triethylamine (2.42 g, 24 mmol), and tetrahydrofuran (36 mL) were added to a reaction flask. Methyl iodide (3.12 g, 22 mmol) was then added to the reaction solution. The reaction was stirred at 25°C for 1.5 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain compound 4 (4.05 g, 66% yield). MS (ESI): (M+H) + Calculated value 308.1, experimental value 308.2.

[0248] Compound 4 (3.3 g, 10.6 mmol) and ethyl acetate (20 mL) were added to a reaction flask, and a solution of hydrogen chloride in ethyl acetate (2.7 mL, 4 M) was added dropwise at 25°C. The reaction was continued for 6 hours. The solvent was removed under reduced pressure to obtain a crude product 5, which was directly used for the next reaction.

[0249] The crude product 5, diglycolic anhydride 6 (1.35 g, 11.7 mmol), triethylamine (2.14 g, 21.2 mmol), and tetrahydrofuran (30 mL) were added to a reaction flask and reacted at 25°C for 1.5 hours. The solvent was removed under reduced pressure, and ether was added to the residue. After filtration, the residue was washed with water and ether to obtain compound 7 (3.4 g, yield 99%). MS (ESI): (M+H) + Calculated value 324.1, experimental value 324.2.

[0250] Compound 7 (3.4 g, 10.5 mmol) and glacial acetic acid (20 mL) were added to a reaction flask. After dissolution, potassium permanganate (2.48 g, 15.7 mmol) was added at 0°C and the temperature was returned to 25°C for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless. Extraction was performed with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain compound 8 (2.4 g, yield 65%). MS (ESI): (M+H)+ Calculated value 356.0, experimental value 356.0.

[0251] 2. Preparation of Compound 045

[0252] At 0°C, 1-(4-aminophenyl)-3-butyn-1-ol (4997 mg, 31 mmol), N-fluorenylmethoxycarbonyl-L-alanine (9651 mg, 31 mmol), EEDQ (11498 mg, 46.5 mmol), and ultra-dry DCM (90 mL) were added to a reaction flask. After 3 hours of reaction, the solvent was removed under reduced pressure, and the residue was slurried in 500 mL of MTBE to obtain compound 10 (9007 mg, 64% yield, dr = 1:1). MS (ESI): (M+H) + Calculated value 455.2, experimental value 455.2.

[0253] Chiral resolution of intermediate 10: Compound 10 (50 g) was subjected to SFC to afford 11 (19 g, retention time 13.55 min) and 12 (20 g, retention time 16.29 min). SFC separation method: Column: DAICEL CHIRALCEL OD (250 mm-50 mm, 10 μm); Mobile phase: A: CO2, B: CO2-ACN / i-PrOH (0.1% NH3H2O); Isocratic elution: B in A for 50%; Flow rate: 200 mL / min; Detector: PDA; Column temperature: 25°C; Back pressure: 100 bar.

[0254] HPLC method: Instrument information: Thermo liquid chromatograph (ADC-U3000-01); Chromatographic column: (4.6*150mm, 5μm); column temperature: 25℃; injection plate temperature: 25℃; mobile phase: A: 10mM NH4FA; B: ACN; flow rate: 0.8mL / min; detection wavelength: 254nm; injection volume: 2μl;

[0255] Gradient conditions:

[0256] The structure and configuration of compound 12 were confirmed by X-RAY and are shown in FIG17 .

[0257] At 0°C, 12 (3632 mg, 8 mmol) and 90 mL of commercially available THF were added to a reaction flask. DBU (1215 mg, 8 mmol) was slowly added with stirring, and the reaction was allowed to proceed for half an hour before being allowed to cool to room temperature. After TLC monitoring of the disappearance of the starting material, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to afford compound 13 (1707 mg, 92% yield). MS (ESI): (M+H) + Calculated value 233.1, experimental value 233.2.

[0258] Compound 13 (1624 mg, 7 mmol), Fmoc-Val-OSu (362 mg, 8.4 mmol), and N,N-dimethylformamide (150 mL) were added to a reaction flask, stirred, and DIEA (1158 uL, 7 mmol) was slowly added dropwise. The reaction was carried out at 25°C for 12 hours. The solvent was removed under reduced pressure, and 50 mL of EA and 50 mL of PE were added for slurrying to precipitate a white solid. This reaction was repeated three times to obtain compound 14. MS (ESI): (M+H) + Calculated value 554.3, experimental value 554.4.

[0259] Compound 14 (1661 mg, 3 mmol), p-nitrophenyl chloroformate (1206 mg, 6 mmol), and THF (150 mL) were added to a reaction flask, stirred, and Py (474 ​​uL, 6 mmol) was added dropwise. The reaction was continued at 65°C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 98:2) to obtain compound 15 (1831 mg, 85% yield). MS (ESI): (M+H) + Calculated value 719.8, experimental value 719.9.

[0260] Compound 15 (1436 mg, 2 mmol), isotecan mesylate 16 (1168.2 mg, 2.2 mmol), and N,N-dimethylformamide (100 mL) were added to a reaction flask, stirred, and DIEA (695 uL, 4 mmol) was slowly added dropwise. The reaction was continued at 25°C for 20 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to obtain compound 17 (1664 mg, 82% yield). MS (ESI): (M+H) + Calculated value 1015.4, experimental value 1015.1.

[0261] At 0°C, 17 (1522 mg, 1.5 mmol) and 40 mL of commercially available THF were added to a reaction flask. DBU (228 mg, 1.5 mmol) was slowly added with stirring. After half an hour of reaction, the mixture was brought to room temperature and the reaction continued for 40 minutes until the starting material disappeared on TLC. The reaction solution was concentrated in vacuo to remove THF, and 15 mL of DCM and 300 mL of PE were added to produce a large amount of yellow-green solid. The solid 18 (1177 mg, 99% yield) was filtered out using a Buchner funnel. MS (ESI): (M+H) + Calculated value 793.3, experimental value 793.6.

[0262] Compound 18 (1031 mg, 1.3 mmol), 8 (554 mg, 1.56 mmol), EDCI (498 mg, 2.6 mmol), HOBT (263 mg, 1.95 mmol), and N,N-dimethylformamide (20 mL) were added to a reaction flask and reacted at 25°C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by C18 purification (60% ACN / 0.05% formic acid in H2O) and lyophilized to obtain compound 19 (1322 mg, 90% yield). MS (ESI): (M+H) + Calculated value 1130.4, experimental value 1130.7.

[0263] Compound 19 (903 mg, 0.8 mmol), compound 20 (702 mg, 1.2 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (34.7 mg, 0.08 mmol) and cuprous bromide (11.4 mg, 0.08 mmol) were added to a reaction flask, the atmosphere was purged with nitrogen three times, THF / DMF / H2O (3.5 mL:0.6 mL:0.4 mL) was added, and the mixture was reacted at 25°C for 0.5 h. The residue was purified by C18 preparative purification (62% ACN / 0.05% formic acid in H2O) to give compound 045 (1276 mg, 93% yield). MS (ESI): (M+H) + Calculated value 1715.7, experimental value 1716.2. 1H NMR (600MHz, DMSO) δ10.71 (s, 1H), 10.08 (s, 1H), 8.48 (d, J = 6.7Hz, 1H), 8.17 ( d,J=8.6Hz,3H),8.09(d,J=8.8Hz,1H),8.03(d,J=8.7Hz,2H),7.89(s,1H),7. 81(d,J=10.8Hz,1H),7.66(d,J=8.4Hz,2H),7.42–7.36(m,3H),6.61(s,1H),6 .02(t,J=6.8Hz,1H),5.55(s,2H),5.38-5.26(m,3H),4.55–4.47(m,3H),4.46– 4.41(m,1H),4.36(q,J=15.6Hz,2H),4.28(s,2H),3.80(s,5H),3.62–3.55(m, 36H),3.52–3.47(m,9H),3.44–3.36(m,2H),3.30-2.29(m,1H),3.19–3.09(m, 1H),2.42(s,3H),2.28–2.19(m,1H),2.17(s,3H),2.17–2.10(m,1H),2.04–1. 89 (m, 2H), 1.42 (d, J = 7.0Hz, 3H), 1.02 (d, J = 6.7Hz, 3H), 0.98 (t, J = 7.2Hz, 6H).

[0264] 2. Preparation of Antibody-Drug Conjugates (ADCs)

[0265] Common Procedure A: Purification of Antibody-Drug Conjugates

[0266] On an AKTA system, a 1 mL Mabselect SuRe prepacked chromatography column was equilibrated with PBS7.0 / EDTA solution (10 mM PB, 137 mM NaCl, 5 mM EDTA, pH 7.0). The antibody-drug conjugate was loaded using a sample loop, and then equilibrated with PBS7.0 / EDTA for another 10 to 20 mL. The column was then eluted with acetate buffer at pH 3.5 and neutralized with 1 / 10 volume of sodium citrate neutralizing solution to obtain the purified antibody-drug conjugate.

[0267] Common Procedure B: Determination of the Drug-Antibody Ratio (DAR) of Antibody-Drug Conjugates

[0268] Dithiothreitol was added to the antibody-drug conjugate to a final concentration of 20 mM, and the sample, which had broken the interchain disulfide bonds of the antibody-drug conjugate, was incubated at 37°C in a water bath for 30 minutes. The sample was then used for HPLC analysis. The HPLC system used was an Agilent Technologies 1260 Infinity HPLC, and the column was a PLRP-S (5 μm particle size; 2.1 mm × 50 mm; Agilent Technologies). The column temperature was 80°C, and the mobile phase A was 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was 0.1% trifluoroacetic acid (TFA) in acetonitrile. The sample volume was 10 μL, and the gradient program was 27%-27% (0-3 minutes), 27%-35% (3-8 minutes), 35%-43% (8-25 minutes), 43%-95% (25-26 minutes), 95%-95% (26-31 minutes), 95%-27% (31-31.5 minutes), and 27%-27% (31.5-40 minutes). Compared to unlinked light chains (L0) and heavy chains (H0), the hydrophobicity of drug-linked light chains (light chains linked to one drug, L1) and heavy chains (heavy chains linked to one drug, H1; heavy chains linked to two drugs, H2; and heavy chains linked to three drugs, H3) increases with the number of linked drugs. Therefore, the elution order was L0, L1, H0, H1, H2, and H3. DAR values ​​were calculated based on peak areas at 280 nm.

[0269] Example 4 Preparation of Antibody-Drug Conjugate DP001-042

[0270] Project 1:

[0271] Antibody reduction:

[0272] The DP001 antibody prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 100 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (8.16 μL; 12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0273] Conjugation of Antibodies to Linker-Drug Compounds:

[0274] To the above solution, a 10 mM DMSO solution of A_10 (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0275] Purification of Antibody-Drug Conjugates:

[0276] The above solution was subjected to purification according to the common procedure A to obtain a solution containing compound DP001-042.

[0277] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0278] The DAR value of DP001-042 was 8.0 when the common procedure B was used for the determination. The results are shown in FIG1 . Example 5 Preparation of Antibody-Drug Conjugate DP001-043

[0279] Project 1:

[0280] Antibody reduction:

[0281] The DP001 antibody prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0282] Conjugation of Antibodies to Linker-Drug Compounds:

[0283] To the above solution, a 10 mM DMSO solution of B_3 (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0284] Purification of Antibody-Drug Conjugates:

[0285] The above solution was subjected to purification according to the common procedure A to obtain a solution containing compound DP001-043.

[0286] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0287] The DAR value of DP001-043 was 8.0 when the common procedure B was used for the determination. The results are shown in FIG2 . Example 6 Preparation of Antibody-Drug Conjugate 43A-DXd

[0288] Project 1:

[0289] Antibody reduction:

[0290] The 43A antibody prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0291] Conjugation of Antibodies to Linker-Drug Compounds:

[0292] To the above solution, a 10 mM DMSO solution of deruxtecan (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and allowed to react at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and allowed to react at room temperature for a further 20 minutes to terminate the coupling reaction.

[0293] Purification of Antibody-Drug Conjugates:

[0294] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound 43A-DXd.

[0295] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0296] DAR measurement was performed using common procedure B. The results are shown in FIG3 , and the DAR value of 43A-DXd was 7.4.

[0297] Example 7 Preparation of Antibody-Drug Conjugate 43A-042

[0298] Project 1:

[0299] Antibody reduction:

[0300] The 43A antibody prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0301] Conjugation of Antibodies to Linker-Drug Compounds:

[0302] To the above solution, a 10 mM DMSO solution of A_10 (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0303] Purification of Antibody-Drug Conjugates:

[0304] The above solution was subjected to purification according to the common procedure A to obtain a solution containing compound 43A-042.

[0305] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0306] DAR was measured using common procedure B. The results are shown in FIG4 , and the DAR value of 43A-042 was 7.6.

[0307] Example 8 Preparation of Antibody-Drug Conjugate 43B-042

[0308] Project 1:

[0309] Antibody reduction:

[0310] The laboratory-prepared 43B antibody medium was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0311] Conjugation of Antibodies to Linker-Drug Compounds:

[0312] To the above solution, a 10 mM DMSO solution of A_10 (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0313] Purification of Antibody-Drug Conjugates:

[0314] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound 43B-042.

[0315] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0316] The DAR measurement was performed using the common procedure B. The results are shown in FIG5 , and the DAR value of 43B-042 was 5.9.

[0317] Example 9 Preparation of Antibody-Drug Conjugate 43A-043

[0318] Project 1:

[0319] Antibody reduction:

[0320] The 43A antibody prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0321] Conjugation of Antibodies to Linker-Drug Compounds:

[0322] To the above solution, a 10 mM DMSO solution of B_3 (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0323] Purification of Antibody-Drug Conjugates:

[0324] The above solution was subjected to purification according to the common procedure A to obtain a solution containing compound 43A-043.

[0325] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0326] The DAR measurement was performed using the common procedure B. The results are shown in FIG6 , and the DAR value of 43A-043 was 7.8.

[0327] Example 10 Preparation of Antibody-Drug Conjugate 43B-043

[0328] Project 1:

[0329] Antibody reduction:

[0330] The laboratory-prepared 43B antibody medium was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0331] Conjugation of Antibodies to Linker-Drug Compounds:

[0332] To the above solution, a 10 mM DMSO solution of B_3 (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0333] Purification of Antibody-Drug Conjugates:

[0334] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound 43B-043.

[0335] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0336] DAR measurement was performed using common procedure B. The results are shown in FIG7 , and the DAR value of 43B-043 was 5.9.

[0337] Example 11: Preparation of Antibody-Drug Conjugate 43A-JSSW-4

[0338] Project 1:

[0339] Antibody reduction:

[0340] The 43A antibody prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and a 10 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (15.64 μL; 2.3 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0341] Conjugation of Antibodies to Drug-Linker Compounds:

[0342] To the above solution, a 10 mM JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) DMSO solution (30.6 μL; 4.5 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and allowed to react at room temperature for a further 20 minutes to terminate the coupling reaction.

[0343] Purification of Antibody-Drug Conjugates:

[0344] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound 43A-JSSW-4.

[0345] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0346] The DAR measurement was performed using the common procedure B, and the DAR value of 43A-JSSW-4 was 3.8.

[0347] Example 12: Preparation of Antibody-Drug Conjugate 43A-JSSW-6

[0348] Project 1:

[0349] Antibody reduction:

[0350] The 43A antibody prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 22.44 μL of 10 mM TCEP (J&K Technologies, Inc.) in water (3.3 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0351] Conjugation of Antibodies to Drug-Linker Compounds:

[0352] To the above solution, a 10 mM JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) DMSO solution (43.5 μL; 6.4 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for another 20 minutes to terminate the coupling reaction.

[0353] Purification of Antibody-Drug Conjugates:

[0354] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound 43A-JSSW-6.

[0355] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0356] The DAR measurement was performed using the common procedure B, and the DAR value of 43A-JSSW-6 was 5.8.

[0357] Example 13: Preparation of Antibody-Drug Conjugate 43A-JSSW-8

[0358] Project 1:

[0359] Antibody reduction:

[0360] The 43A antibody prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0361] Conjugation of Antibodies to Drug-Linker Compounds:

[0362] To the above solution, a 10 mM JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) DMSO solution (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and allowed to react at room temperature for a further 20 minutes to terminate the coupling reaction.

[0363] Purification of Antibody-Drug Conjugates:

[0364] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound 43A-JSSW-8.

[0365] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0366] The DAR measurement was performed using the common procedure B, and the DAR value of 43A-JSSW-8 was 7.5.

[0367] Example 14: Preparation of Antibody-Drug Conjugate 43B-JSSW-6

[0368] Project 1:

[0369] Antibody reduction:

[0370] The laboratory-prepared 43B antibody medium was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0371] Conjugation of Antibodies to Drug-Linker Compounds:

[0372] To the above solution, a 10 mM JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) DMSO solution (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and allowed to react at room temperature for a further 20 minutes to terminate the coupling reaction.

[0373] Purification of Antibody-Drug Conjugates:

[0374] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound 43B-JSSW-6.

[0375] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0376] The DAR measurement was performed using the common procedure B, and the DAR value of 43B-JSSW-6 was 5.6.

[0377] Example 15: Preparation of Antibody-Drug Conjugate 43B-045-4

[0378] Project 1:

[0379] Antibody reduction:

[0380] The 43B antibody medium prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and a 10 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (15.64 μL; 2.3 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0381] Conjugation of Antibodies to Drug-Linker Compounds:

[0382] To the above solution, a 10 mM DMSO solution of compound 045 (29.92 μL; 4.4 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0383] Purification of Antibody-Drug Conjugates:

[0384] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound 43B-045-4.

[0385] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0386] The DAR measurement was performed using the common procedure B, and the DAR value of 43B-045-4 was 4.0.

[0387] Example 16: Preparation of Antibody-Drug Conjugate 43B-045-6

[0388] Project 1:

[0389] Antibody reduction:

[0390] The laboratory-prepared 43B antibody medium was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0391] Conjugation of Antibodies to Drug-Linker Compounds:

[0392] To the above solution, a 10 mM DMSO solution of compound 045 (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technologies, Inc.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0393] Purification of Antibody-Drug Conjugates:

[0394] The above solution was purified using common procedure A to obtain a solution containing compound 43B-045-6. Determination of the Drug-Antibody Conjugate Ratio (DAR) of Antibody-Drug Conjugates:

[0395] The DAR value of 43B-045-6 was 5.7 when the DAR measurement was performed using the common procedure B.

[0396] Example 17: Preparation of Antibody-Drug Conjugate 43A-045-8

[0397] Project 1:

[0398] Antibody reduction:

[0399] The 43A antibody prepared in the laboratory was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technology Co., Ltd.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0400] Conjugation of Antibodies to Drug-Linker Compounds:

[0401] To the above solution, a 10 mM DMSO solution of compound 045 (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and reacted at room temperature for 30 minutes to attach the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technologies, Inc.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0402] Purification of Antibody-Drug Conjugates:

[0403] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound 43A-045-8.

[0404] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0405] The DAR value of 43A-045-8 was 7.9 when the DAR measurement was performed using the common procedure B.

[0406] Example 18: Preparation of Antibody-Drug Conjugate M30-DXd-4

[0407] Project 1:

[0408] Antibody reduction:

[0409] The laboratory-prepared M30 antibody medium was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and a 10 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (15.64 μL; 2.3 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0410] Conjugation of Antibodies to Drug-Linker Compounds:

[0411] To the above solution, a 10 mM DMSO solution of deruxtecan (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) (30.6 μL; 4.5 equivalents per antibody molecule) was added at room temperature, mixed, and allowed to react at room temperature for 30 minutes to attach the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and allowed to react at room temperature for a further 20 minutes to terminate the coupling reaction.

[0412] Purification of Antibody-Drug Conjugates:

[0413] The above solution was subjected to purification according to the common procedure A to obtain a solution containing the compound M30-DXd-4.

[0414] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0415] The DAR value of M30-DXd-4 was 4.2 when the DAR measurement was performed using the common procedure B.

[0416] Example 19: Hydrophobicity Detection of Antibody-Drug Conjugates

[0417] The hydrophobicity of 43A-042, 43B-042, 43A-043, 43B-043, and 43A-DXd was compared using hydrophobic interaction chromatography. Detection was performed on an e2695 HPLC using a TSKgel Butyl-NPR column (4.6 mm x 10 cm, Cat. No. 0042168 / TOSOH). Mobile phase A consisted of 1.0 M ammonium sulfate in 25 mM phosphate buffer, and mobile phase B consisted of 25% isopropanol in 75% 25 mM phosphate buffer. The flow rate was 0.8 ml / min, the detection wavelength was 280 nm, the column temperature was 30°C, and the mobile phase gradient was: 100% to 0% A from 0 to 20 minutes, 0% to 0% A from 20 to 23 minutes, 0% to 100% A from 23 to 24 minutes, and 100% to 100% A from 24 to 28 minutes.

[0418] The results, as shown in Figure 8, show that 43A-042, 43B-042, 43A-043, 43B-043, and 43A-DXd all exhibit good hydrophilicity. 43A-043 exhibits superior hydrophilicity to 43A-DXd and 43A-042, which have similar DAR values, with retention times of 7.130 min, 8.809 min, and 9.189 min, respectively. Similarly, 43B-043 exhibits stronger hydrophilicity than 43B-042, which has the same DAR value, with retention times of 6.426 min and 8.643 min, respectively.

[0419] Example 20: Determination of Antibody Affinity by Biomembrane Interference

[0420] The affinity of anti-B7H3 antibodies 43A and 43B for human B7H3 was determined using a ForteBio Octet red 96e instrument (Sartorius). Protein A capture sensors were immobilized with 5 μg / ml of each test sample. B7H3 antigen was serially diluted, starting at 100 nM and followed by a two-fold dilution gradient across seven steps. The buffer system was PBS pH 7.4 + 0.02% Tween 20. The following steps were used: ① Baseline 60 s, ② Loading 800 s (antibody immobilization, threshold set to 0.50 nM), ③ Baseline 2 120 s, ④ Association 120 s, ⑤ Dissociation 300 s, and ⑥ Regeneration 300 s. Blank buffer was used for blank analysis, and the baseline was aligned to the y-axis. Savitzky-Golay fitting was performed in Octet data analysis software. The fitting curves of 43A and 43B are shown in Figures 9 and 10, respectively, and the experimental results are shown in Table 1.

[0421] Table 1 Affinity of different antibodies to human B7H3

[0422] Example 21: Cell binding activity assay

[0423] The affinity of anti-B7H3 antibodies 43A and 43B was tested using the B7H3-positive human melanoma cell line A375, with M30 antibody as a positive reference. The sample was diluted to 15,000 ng / mL with 2% BSA in PBS and then serially diluted through 11 steps to a sample concentration of 0.014 to 15,000 ng / mL. A375 cells were centrifuged at 500 × g for 5 minutes, washed three times with 2% BSA in PBS, and incubated with the various diluted samples at 4°C for 2 hours. After washing, the cells were incubated with goat anti-human Alexa Fluor 488 fluorescent dye for 1 hour at 4°C in the dark. After washing, the cells were resuspended and the mean fluorescence intensity (MFI) was measured using an Attune NxT flow cytometer (Thermo Fisher Scientific, Inc.).

[0424] The experimental results are shown in Figure 11. Both 43A and 43B can bind to B7H3-positive A375 cells with high affinity. 50 They were 54.5ng / ml and 61.2ng / ml respectively, both significantly better than M30's 125.3ng / ml.

[0425] The conjugate of antibody 43B, 43B-045-6, also binds to B7H3-positive A375 cells with high affinity. 50 It is 37.78 ng / ml.

[0426] Example 22 Endocytic activity study

[0427] M30, 43A, and 43B antibodies were incubated with human melanoma A375 cells in a refrigerator at 4°C for 2 hours. After incubation, the cells were washed twice with PBS, supplemented with culture medium, and then placed in a 37°C, 5% CO2 incubator for internalization. Cells were harvested after the indicated incubation times. They were then stained with goat anti-human Alexa Fluor 488 fluorescent dye and incubated at 4°C in the dark for 60 minutes. After incubation, the cells were washed twice with assay buffer (2% FBS / PBS) and resuspended in buffer. Flow cytometry was then used to measure the median fluorescence signal of the corresponding wells in the assay plate using a plate reader.

[0428] Internalization rate (%) = (initial average median fluorescence value - average median fluorescence value at different time points) / initial average median fluorescence value × 100%.

[0429] The experimental results are shown in Table 2. After binding to A375 cells, 43A and 43B can be rapidly internalized into the cells, and the internalization rate is faster than that of M30.

[0430] Table 2 Antibody endocytosis rate at different time points

[0431] Example 23 Antigen Binding Activity Detection

[0432] Dilute the His-tagged human B7H3 protein (purchased from Beijing Biopsies Biotechnology Co., Ltd.) to 1 μg / mL with coating solution, add 100 μL to each well, and incubate at 4°C overnight; add 100 μL of blocking solution to each well of the enzyme-labeled plate containing the coating solution, shake at room temperature for 2 hours, and then wash the plate 3 times with washing buffer. Gradient dilution of samples and reference materials, add 100 μL to each well, shake at room temperature for 2 hours, wash 4 times with washing buffer, add enzyme-labeled antibody, dilute goat anti-human IgG-HRP with sample diluent, add 100 μL to each well, shake at room temperature for 1 hour, wash the plate 4 times with washing buffer, add substrate solution for color development, add 100 μL of freshly prepared TMB reaction solution to each reaction well, react at room temperature for 5-10 minutes, add 100 μL of stop solution to each well and mix gently to terminate the reaction. Use an enzyme reader to measure the absorbance at a wavelength of 450 / 650 nm, and use software to perform four-parameter fitting, where the C value is the half-effective concentration EC 50 The binding activity of the corresponding antibody-drug conjugates was tested using antibodies 43A and 43B as reference products. The experimental results are shown in Table 3, indicating that the binding activity of the antibodies was comparable to that of the corresponding antibody-drug conjugates.

[0433] Table 3 Summary of human B7H3 protein binding activity

[0434] Example 24 Inhibitory Effect of Antibody-Drug Conjugates on the Growth of SK-OV-3 and SK-BR-3 Cells in Vitro

[0435] The target cells were collected and resuspended into single cell suspension, and the cell viability and cell count were determined by trypan blue staining; the cell density was adjusted to 1×10 5 cells / mL; add 100 μL per well to a 96-well black flat-bottom cell culture plate; use the commercially available Enhertu (anti-HER2 antibody-drug conjugate) as a reference substance, add 20 μL per well of the diluted test sample to a 96-well black flat-bottom cell culture plate seeded with cells; place in a cell culture incubator (37°C, 5% CO2) and incubate for 168±8 hours; add 20 μL per well of resazurin sodium solution (0.03%); incubate at 37°C for 3-4 hours, read the fluorescence value at 550nm / 610nm on a microplate reader, and use Prism or similar plotting software to plot the half-inhibitory concentration (IC) of the reference substance and sample. 50 .

[0436] The experimental results, shown in Figures 12 and 13, demonstrate that DP001-042 exhibits significantly superior inhibitory activity to Enhertu in vitro assays targeting human ovarian adenocarcinoma cells (SK-OV-3) and human breast cancer cells (SK-BR-3). The half-inhibitory concentration (CI50) data are summarized in Table 4.

[0437] Table 4 In vitro growth inhibitory activity of antibody-drug conjugates

[0438] Example 25 Inhibitory effect of antibody-drug conjugates on the growth of human melanoma A375 cells in vitro

[0439] The target cells were collected and resuspended into single cell suspension, and the cell viability and cell count were determined by trypan blue staining; the cell density was adjusted to 1×10 5 cells / mL; add 100 μL per well to a 96-well black flat-bottom cell culture plate; use 43A-DXd as a reference substance, add 20 μL per well of the diluted test substance to a 96-well black flat-bottom cell culture plate seeded with cells; place in a cell culture incubator (37°C, 5% CO2) and incubate for 168±8 hours; add 20 μL per well of resazurin sodium solution (0.03%); incubate at 37°C for 3-4 hours, read the fluorescence value at 550nm / 610nm on a microplate reader, and use Prism or similar plotting software to plot the half-inhibitory concentration (IC) of the reference substance and sample. 50 .

[0440] In vitro experiments on human melanoma A375 cells revealed that 43A-042, 43B-042, 43A-DXd, 43A-043, and 43B-043 all exhibited significant cell growth inhibitory activity. The results are shown in Tables 5 and 6 and Figures 14 and 15.

[0441] Table 5 In vitro growth inhibitory activity of antibody-drug conjugates

[0442] Table 6 In vitro growth inhibitory activity of antibody-drug conjugates

[0443] Example 26 Antitumor Experiment of Antibody-Drug Conjugates on Calu-6 Human Lung Cancer Cell Transplanted Tumors in Mice

[0444] In this experiment, female NOD / SCID mice of appropriate age were inoculated with human lung cancer cells Calu-6. After the tumors grew to a volume of about 100-200 mm, 3 At the same time, 48 animals with good tumor growth were selected and divided into 6 groups according to tumor volume. The animal groups and dosage regimens are shown in Table 7.

[0445] Table 7 Animal grouping and dosage regimen

[0446] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after drug administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows:

[0447] Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0448] On the 25th day after administration, the mice were asphyxiated with carbon dioxide, and the tumors were removed and weighed.

[0449] At the experimental endpoint, compared with the vehicle group, all antibody-drug conjugates were able to significantly inhibit tumor growth. The tumor weight inhibition rate of 43B-042 was as high as 84.8%. The efficacy of 43B-043, 43A-042, and 43A-043 were all better than that of 43A-DXd. Detailed information is shown in Figure 16 and Table 8.

[0450] Table 8 Tumor parameters of each group after 25 days of drug administration

[0451] Example 27 Inhibitory Effect of Antibody-Drug Conjugates on the Growth of Human Melanoma Cells A375 in Vitro

[0452] The target cells were collected and resuspended into single cell suspension, and the cell viability and cell count were determined by trypan blue staining; the cell density was adjusted to 1×10 5 cells / mL; add 100 μL per well to a 96-well black flat-bottom cell culture plate; add 20 μL per well of the diluted test sample to the 96-well black flat-bottom cell culture plate seeded with cells; place in a cell culture incubator (37°C, 5% CO2) and incubate for 168±8 hours; add 20 μL per well of resazurin sodium solution (0.03%); incubate at 37°C for 3-4 hours, read the fluorescence value at 550nm / 610nm on a microplate reader, and use Prism or similar plotting software to plot the half-inhibitory concentration (IC) of the test sample. 50 .

[0453] In an in vitro experiment on human melanoma A375 cells, 43A-JSSW-4, 43A-JSSW-6, 43A-JSSW-8, 43B-JSSW-6, 43B-045-4, 43B-045-6, and 43A-045-8 all exhibited good cell growth inhibitory activity. The results are shown in Table 9.

[0454] Table 9 In vitro growth inhibitory activity of antibody-drug conjugates

[0455] Example 28 Antitumor Experiment of Antibody-Drug Conjugates on Human Melanoma Cell A375 Transplanted Tumors in Mice

[0456] In this experiment, female NU / NU mice of appropriate age were inoculated with human melanoma cells A375. The tumors were grown to a volume of about 100-200 mm. 3 At the same time, 28 animals with good tumor growth were selected and divided into 4 groups according to the tumor volume. The animal grouping and dosage regimen are shown in Table 10.

[0457] Table 10 Animal grouping and dosage regimen

[0458] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after drug administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows:

[0459] Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0460] At the experimental endpoint, compared with the vehicle group, all antibody-drug conjugates were able to significantly inhibit tumor growth. The tumor inhibition rates of 43A-JSSW-4, 43A-JSSW-6, and 43A-JSSW-8 were 53.6%, 65.2%, and 87.7%, respectively. Detailed information is shown in Figure 18 and Table 11.

[0461] Table 11 Efficacy of ADC on A375 xenograft tumors in tumor-bearing mice

[0462] Example 29 Antitumor Experiment of Antibody-Drug Conjugates on Calu-6 Human Lung Cancer Cell Transplanted Tumors in Mice

[0463] In this experiment, female NOD / SCID mice of appropriate age were inoculated with human lung cancer cells Calu-6. The tumors were grown to a volume of about 100-200 mm. 3 At the same time, 35 animals with good tumor growth were selected and divided into 5 groups according to the tumor volume. The animal grouping and dosage regimen are shown in Table 12.

[0464] Table 12 Animal grouping and dosage regimen

[0465] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after drug administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows:

[0466] Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0467] At the experimental endpoint, all antibody-drug conjugates were able to significantly inhibit tumor growth compared with the vehicle group. Detailed information is shown in Figure 19 and Table 13.

[0468] Table 13 Efficacy of ADC on Calu-6 xenograft tumors in tumor-bearing mice

[0469] Example 30 Antitumor Experiment of Antibody-Drug Conjugates on Human Melanoma Cell A375 Transplanted Tumors in Mice

[0470] In this experiment, female NU / NU mice of appropriate age were inoculated with human melanoma cells A375. The tumors were grown to a volume of about 100-200 mm. 3 When the tumors were well grown, animals were selected and divided into groups evenly according to the tumor volume. The animal groups and dosage regimens are shown in Table 14.

[0471] Table 14 Animal grouping and dosage regimen

[0472] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after drug administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows:

[0473] Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0474] At the experimental endpoint, all antibody-drug conjugates were able to significantly inhibit tumor growth compared with the vehicle group. Detailed information is shown in Figure 20 and Table 15.

[0475] Table 15 ADC efficacy against A375 xenograft tumors in tumor-bearing mice

[0476] Note: On day 22, the average tumor volume of animals in the vehicle group reached 3000 mm 3 , and euthanasia was performed according to animal welfare.

[0477] Example 31 Antitumor Experiment of Antibody-Drug Conjugate on Human Esophageal Squamous Cell Carcinoma KYSE-150 Mouse Xenografts

[0478] In this experiment, female NU / NU mice of appropriate age were inoculated with human esophageal squamous cell carcinoma cells KYSE-150. 3 Around 3 d, animals with good tumor growth were selected and divided into groups evenly according to tumor volume. The animal groups and dosage regimens are shown in Table 16.

[0479] Table 16 Animal grouping and dosage regimen

[0480] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after drug administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows:

[0481] Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0482] At the experimental endpoint, 43B-JSSW-6 was able to significantly inhibit tumor growth compared with the vehicle group. Detailed information is shown in Figure 21 and Table 17.

[0483] Table 17 Efficacy of ADC on KYSE-150 xenografts in tumor-bearing mice

[0484] Example 32 Antitumor Experiment of Antibody-Drug Conjugates on Human Non-Small Cell Lung Cancer SK-MES-1 Mouse Xenografts

[0485] In this experiment, female NU / NU mice of appropriate age were inoculated with human non-small cell lung cancer SK-MES-1 and the tumor volume grew to about 100 mm. 3 Around 3 d, animals with good tumor growth were selected and divided into groups evenly according to tumor volume. The animal groups and dosage regimens are shown in Table 18.

[0486] Table 18 Animal grouping and dosage regimen

[0487] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after drug administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows:

[0488] Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0489] At the experimental endpoint, 43B-JSSW-6 was able to significantly inhibit tumor growth compared with the vehicle group. Detailed information is shown in Figure 22 and Table 19.

[0490] Table 19 ADC efficacy against SK-MES-1 xenografts in tumor-bearing mice

[0491] Example 33 Antitumor Experiment of Antibody-Drug Conjugates on Human Hep3B Hepatocellular Carcinoma Transplanted Tumors in Mice

[0492] In this experiment, female NU / NU mice of appropriate age were inoculated with human hepatoma cell line Hep3B. When the tumor volume grew to about 100-200 mm,3 At the same time, animals with good tumor growth were selected and divided into 3 groups evenly according to tumor volume. The animal grouping and dosage regimen are shown in Table 20.

[0493] Table 20 Animal grouping and dosage regimen

[0494] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after drug administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows:

[0495] Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0496] At the experimental endpoint, compared with the vehicle group, the test group was able to significantly inhibit tumor growth. Detailed information is shown in Figure 23 and Table 21.

[0497] Table 21 Efficacy of ADC on Hep3B xenografts in tumor-bearing mice

[0498] Example 34 Antitumor Experiment of Antibody-Drug Conjugates on Calu-6 Human Lung Cancer Xenografts in Mice

[0499] In this experiment, NOD / SCID mice of appropriate age were inoculated with human lung cancer cells Calu-6. When the tumor volume grew to about 100-200 mm, 3 At the same time, 24 animals with good tumor growth were selected and divided into 4 groups evenly according to tumor volume. The animal grouping and dosage regimen are shown in Table 22.

[0500] Table 22 Animal grouping and dosage regimen

[0501] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after drug administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows:

[0502] Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0503] At the experimental endpoint, compared with the vehicle group, the test group was able to significantly inhibit tumor growth, and the efficacy of 43B-JSSW-6 and 43B-045-4 was significantly better than the positive reference M30-DXd-4. Detailed information is shown in Figure 24 and Table 23.

[0504] Table 23 Efficacy of ADC on Calu-6 xenograft tumors in tumor-bearing mice

Claims

1. An antibody-drug conjugate having a structure represented by formula (I) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: A-(LD) d (I) wherein A is selected from an antibody (such as a monoclonal antibody) or an antigen-binding fragment thereof; L is a linker part, one end of which is connected to A and the other end is connected to the biologically active molecule D; D is a bioactive molecule; d represents the molar ratio of the bioactive molecule to A (also known as DAR, i.e., drug-antibody coupling ratio), which is an integer or decimal selected from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); When d is a decimal, it refers to the average number of linker-bioactive molecules (LD) conjugated per A.

2. The antibody drug conjugate having the structure shown in formula (I) according to claim 1 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is selected from antibodies or antigen-binding fragments thereof targeting HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4), EGFR, dll3, TROP2, B7H3, B7H4, TF (Tissue factor), c-Met, CD20, CD22, CD30, CD33, CD44, CD47, CD56, CD70, CD73, CD79b, CD105, CEA, A33, Cripto, EphA2, G250, MUCl, Lewis Y, EDB-FN, VEGFR, VEDF, PD-1, PD-L1, MET, RET, GPNMB, Integrin, PSMA, Tenascin-C, SLC44A4, Claudin18.2 or Mesothelin.

3. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 2, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementary determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementary determining region 1 (HCDR1) is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementary determining region 2 (HCDR2) is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain complementary determining region 3 (HCDR3) is shown in SEQ ID NO: 3; the light chain comprises three complementary determining regions (CDRs) of the light chain, wherein the amino acid sequence of the light chain complementary determining region 1 (LCDR1) is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementary determining region 2 (LCDR2) is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementary determining region 3 (LCDR3) is shown in SEQ ID NO: 6, wherein the CDRs are determined according to the Kabat numbering convention.

4. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 3, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the anti-B7H3 antibody or the antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the variable region (HV) sequence of the heavy chain is shown in SEQ ID NO:7, and the variable region (LV) sequence of the light chain is shown in SEQ ID NO:

8.

5. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 4, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antibody targeting B7H3, wherein the anti-B7H3 antibody further comprises a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

6. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 5, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antigen-binding fragment targeting B7H3, and the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2.

7. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 6, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antibody or an antigen-binding fragment thereof targeting B7H3, wherein the anti-B7H3 antibody or the antigen-binding fragment thereof is a humanized or fully human antibody or the antigen-binding fragment thereof.

8. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 7, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein A is an antibody targeting B7H3 or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody comprises a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:9 or SEQ ID NO:10, and the amino acid sequence of the light chain is shown in SEQ ID NO:

11.

9. The antibody drug conjugate having the structure shown in formula (I) according to claim 1 and its tautomer, mesomer, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance, wherein A is selected from 43A or 43B targeting B7H3, DP001 targeting HER2, sacituzumab targeting TROP2, Alsevalimab targeting B7H4, Tisotumab targeting TF, L19A targeting EDB-FN, Zolbetuximab targeting Claudin18.2 or their biosimilars, wherein 43A comprises two identical heavy chains and light chains, and the amino acid sequence of the heavy chain is as follows As shown in SEQ ID NO:9, the light chain amino acid sequence is shown in SEQ ID NO:11; 43B includes two identical heavy chains and light chains, the heavy chain amino acid sequence is shown in SEQ ID NO:10, and the light chain amino acid sequence is shown in SEQ ID NO:

11.

10. The antibody drug conjugate having the structure shown in formula (I) according to claims 1-9 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein -L- is selected from -L1-L2-L3-L4-L5-, wherein L1 is a covalent linking unit, which is covalently linked to A, L2 is an extension unit, L3 is selected from a bond or an amino acid residue optionally substituted by a polar hydrophilic group, L4 is selected from a peptide residue consisting of 2-8 amino acids, and L5 is a bond or a self-cleaving fragment or a self-cleaving fragment substituted by a polar hydrophilic group.

11. The antibody drug conjugate having the structure shown in formula (I) according to claim 10, and its tautomer, mesomer, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance, wherein L1 is selected from Where * indicates connection with A; L2 is selected from -L 2a -、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -alkynylene-C1-C6 alkylene-, -phenylene-, -phenylene-C1-C3 alkylene-, 1-50 (preferably 9-30, more preferably 9-26, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, each of the alkylene, cycloalkylene and heterocyclylene is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 1-6 atoms of heteroalkyl, C1-C6 alkoxy, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene contains 1-12 (preferably 1-8, more preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene are selected from one or more of N, O or S (preferably O); L3 is selected from a bond or an amino acid residue substituted with a polar hydrophilic group, wherein the polar hydrophilic group comprises a sugar residue and its derivatives or a polyethylene glycol residue and its derivatives or a polysarcosine residue and its derivatives or a combination thereof; L4 is selected from a peptide residue consisting of 2-8 amino acids selected from phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine; L5 is selected from a key, wherein R3 is selected from hydrogen, mSelect An integer from 3 to 50, preferably an integer from 8 to 24, further preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, * represents connection with D; Preferably, L5 is selected from 12. The antibody drug conjugate having the structure shown in formula (I) according to claim 11, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L2 is selected from -L 2a -、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from -C1-C6 alkylene-, -C1-C3 alkylene-C3-C6 cycloalkylene-, -ethynylene-C1-C6 alkylene-, -(CH2CH2O) s -、-(CH2CH2O) s C 1-3 Alkylene-, -C 1-3 Alkylene (CH2CH2O) s C 1-3 Alkylene-, phenylene, phenylenemethylene, wherein s is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

13. The antibody drug conjugate having the structure shown in formula (I) according to claims 11-12 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L2 is selected from -L 2a -、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from methylene, ethylene, n-propylene, isopropylene, n-pentylene, methylene cyclohexylene, ethynylene methylene, ethynylene ethylene, ethynylene n-propylene, ethynylene n-butylene, ethynylene n-pentylene, ethynylene n-hexylene, phenylene, phenylene methylene, -(CH2CH2O) s CH2-、-(CH2CH2O) s CH2CH2-、-CH2(OCH2CH2) s -、-CH2CH2(OCH2CH2) s -、-CH2CH2(OCH2CH2) s CH2-, -CH2CH2(OCH2CH2) s CH2CH2-、-CH2(OCH2CH2) s CH2-, wherein s is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

14. The antibody drug conjugate having the structure shown in formula (I) according to claims 11-13 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L2 is selected from: -(CH2CH2O) s CH2C(O)-、-(CH2CH2O) s CH2CH2-、 wherein s is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

15. The antibody drug conjugate having the structure shown in formula (I) according to claims 11-14 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L3 is selected from glutamic acid substituted with a polar hydrophilic group, wherein the polar hydrophilic group comprises a sugar residue and its derivatives, a polyethylene glycol residue and its derivatives, a polysarcosine residue and its derivatives, or a combination thereof.

16. The antibody drug conjugate having the structure shown in formula (I) according to claims 11-15 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L3 is selected from: Wherein HG is selected from a group comprising sugar residues and derivatives thereof, a group comprising polyethylene glycol residues and derivatives thereof, a group comprising polysarcosine residues and derivatives thereof, or a combination thereof, wherein * indicates connection with L2.

17. The antibody drug conjugate having the structure shown in formula (I) according to claims 11-16 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein HG is selected from: -NH(CH2CH2O)nCH3, wherein n and q are each independently selected from 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 47,48,49,50), preferably an integer of 1-24, more preferably 3-12.

18. The antibody drug conjugate having the structure shown in formula (I) according to claims 11-17 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein L4 is selected from: * indicates connection to L3.

19. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 9, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein -L- is selected from: Wherein s is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), HG is as defined in claim 17, and * indicates connection with A.

20. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 9, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein -L- is selected from -L1-L2-L4-L5-, wherein L1, L2, L4, and L5 are defined as in claims 11 to 18.

21. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 9, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein -L- is selected from: in, m is selected from an integer of 3-50, preferably an integer of 8-24, and more preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.

22. The antibody-drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 21, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein D is selected from topoisomerase I inhibitors (such as camptothecin derivatives), microtubule inhibitors (such as MMAE, MMAF, eribulin), and DNA damaging agents (such as pyrrolobenzodiazepines).

23. The antibody drug conjugate having the structure shown in formula (I) according to any one of claims 1 to 22, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances, wherein D is selected from: Wherein R1 and R2 are each independently selected from H, C1-3 alkyl (preferably methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), or R1 and R2 together with the carbon atoms connecting them form a 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutane, cyclopentyl, cyclohexyl).

24. The antibody drug conjugate having a structure as shown in formula (I) according to any one of claims 1 to 23, and its tautomer, mesomer, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance, which has the following structure: in, n is selected from an integer of 1-50, preferably 1-24, and more preferably 3-12, d is selected from an integer or decimal from 1 to 12, s is selected from an integer of 1-12, m is selected from an integer of 3-50, preferably an integer of 8-24, and more preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.

25. A pharmaceutical composition comprising the antibody drug conjugate having a structure represented by formula (I) according to any one of claims 1 to 24, and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products.

26. Use of the antibody drug conjugate having the structure shown in formula (I) according to any one of claims 1 to 24 and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled products in the preparation of a medicament for treating a proliferative disease.

27. The use of claim 26, wherein the proliferative disease is preferably a disease associated with abnormal expression of HER2 and B7H3, including cancer, and the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma or relapsed anaplastic large cell lymphoma).

28. A linker-drug compound of the structure represented by formula (II) and its tautomers, mesomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, solvates or isotope-labeled substances: L1'-L2-L3-L4-L5-D (II) Wherein L1' is selected from L2, L3, L4, L5 and D are defined the same as in formula (I).

29. The linker-drug compound of the structure represented by formula (II) according to claim 28 and its tautomer, mesomer, racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, hydrate, solvate or isotope-labeled substance, which is selected from: in, n is selected from an integer of 1-50, preferably 1-24, and more preferably 3-12, d is selected from an integer or decimal of 1 to 12, and s is selected from an integer of 1-12.

30. An antibody or antigen-binding fragment thereof targeting B7H3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, wherein the heavy chain comprises three complementary determining regions (CDRs), wherein the amino acid sequence of the heavy chain complementary determining region 1 (HCDR1) is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain complementary determining region 2 (HCDR2) is shown in SEQ ID NO: 2, and the amino acid sequence of the heavy chain complementary determining region 3 (HCDR3) is shown in SEQ ID NO: 3; and the light chain comprises three complementary determining regions (CDRs) of the light chain, wherein the amino acid sequence of the light chain complementary determining region 1 (LCDR1) is shown in SEQ ID NO: 4, the amino acid sequence of the light chain complementary determining region 2 (LCDR2) is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain complementary determining region 3 (LCDR3) is shown in SEQ ID NO: 6, wherein the CDRs are determined according to the Kabat numbering convention.

31. The antibody or antigen-binding fragment thereof targeting B7H3 according to claim 30, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, the variable region (HV) sequence of the heavy chain is as shown in SEQ ID NO:7, and the variable region (LV) sequence of the light chain is as shown in SEQ ID NO:

8.

32. The antibody or antigen-binding fragment thereof targeting B7H3 according to claims 30-31, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and / or a light chain, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:9 or SEQ ID NO:10, and the variable region (LV) sequence of the light chain is as shown in SEQ ID NO:

11.

33. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof targeting B7H3 according to claims 30-31.

Images