Anti-b7-h4 antibody-drug conjugate and application thereof

Abstract

The present application relates to an anti-B7-H4 antibody-drug conjugate and an application thereof. Specifically, provided is an antibody-drug conjugate as represented by formula (I), or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein formula (I) is Ab-(L-D)a.

Description

Anti-b7-h4 antibody drug conjugates and uses thereof

[0001] This application is based on and claims priority to CN application No. 202410657156.0, filed on May 24, 2024, the disclosure of which is incorporated herein in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of medicine, in particular to anti-B7-H4 antibody drug conjugates and uses thereof. BACKGROUND

[0003] B7-H4 (B7 homolog 4), also known as V-set domain-containing T-cell activation inhibitor 1 (VTCN1), B7S1, B7x, is a member of the B7 family of immunomodulatory proteins, which inhibits T cell function and negatively regulates T cell immune response by acting on unknown receptors on T cells, and plays an extremely important role in tumor immune escape. B7-H4 has a relatively low expression level in normal tissues, but is highly expressed in various solid tumors, such as breast cancer, ovarian cancer, endometrial cancer, and cholangiocarcinoma, and is associated with poor prognosis of various tumors.

[0004] Currently, there are various B7-H4 targeted therapies under development, including monoclonal antibodies, bispecific antibodies, CAR-T, antibody-drug conjugates (ADC), etc., but there is no approved drug. Among them, antibody-drug conjugates target small molecule toxins (payloads) into tumor cells expressing B7-H4 protein through specific antibodies of B7-H4, while retaining the tumor-killing properties of small molecule toxins, selectively reducing off-target toxic side effects of small molecule toxins, and have great potential in improving the risk-benefit ratio of anti-tumor therapy, and are expected to bring greater benefits to patients. SUMMARY

[0005] The first aspect of the present application provides an antibody drug conjugate represented by formula (I) or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, Ab-(L-D) a Formula (I)

[0006] wherein:

[0007] Ab is an antibody or an antigen-binding fragment thereof;

[0008] L is L 1 -L 2 -L3 -L 4 wherein L 1 is attached to Ab, L 4 is attached to D;

[0009] L 1 is selected from R a is selected from hydrogen, Ci-C6alkyl, Ci-C6deuteroalkyl, C3-C8cycloalkyl, and C3-C8deutero cycloalkyl;

[0010] L 2 is selected from -(CH2) m -X1-X2-(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)- and -(Ci-C4)alkylene-C(=O)-, wherein,

[0011] X1is selected from a bond, C3-C8cycloalkyl, 3-8 membered heterocyclyl, C6-C 10 aryl, and 5-9 membered heteroaryl,

[0012] X2is selected from a bond, -O-, -NH-, -C(=O)-, and -C(=O)NH-,

[0013] m, n, p, and q are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,

[0014] said -(CH2) m -X1-X2-(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)- and -(Ci-C4)alkylene-C(=O)- are optionally substituted with one or more groups selected from hydrogen, halogen, hydroxyl, amino, -(Ci-C4)alkylene-hydroxyl, and -O-(Ci-C4)alkylene-hydroxyl;

[0015] L 3 is selected from an amino acid residue and a peptide residue consisting of 2-10 amino acid residues;

[0016] L 4 is wherein the * end is attached to D;

[0017] D is

[0018] R 1 and R 2each independently is selected from hydrogen, deuterium, halogen, hydroxyl, amino, C1-C6alkyl, C1-C6deuterated alkyl, C1-C6halogenated alkyl, and C3-C8cycloalkyl, or,

[0019] R 1 and R 2 together with the carbon atom to which they are attached form a C3-C6cycloalkyl or a 3-6 membered heterocyclyl;

[0020] R 3 and R 4 each independently is selected from hydrogen, deuterium, halogen, C1-C6alkyl, C1-C6deuterated alkyl, and C1-C6halogenated alkyl, or,

[0021] R 3 and R 4 together with the carbon atom to which they are attached form a 5-6 membered heterocyclyl, which is optionally substituted with one or more groups selected from hydrogen, deuterium, halogen, hydroxyl, amino, C1-C6alkyl, C1-C6deuterated alkyl, and C1-C6halogenated alkyl;

[0022] a is any numerical value between 1-10.

[0023] In some embodiments, the R a is selected from hydrogen, C1-C4alkyl, and C1-C4deuterated alkyl.

[0024] In some embodiments, the R a is selected from hydrogen, methyl, ethyl, isopropyl, deuterated methyl, deuterated ethyl, and deuterated isopropyl.

[0025] In some embodiments, the L 1 is selected from 2 .

[0026] In some embodiments, the L 1 is selected from wherein the * end is attached to L 2 .

[0027] In some embodiments, the L 1 is selected from wherein the * end is attached to L 2 .

[0028] In some embodiments, the X1is selected from a bond, a 5-6 membered heterocyclyl, and a 5-6 membered heteroaryl.

[0029] In some embodiments, the X1is selected from a bond, a 5-6 membered heterocyclyl, and a 5-6 membered heteroaryl, the 5-6 membered heterocyclyl containing 1 or 2 heteroatoms selected from N and O, the 5-6 membered heteroaryl containing 1, 2, or 3 heteroatoms selected from N and O.

[0030] In some embodiments, said X1is selected from a bond, pyrrolidinyl, tetrahydrofuranyl, 1,3-dioxolanyl, 1,3-dioxanyl, pyridinyl, and pyrimidinyl.

[0031] In some embodiments, said X1is selected from 1,3-dioxolanyl, 1,3-dioxanyl, and pyridinyl.

[0032] In some embodiments, said X2is selected from a bond and -C(=0)NH-.

[0033] In some embodiments, said X2is -C(=0)NH-.

[0034] In some embodiments, said m is 0, 1, 2, or 3.

[0035] In some embodiments, said m is 1 or 2.

[0036] In some embodiments, said n is 0, 1, 2, or 3.

[0037] In some embodiments, said n is 1.

[0038] In some embodiments, said p is 0, 3, 4, 5, 6, 7, 8, 9, or 10.

[0039] In some embodiments, said p is 4 or 8.

[0040] In some embodiments, said q is 0, 1, 2, or 3.

[0041] In some embodiments, said q is 1.

[0042] In some embodiments, said q is 0.

[0043] In some embodiments, said L 2 is selected from -(CH2) m -X2-(CH2) n -(CH2OCH2) p -(CH2) q -C(=0)-.

[0044] In some embodiments, said L 2 is selected from -(CH2) m -X2-(CH2) n -(CH2OCH2) p -C(=0)-, -(CH2) m -X1-X2-(CH2) n -(CH2OCH2) p -(CH2)q -C(=O)-, -X1-(CH2OCH2) p -C(=O)-, -(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)- and -(C1-C4)alkylene-C(=O)-, said -(CH2) m -X2-(CH2) n -(CH2OCH2) p -C(=O)-, -(CH2) m -X1-X2-(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)-, -X1-(CH2OCH2) p -C(=O)-, -(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)- and -(C1-C4)alkylene-C(=O)- optionally substituted with one or more groups selected from hydrogen, hydroxyl, amino, -methylene-hydroxyl, -0-ethylene-hydroxyl.

[0045] In some embodiments, the L 2 selected from -CH2CH2-C(=O)NH-CH2-(CH2OCH2)8-CH2-C(=O)-, -CH2-C(=O)-, -CH2CH2-C(=O)-, -CH(CH3)-C(=O)-, -CH(CH2CH3)-C(=O)-, -CH(CH3)CH2-C(=O)-, and -CH2CH(CH3)-C(=O)-, said -CH2CH2-C(=O)NH-CH2-(CH2OCH2)8-CH2-C(=O)-, -CH2-C(=O)-, -CH2CH2-C(=O)-, -CH(CH3)-C(=O)-, -CH(CH2CH3)-C(=O)-, -CH(CH3)CH2-C(=O)-, and -CH2CH(CH3)-C(=O)- optionally substituted with one or more groups selected from H, OH, NH2, CH2OH, and -0-CH2CH2OH.

[0046] In some embodiments, the L 2 selected from -CH(CH2OH)-C(=O)-, -CH(CH2OCH2CH2OH)-C(=O)-,

[0047] In some implementations, the L 2 It is -CH(CH2OH)-C(=O)-.

[0048] In some implementations, the L 2 The C (=O) terminal and L 3 Connected.

[0049] In some implementations, the L 2 for

[0050] In some implementations, the L 2 for Where * end and L 3 Connected.

[0051] In some implementations, the L 3 A peptide residue consisting of 2-4 (preferably 4) amino acid residues, wherein the amino acids are selected from glycine, phenylalanine, valine, alanine, lysine, citrulline, serine, glutamic acid, and aspartic acid. In some embodiments, the amino acids are selected from glycine and phenylalanine.

[0052] In some implementations, the L 3 It is glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) (SEQ ID NO:12).

[0053] In some implementations, the L 3 The C (=O) terminal and L 4 Connected.

[0054] In some implementations, the L 3 for

[0055] In some implementations, the L 3 for Where * end and L 4 Connected.

[0056] In some implementations, the R 1 and R 2 Each is independently selected from hydrogen, deuterium, C1-C4 alkyl, C1-C4 deuterated alkyl, C1-C4 haloalkyl and C3-C6 cycloalkyl.

[0057] In some implementations, the R 1 and R 2 Each is independently selected from hydrogen, deuterium, methyl, deuterated methyl, halomethyl and cyclopropyl.

[0058] In some embodiments, the R 1 and R 2 are each independently hydrogen.

[0059] In some embodiments, the R 1 and R 2 together with the carbon atom to which they are attached form a cyclopropyl, cyclobutyl, oxetanyl, azetidinyl.

[0060] In some embodiments, the R 1 and R 2 together with the carbon atom to which they are attached form a cyclopropyl, cyclobutyl.

[0061] In some embodiments, the R 3 and R 4 are each independently selected from hydrogen, deuterium, halogen, C1-C4 alkyl, C1-C4 deuterated alkyl, and C1-C4 halogenated alkyl.

[0062] In some embodiments, the R 3 and R 4 are each independently selected from hydrogen, deuterium, fluorine, chlorine, methyl, deuterated methyl, and halogenated methyl.

[0063] In some embodiments, the R 3 is methyl.

[0064] In some embodiments, the R 4 is fluorine.

[0065] In some embodiments, the R 3 and R 4 together with the carbon atom to which they are attached form a 5-6 membered heterocyclyl containing 1 or 2 heteroatoms selected from O, the 5-6 membered heterocyclyl being optionally substituted with one or more groups selected from hydrogen, deuterium, halogen, and C1-C4 alkyl.

[0066] In some embodiments, the R 3 and R 4 together with the carbon atom to which they are attached form the is optionally substituted with 1, 2, or 3 groups selected from hydrogen, deuterium, and fluorine.

[0067] In some embodiments, the D is

[0068] In some embodiments, each L is independently selected from:

[0069] In some embodiments, each L-D is each independently selected from:

[0070] In some embodiments, the Ab is an anti-B7-H4 antibody or antigen binding fragment thereof.

[0071] In some embodiments, the antibody or antigen binding fragment thereof comprises:

[0072] (a) the following 3 heavy chain variable region (VH) complementarity determining regions (CDRs):

[0073] (i) a VH CDR1 that has a sequence of a CDR1 contained in a VH as set forth in SEQ ID NO: 7 or SEQ ID NO: 8, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR1 contained in the VH;

[0074] (ii) a VH CDR2 that has a sequence of a CDR2 contained in a VH as set forth in SEQ ID NO: 7 or SEQ ID NO: 8, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR2 contained in the VH; and

[0075] (iii) a VH CDR3 that has a sequence of a CDR3 contained in a VH as set forth in SEQ ID NO: 7 or SEQ ID NO: 8, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR3 contained in the VH;

[0076] and / or

[0077] (b) the following 3 light chain variable region (VL) CDRs:

[0078] (iv) a VL CDR1 that has a sequence of a CDR1 contained in a VL as set forth in SEQ ID NO: 9, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR1 contained in the VL;

[0079] (v) a VL CDR2 that has the sequence of CDR2 contained in the VL set forth in SEQ ID NO: 9, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of CDR2 contained in the VL set forth in SEQ ID NO: 9; and

[0080] (vi) a VL CDR3 that has the sequence of CDR3 contained in the VL set forth in SEQ ID NO: 9, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of CDR3 contained in the VL set forth in SEQ ID NO: 9.

[0081] In some embodiments, the substitution in any of (i)-(vi) is a conservative substitution.

[0082] In some embodiments, the CDR1, CDR2, and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2, and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering system.

[0083] In some embodiments, the CDR1, CDR2, and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2, and CDR3 contained in the light chain variable region (VL) are defined by the Kabat numbering system.

[0084] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0085] (a) the sequences of CDR1, CDR2, and CDR3 contained in the VH set forth in SEQ ID NO: 7 or SEQ ID NO: 8; and / or

[0086] (b) the sequences of CDR1, CDR2, and CDR3 contained in the VL set forth in SEQ ID NO: 9.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0088] (a) the following 3 heavy chain variable region (VH) CDRs:

[0089] (i) a VH CDR1 that consists of the sequence of SEQ ID NO: 1, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to SEQ ID NO: 1,

[0090] (ii) a VH CDR2 consisting of the sequence set forth in SEQ ID NO:2, or a sequence having one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:2, and

[0091] (iii) a VH CDR3 consisting of the sequence set forth in SEQ ID NO:3, or a sequence having one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:3;

[0092] and / or

[0093] (b) the following 3 light chain variable region (VL) CDRs:

[0094] (iv) a VL CDR1 consisting of the sequence set forth in SEQ ID NO:4, or a sequence having one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:4,

[0095] (v) a VL CDR2 consisting of the sequence set forth in SEQ ID NO:5, or a sequence having one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:5, and

[0096] (vi) a VL CDR3 consisting of the sequence set forth in SEQ ID NO:6, or a sequence having one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:6.

[0097] In some embodiments, the substitution in any of (i)-(vi) is a conservative substitution.

[0098] In some embodiments, the VH of the antibody or antigen-binding fragment thereof comprises a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 2; and, a VH CDR3 as set forth in SEQ ID NO: 3; and / or, the VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 4; a VL CDR2 as set forth in SEQ ID NO: 5; and, a VL CDR3 as set forth in SEQ ID NO: 6.

[0099] In some embodiments, the VH of the antibody or antigen-binding fragment thereof comprises a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO: 2; and, a VH CDR3 as set forth in SEQ ID NO: 3; and, the VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 4; a VL CDR2 as set forth in SEQ ID NO: 5; and, a VL CDR3 as set forth in SEQ ID NO: 6.

[0100] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 9.

[0101] In some embodiments, the amino acid sequence of the heavy chain of the antibody is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain of the antibody is set forth in SEQ ID NO: 11.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof is linked to L through a thiol group thereof.

[0103] In some embodiments, each a is independently any number between 1-8.

[0104] In some embodiments, each a is independently any number between 3-8.

[0105] In some embodiments, each a is independently any number between 3-6, such as 3-4, 4-5, 5-6, or such as about 3.1, about 3.3, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.9, about 5.1, about 5.3, about 5.5. Such as about 3.0, about 3.2, about 3.4, about 4.8, about 5.0, about 5.2, about 5.4.

[0106] In some embodiments, the antibody drug conjugate of Formula (I) is selected from:

[0107] wherein,

[0108] Ab and a are as defined in any embodiment;

[0109] In some embodiments, in MH-ADC2, MH-ADC4, MH-ADC6, MH-ADC8, the Ab is linked to the e position and / or the f position via its thiol group.

[0110] In some embodiments, in MH-ADC2, MH-ADC4, MH-ADC6, MH-ADC8, the Ab is linked to the e position and / or the f position via its thiol group.

[0111] In some embodiments, in MH-ADC2, MH-ADC4, MH-ADC6, MH-ADC8, one thiol group on the Ab is linked to the e position.

[0112] In some embodiments, in MH-ADC2, MH-ADC4, MH-ADC6, MH-ADC8, one thiol group on the Ab is linked to the f position.

[0113] In some embodiments, in MH-ADC2, MH-ADC4, MH-ADC6, MH-ADC8, one or more thiol groups on the Ab are linked to the e position of one or more linkers, and another or further thiol group(s) are linked to the f position of another or further linker(s).

[0114] In some embodiments, in MH-ADC2, MH-ADC4, MH-ADC6, MH-ADC8, one or more thiol groups on the Ab are linked to the e position, and another or further thiol group(s) are linked to the f position.

[0115] In some embodiments, in MH-ADC2, MH-ADC4, MH-ADC6, MH-ADC8, all thiol groups on the Ab are linked to the e position.

[0116] In some embodiments, in MH-ADC2, MH-ADC4, MH-ADC6, MH-ADC8, all thiol groups on the Ab are linked to the f position.

[0117] A second aspect of the present application provides a pharmaceutical composition comprising the antibody drug conjugate according to the first aspect of the present application, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and optionally one or more pharmaceutically acceptable excipients.

[0118] The third aspect of the present application provides a composition comprising at least one antibody drug conjugate according to the first aspect of the present application, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[0119] In some embodiments, the composition comprises antibody drug conjugate MH-ADC1, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and antibody drug conjugate MH-ADC2, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[0120] In some embodiments, the composition comprises antibody drug conjugate MH-ADC3, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and antibody drug conjugate MH-ADC4, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[0121] In some embodiments, the composition comprises antibody drug conjugate MH-ADC5, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and antibody drug conjugate MH-ADC6, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[0122] In some embodiments, the composition comprises antibody drug conjugate MH-ADC7, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and antibody drug conjugate MH-ADC8, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[0123] The fourth aspect of the present application provides use of the antibody drug conjugate according to the first aspect of the present application, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the pharmaceutical composition according to the second aspect of the present application, or the composition according to the third aspect of the present application in the manufacture of a medicament for acting on B7-H4 target.

[0124] The fifth aspect of the present application provides use of the antibody drug conjugate according to the first aspect of the present application, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the pharmaceutical composition according to the second aspect of the present application, or the composition according to the third aspect of the present application in the manufacture of a medicament for treating and / or preventing a disease.

[0125] In some embodiments, the disease is a B7-H4 related disease.

[0126] In some embodiments, the disease is a disease associated with abnormal expression of B7-H4.

[0127] In some embodiments, the disease is a cancer or an autoimmune disease.

[0128] In some embodiments, the disease is selected from breast cancer, ovarian cancer, endometrial cancer, and cholangiocarcinoma.

[0129] Definitions of terms

[0130] In the present application, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art, unless otherwise indicated. Also, for better understanding of the present application, the definitions and explanations of relevant terms are provided below.

[0131] In the present application, the term "and / or" should be considered as a specific disclosure of each of the two or more stated features or elements, with or without the other features or elements. Thus, the term "and / or" used in a phrase such as "A and / or B" herein is intended to encompass the "A and B", "A or B", "A" (alone), and "B" (alone).

[0132] It should be understood that wherever aspects are described herein with the language "comprising" or "including" it is also contemplated that "consisting essentially of" and / or "consisting of" aspects are also provided.

[0133] The stereoisomers in the compounds described in the present application, when specifically designated as (R)- or (S)-isomer in the chemical name, should be understood as the major configuration of (R)-isomer or (S)-isomer, respectively. Any asymmetric carbon atom can exist in the (R)-, (S)-, or (R, S)-configuration, preferably in the (R)- or (S)-configuration.

[0134] In the present application, the term "prodrug" refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide a compound of the present application. Prodrugs are inactive until they are converted to active compounds under biological conditions, or they have no or only lower activity in their non-reacted form. Prodrugs can generally be prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, ed., 5th ed.).

[0135] In the present application, the term "pharmaceutically acceptable salt" refers to (i) salts of acidic functions present in the compounds provided in the present application with appropriate inorganic or organic cations (bases) and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, and the like; alkaline earth metal salts, such as calcium salts, magnesium salts, and the like; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, and the like; inorganic base salts, such as ammonium salts; organic base salts, such as tertiary octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts, and (ii) salts of basic functions present in the compounds provided in the present application with appropriate inorganic or organic anions (acids) and includes, but is not limited to, hydrogen halide salts, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, and the like; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, and the like; lower alkylsulfonic acid salts, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, and the like; arylsulfonic acid salts, such as benzenesulfonates, p-toluenesulfonates, and the like; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, and the like; amino acid salts, such as glycine salts, trimethylglycine salts, arginine salts, ornithine salts, glutamic acid salts, aspartic acid salts, and the like.

[0136] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of the basic compound with a suitable acid affording a pharmaceutically acceptable anion, or by reacting a sufficient amount of the acidic compound with a suitable base affording a pharmaceutically acceptable cation.

[0137] In the present application, "solvate" or "solvate" are used interchangeably to refer to a compound that exists in combination with one or more solvent molecules. The combination can include stoichiometric amounts of solvent molecules, for example, one or two molecules of water for hydrates, or it can include any amount of solvent molecules, for example, methanol or ethanol can form "alcohols" which can also be stoichiometric or non-stoichiometric. The term "solvate" as used herein refers to a solid form, i.e., a compound in solution in a solvent, although it can be solvated, is not a solvate as the term is used herein.

[0138] In the present application, the phrases "each independently selected from" and "each is independently selected from" are used interchangeably throughout the present application and are to be interpreted in their broadest context unless otherwise explicitly indicated herein. They mean that the recited options for the same or different symbols in different groups are independent of each other.

[0139] In various portions of the present application, substituents of the compounds of the present application are presented according to group type or range. It is specifically intended that the present application include each and every independent combination of subgroups from a particular group type or range.

[0140] In the present application, the term "alkyl" refers to a straight or branched chain monovalent saturated hydrocarbon group, for example, C1-C6 alkyl refers to having 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms; C1-C4 alkyl refers to having 1 to 4 carbon atoms, such as 1, 2, 3, or 4 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, and the like.

[0141] In the present application, the term "alkylene" refers to a straight or branched chain divalent saturated hydrocarbon group, for example, C1-C4 alkylene refers to having 1 to 4 carbon atoms, such as 1, 2, 3, or 4 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, and the like.

[0142] In the present application, the term "cycloalkyl" refers to a monovalent saturated hydrocarbon group consisting of carbon atoms, for example, C3-C8 cycloalkyl refers to consisting of 3-8 (e.g., 3, 4, 5, 6, 7, or 8) carbon atoms, including C3-C6 cycloalkyl, C3 cycloalkyl, C4 cycloalkyl, C5 cycloalkyl, C6 cycloalkyl, and the like. The cycloalkyl groups include monocyclic, bicyclic, or polycyclic rings, including spiro, fused, or bridged rings. Non-limiting examples include, but are not limited to, cyclobutyl, cyclopentyl, or cyclohexyl, and the like.

[0143] In the present application, the term "heterocyclyl" refers to a saturated or partially unsaturated cyclic group consisting of ring atoms, wherein 1, 2, 3, or 4 of the ring atoms are heteroatoms, and the remainder are carbon atoms; preferably the heteroatoms are selected from N, O, or S, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized; and preferably the carbon atoms are optionally substituted with =0. For example, 3-8 membered heterocyclyl refers to consisting of 3-8 (e.g., 3, 4, 5, 6, 7, or 8) ring atoms, 5-6 membered heterocyclyl refers to consisting of 5 or 6 ring atoms, including 3-7 membered heterocyclyl, 4-7 membered heterocyclyl, 5-6 membered heterocyclyl, and the like. Non-limiting examples include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, and the like.

[0144] In the present application, the term "aryl" refers to an unsaturated carbocyclic group having a conjugated pi-electron system, for example, C6-Ci0aryl. Non-limiting examples include, but are not limited to, phenyl, naphthyl. 10 An aryl group consists of 6 to 10 carbon atoms. Non-limiting examples include, but are not limited to, phenyl, naphthyl.

[0145] In the present application, the term "heteroaryl" refers to an unsaturated group having a conjugated pi-electron system consisting of ring atoms, of which 1, 2, 3, or 4 are heteroatoms, the rest being carbon atoms; preferably the heteroatoms are selected from N, O, or S, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized. For example, a 5-9 membered heteroaryl group consists of 5 to 9 (e.g., 5, 6, 7, 8, or 9) ring atoms, including 5-6 membered heteroaryl groups, and the like. The heteroaryl groups include monocyclic and polycyclic rings, examples of which include, but are not limited to, imidazolyl, pyridinyl, quinolinyl, or isoquinolinyl, and the like.

[0146] In the present application, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0147] In the present application, the term "halo" refers to a group that is modified by one or more halogens, for example, 1, 2, 3, 4, 5, or 6 halogens. For example, "haloalkyl" refers to a group in which any of the aforementioned alkyl groups (e.g., Ci-C6alkyl, Ci-C4alkyl, and the like) has one or more hydrogen atoms replaced by a halogen, non-limiting examples of which include, but are not limited to, CF3, CHF2, or CF2CF3, and the like.

[0148] In the present application, the term "deutero" refers to a group that is modified by one or more deuterium, for example, 1, 2, 3, 4, 5, or 6 deuterium. For example, "deuteroalkyl" refers to a group in which any of the aforementioned alkyl groups (e.g., Ci-C6alkyl, Ci-C4alkyl, and the like) has one or more hydrogen atoms replaced by a deuterium atom, for example, monodeuteromethyl, dideuteromethyl, trideuteromethyl, and the like. For example, "deutero cycloalkyl" refers to a group in which any of the aforementioned cycloalkyl groups (e.g., C3-C8cycloalkyl, C3-C6cycloalkyl, and the like) has one or more hydrogen atoms replaced by a deuterium atom, for example, monodeutero cyclopropyl, dideutero cyclopropyl, trideutero cyclopropyl, and the like.

[0149] In the present application, the term "amino acid residue" refers to the incomplete amino acid structure that remains after the amino group of an amino acid loses one hydrogen and the carboxyl group loses one hydroxyl.

[0150] In the present application, the term "antibody" is interpreted in its broadest context to include intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies formed from at least two intact antibodies, e.g., bispecific antibodies, as long as they exhibit the desired biological activity. In the present application, "antibody" and "immunoglobulin" are used interchangeably.

[0151] The term "monoclonal antibody" as used herein refers to antibodies that are derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single determinant (epitope) of an antigen, in contrast to polyclonal antibodies which typically include different antibodies directed against different determinants (epitopes). In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a single population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0152] In certain embodiments of the application, the monoclonal antibodies also specifically include chimeric antibodies, in which a portion of the heavy and / or light chain is identical with or homologous to a corresponding portion of some, but not all of the antibodies, and the remainder of the chain is identical with or homologous to a corresponding portion of some other, but not all of the antibodies, so long as the antibodies exhibit the desired biological activity (see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., 1984, PNAS, 81 :6851-6855). Chimeric antibodies of the application include primatized antibodies comprising variable region antigen binding sequences from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences.

[0153] The monoclonal antibodies of the application can be produced by a variety of methods. For example, the monoclonal antibodies used in the application can be obtained by the hybridoma method using a number of species, including mouse, hamster, rat and human cells (see, e.g., Kohler et al., 1975, Nature, 256:495), or produced by recombinant DNA techniques (see, e.g., U.S. Patent No. 4,816,567), or isolated from phage antibody libraries (see, e.g., Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:581-597).

[0154] In the present application, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these regions can be defined according to various numbering systems known in the art, for example, as defined by the numbering system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or IMGT (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill in the art will readily be able to identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well-known to those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0155] In the present application, the term "antigen-binding fragment" of an antibody refers to a polypeptide that comprises a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some instances, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.

[0156] In the present application, the term "Fd fragment" means an antibody fragment consisting of a VH and CHI domain; the term "Fv fragment" means an antibody fragment consisting of a VL and VH domain of a single arm of an antibody; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341 :544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CHI domain; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments connected by a disulfide bridge over the hinge region; the term "Fab' fragment" means one of the two Fab' fragments formed by reducing a F(ab')2 fragment by breaking the disulfide bond over the F(ab')2 hinge region.

[0157] In some cases, an antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which a VL and VH domain are paired by a linker that enables it to be produced as a single polypeptide chain, forming a monovalent molecule (see, e.g., Bird et al., Science 242:423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 5883 (1988)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS (SEQ ID NO: 13) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4(SEQ ID NO: 14) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present application are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31 :94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0158] In the present application, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the essential properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue for an amino acid residue with similar side chains, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a preference is given to substituting the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0159] As understood by one skilled in the art, in the antibody drug conjugates of the present application, the antibody is linked to the linker-payload through -S- and -S- is not an additional externally attached thiol, but rather the thiol contained in the antibody itself after the antibody has been reduced and disulfide bonds have been opened.

[0160] In the present application, the drug antibody ratio (DAR) refers to the number of drug molecules (e.g., a in Formula I) conjugated to the antibody. The number of drug molecules contained in the antibody drug conjugates described herein can be an integer or a fraction. Whether it is an integer or a fraction, it refers to the average number of drug molecules conjugated per antibody. "a is any number between 1 and 10" means that a can be any integer selected from 1 to 10 (including endpoints 1 and 10), or any fraction selected from 1 to 10, such as 3.9 or 4.0. At the same time, those skilled in the art can understand that even if the same preparation method is used, the DAR values of antibody drug conjugates prepared in different batches are not necessarily exactly the same, for example, they can fluctuate within a range of not more than 0.5 up and down.

[0161] In the present application, the term "about" can be understood as within + / - 10%, + / - 9%, + / - 8%, + / - 7%, + / - 6%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, + / - 1%, + / - 0.5%, + / - 0.4%, + / - 0.3%, + / - 0.2%, + / - 0.1% of the value. Unless otherwise apparent from the context, all numerical values provided herein are modified by the term "about".

[0162] In the present application, the pharmaceutical excipient refers to the excipients and additional agents used in the production of pharmaceuticals and the dispensing of prescriptions, which refers to substances other than active ingredients that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations. In addition to excipients, acting as carriers, improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, controlled release, etc., which are important ingredients that may affect the quality, safety and effectiveness of pharmaceuticals. According to its source, it can be divided into natural, semi-synthetic and synthetic. According to its function and use, it can be divided into: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, humectants, osmotic pressure regulators, stabilizers, flow aids, flavorings, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculants, filter aids, release retardants, etc. According to its administration route, it can be divided into oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations for different administration routes, and has different functions and uses.

[0163] In the present application, the pharmaceutical composition can be prepared into various suitable dosage forms according to the administration route. For example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, eye preparations, pills, implants, aerosols, powder sprays, spray preparations, etc. Among them, the pharmaceutical composition or suitable dosage form can contain 0.01 mg to 1000 mg of the antibody drug conjugate of the present application or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate.

[0164] In the present application, the term "treatment" aims to alleviate, reduce, improve or eliminate the disease state or condition targeted. A subject is successfully "treated" if the subject exhibits an observable and / or detectable reduction or improvement in one or more indicators and symptoms, following administration of a therapeutic amount of the ligand conjugate drug or its racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, or mixture of the foregoing, according to the methods described herein. It should also be understood that the treatment of the disease state or condition includes not only complete treatment, but also less than complete treatment, but achieving some biologically or medically relevant result.

[0165] In the present application, the term "prevention" aims to avoid, reduce, prevent or delay the occurrence of a disease or disease-related symptoms, and such disease or disease-related symptoms have not yet occurred before the administration of the relevant drug. "Prevention" does not require complete prevention of the occurrence of a disease or disease-related symptoms, for example, reducing the risk of a subject developing a particular disease or disease-related symptoms after administration of the relevant drug, or reducing the severity of the relevant symptoms that occur later, can also be considered as "preventing" the occurrence or development of the disease. Beneficial effects

[0166] 1) The antibody-drug conjugate of the present application has a binding effect on tumor cell lines expressing B7H4, especially excellent binding activity on tumor cell lines with high expression of B7H4;

[0167] 2) The antibody-drug conjugate of the present application exhibits endocytosis activity in tumor cell lines expressing B7H4, especially excellent endocytosis activity in tumor cell lines with high expression of B7H4;

[0168] 3) The antibody-drug conjugate of the present application has excellent proliferation inhibition activity on tumor cell lines expressing B7H4, especially overexpressing B7H4;

[0169] 4) The antibody-drug conjugate of the present application has a target-dependent killing effect;

[0170] 5) The antibody-drug conjugate of the present application has excellent in vivo proliferation inhibition effect;

[0171] 6) The antibody-drug conjugate of the present application has excellent pharmacokinetic properties;

[0172] 7) The antibody-drug conjugate of the present application has good plasma stability. BRIEF DESCRIPTION OF DRAWINGS

[0173] Figure 1. Binding curves of antibody and antibody-drug conjugate to human breast cancer tumor cell line MX-1.

[0174] Figure 2. Endocytosis activity of antibody and antibody-drug conjugate in human breast cancer tumor cell line MX-1.

[0175] Figure 3. Anti-tumor effect of MH-ADC2 in MX-1 nude mouse subcutaneous tumor model.

[0176] Figure 4. Total antibody and ADC detection results of MH-ADC2 in serum after single intravenous administration in rats.

[0177] Figure 5. Total antibody and ADC detection results of MH-ADC2 in serum after single intravenous administration in cynomolgus monkeys.

[0178] SEQUENCE INFORMATION

[0179] The sequence information involved in the present application is shown in Table 1 below.

[0180] Table 1 DETAILED DESCRIPTION

[0181] The present application is further described below by way of specific embodiments, but this is not a limitation on the present application. Those skilled in the art can make various modifications or improvements according to the teachings of the present application without departing from the basic idea and scope of the present application. The reagents or instruments used are not specified by the manufacturer, but are conventional products that can be obtained commercially.

[0182] In the following examples, the abbreviations of some drugs and their corresponding structural formulas are as follows:

[0183] Deruxtecan:

[0184] Among them, Deruxtecan (i.e. MC-GGFG-DXd) can be prepared according to CN104755494A Example 58.

[0185] In the following examples, the 2A7-H1 antibody used is humanized to the VH of the 2A7 antibody in patent WO2007067991A2 (1 amino acid mutation: V67F) and the Fc is mutated (L234A, L235A). The heavy chain sequence of the 2A7-H1 antibody (SEQ ID NO: 10), the light chain sequence (SEQ ID NO: 11), the heavy chain variable region sequence (SEQ ID NO: 8), the light chain variable region sequence (SEQ ID NO: 9), the heavy chain CDR1 sequence (SEQ ID NO: 1), the heavy chain CDR2 sequence (SEQ ID NO: 2), the heavy chain CDR3 sequence (SEQ ID NO: 3), the light chain CDR sequence (SEQ ID NO: 4), the light chain CDR2 sequence (SEQ ID NO: 5) and the light chain CDR3 sequence (SEQ ID NO: 6) are shown in Table 1. The CDR region sequence is defined using the Kabat numbering system, and any other CDR region sequence determination method known in the art can be used to identify the amino acid residues in the CDR region of the variable region.

[0186] Example 1. Preparation of linker-drugs

[0187] Example 1.1

[0188] First step

[0189] Dissolve 1a (10.00 g, 95.20 mmol) in acetonitrile (100 mL), add tert-butyl dimethylchlorosilane (15.06 g, 99.92 mmol), cool the mixture to 0 °C, add 1,8-diazobicyclo[5.4.0]undec-7-ene (13.73 g, 90.21 mmol) dropwise, after the addition is complete, restore the reaction system to room temperature and stir for 16 h. Solid precipitates, filter the reaction system directly, and collect the filter cake to obtain 1b (11.50 g), yield: 55%.

[0190] MS-ESI calculated value [M+H] + = 220, found 220.

[0191] Second step

[0192] Add 1b (5.55 g, 25.33 mmol), acetone (55.5 mL), and maleic anhydride (2.48 g, 25.33 mmol) sequentially to the reaction flask, and stir at room temperature for 2 h. The reaction solution was directly concentrated to obtain a yellow oil (8.25 g). Dissolve the yellow oil in toluene (82 mL), add triethylamine (5.12 g, 50.66 mmol), and reflux the mixture at 120 °C for 2 h. Concentrate the reaction solution directly under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 1c (1.53 g), yield: 33%.

[0193] MS-ESI calculated value [MH] - =184, the actual measured value is 184.

[0194] 1 H NMR (400MHz, DMSO-d6) δ7.00 (s, 2H), 6.05 (s, 1H), 4.30 (dd, J = 9.6, 5.6Hz, 1H), 3.98 (dd, J = 10.8, 5.6Hz, 1H), 3.84 (dd, J = 10.8, 10.8Hz, 1H).

[0195] Step 3

[0196] 1d (10.00 g, 13.59 mmol) was dissolved in THF (450 mL), and wet Pd / C (2 g, 20% w / w) was added. The mixture was stirred for 66 h under a hydrogen atmosphere. The reaction solution was filtered, and the filter cake was washed with a DCM / MeOH mixed solvent. The filtrates were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 1e (6.10 g), yield: 69%.

[0197] MS-ESI calculated value [M+H] + =668, the actual measured value is 668.

[0198] Step 4

[0199] Under a nitrogen atmosphere, 1f (2.70 g, 5.97 mmol) and trifluoroacetic acid (680 mg, 5.97 mmol) were dissolved in N,N-dimethylformamide (54 mL), and stirred in an ice bath for 10 min. Then, 1e (5.40 g, 8.36 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.30 g, 11.94 mmol), and 2,4,6-trimethylpyridine (722 mg, 5.97 mmol) were added to the reaction solution, and the reaction was continued at 0℃–10℃ with stirring for 2 h. HCl was then added dropwise to the reaction solution in an ice bath. aq(0.05N, 54 mL), solid precipitated, the suspension was diluted with 2-methyltetrahydrofuran (100 mL), stirred until dissolved, separated, the aqueous phase was extracted with 2-methyltetrahydrofuran (100 mL x 2), the combined organic phase was washed with saturated brine (50 mL x 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (MeOH:DCM = 0-100%) to give 1 g (6.00 g), yield: 84%.

[0200] MS-ESI calculated value [M+H] + = 1079, measured value 1079.

[0201] Fifth step

[0202] 1 g (6.00 g, 5.56 mmol) was dissolved in a mixed solvent of DCM / MeOH (120 mL / 12 mL), diethylamine (24 mL) was added dropwise under ice bath, after the addition was completed, the natural temperature was increased to room temperature, and the reaction was stirred for 4 h. The reaction solution was directly concentrated to give a brown solid crude product, methyl tert-butyl ether was added to pulp, filtered, the filter cake was washed with methyl tert-butyl ether for 3 times, and the filtrate was concentrated to give a yellow solid crude product. The crude product (1 g) was purified by preparative liquid chromatography to give 1h (150 mg), and the remaining crude product was purified by silica gel column chromatography (MeOH:DCM = 0-100%) to give 1h (800 mg), combined yield: 59%.

[0203] MS-ESI calculated value [M+H] + = 857, measured value 857.

[0204] Sixth step

[0205] 1c (65 mg, 0.234 mmol), 1h (0.20 g, 0.23 mmol) and N,N-dimethylformamide (4 mL) were sequentially added to a reaction bottle, stirred to dissolve, placed in an ice bath, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.13 g, 0.35 mmol) and 2,4,6-trimethylpyridine (85 mg, 0.70 mmol) were added, and the mixed system was stirred at 0-5°C for 1 h. The reaction solution was adjusted to pH = 4-5 with HCl aq (0.5N), and purified by preparative liquid chromatography to give compound 1 (70 mg), yield: 14%.

[0206] MS-ESI calculated value [M+H] + = 1024, measured value 1024.

[0207] 1H NMR (400MHz, DMSO-d6) δ8.65(t,J=6.8Hz,1H),8.60(d,J=8.8Hz,1H),8.29(q,J= 5.6Hz,2H),8.09(d,J=8.0Hz,1H),7.99(t,J=5.6Hz,1H),7.81(d,J=11.2Hz,1H), 7.80(s,1H),7.29-7.14(m,5H),7.04(s,2H),6.68(s,1H),5.91(d,J=16.8Hz,1H) ,5.71-5.60(m,1H),5.56-5.45(m,2H),5.36(d,J=19.6Hz,1H),5.05(t,J=5.6Hz, 1H),4.68(d,J=6.4Hz,2H),4.59(dd,J=9.2,6.0Hz,1H),4.52-4.43(m,1H),4.17 -4.04(m,2H),4.00-3.92(m,1H),3.98-3.80(m,1H),3.78-3.68(m,5H),3.64-3.5 4(m,2H),3.20-3.09(m,1H),3.00(dd,J=14.0,4.4Hz,1H),2.73(dd,J=13.6,9.6H z,1H),2.39(s,3H),2.26-2.17(m,2H),1.94-1.83(m,2H),0.86(t,J=7.2Hz,3H).

[0208] Example 1.2

[0209] first step

[0210] Compounds 2b (342 mg, 0.81 mmol) and 2a (500 mg, 0.85 mmol) were dissolved in a mixed solvent of acetonitrile (3.42 mL) and water (6.84 mL). The mixture was cooled to 0 °C, and N,N-diisopropylethylamine (83 mg, 0.64 mmol) was added dropwise with stirring. After the addition was complete, the reaction mixture was stirred for 3 h. The reaction solution was directly purified by preparative chromatography to give 2c (283 mg), yield: 39%.

[0211] MS-ESI calculated value [M+Na] + =1020, the actual measured value is 1020.

[0212] Step 2

[0213] Compound 2c (85 mg, 0.19 mmol) was dissolved in N,N-dimethylformamide (1.70 mL), trifluoroacetic acid (22 mg, 0.19 mmol) was added, the mixture was cooled to 0 °C, 1f (226 mg, 0.23 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (72 mg, 0.38 mmol) and 2,4,6-trimethylpyridine (23 mg, 0.19 mmol) were added in turn, after the addition was completed, the reaction system was kept at 0 °C-10 °C and stirred for 1 h. The reaction solution was adjusted to pH 6-7 with HCl (0.05 M) and purified by preparative liquid phase to obtain compound 2 (134 mg) with a yield of 49%. (aq) (0.05M) to pH 6-7 and purified by preparative liquid phase to obtain compound 2 (134 mg) with a yield of 49%.

[0214] 1 H NMR (400 MHz, DMSO-d6) δ 8.66 (t, J = 6.4 Hz, 1H), 8.60 (d, J = 8.8 Hz, 1H), 8.28 (t, J = 6.0 Hz, 1H), 8.15 (t, J = 5.6 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 8.04-7.95 (m, 2H), 7.84-7.77 (m, 2H), 7.28-7.14 (m, 5H), 6.99 (s, 2H), 6.69 (s, 1H), 5.91 (d, J = 16.8 Hz, 1H), 5.70-5.62 (m, 1H), 5.56-5.45 (m, 2H), 5.35 (d, J = 18.8 Hz, 1H), 4.68 (d, J = 6.8 Hz, 2H), 4.51-4.42 (m, 1H), 4.17-4.04 (m, 2H), 3.80-3.55 (m, 12H), 3.55-3.42 (m, 32H), 3.17-3.10 (m, 2H), 3.06-2.98 (m, 1H), 2.80-2.71 (m, 1H), 2.41-2.28 (m, 7H), 2.27-2.15 (m, 2H), 1.94-1.82 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H).

[0215] Example 1.3

[0216] First step

[0217] Dissolve 3a (14.72 g, 150.16 mmol) in acetone (118 mL), add 3b (20.00 g, 150.16 mmol) under ice bath, stir the mixture for 5 min, then monitor the reaction by TLC until it is complete. Concentrate the reaction mixture to get a solid. Dissolve the solid in acetic anhydride (28 mL), add sodium acetate (24.63 g, 300.32 mmol), and heat the reaction mixture to 90 °C for 2 h. Filter the reaction mixture to remove the insoluble solid, rinse the filter cake with toluene, and concentrate the filtrate under reduced pressure to get the crude product. Purify the crude product by silica gel column chromatography (EA: Hexanes = 0-100%) to get 3c (17.43 g) in 43% yield.

[0218] MS-ESI calculated [M+Na] = 236, found 236. +

[0219] Second Step

[0220] Dissolve 3c (12.95 g, 60.74 mmol) and 3d (9.00 g, 60.74 mmol) in toluene (180 mL), add p-toluenesulfonic acid (2.10 g, 12.15 mmol), and heat the mixture to 90 °C for 2 h. Cool the reaction mixture to room temperature, concentrate under reduced pressure to get a residue, dissolve the residue in ethyl acetate, wash the organic phase sequentially with saturated sodium bicarbonate and saturated brine (200 mL x 1), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to get the crude product. Purify the crude product by silica gel column chromatography (EA: Hexanes = 0-100%) to get 3e (5.53 g) in 33% yield.

[0221] MS-ESI calculated [M+H] = 270, found 270. +

[0222] Third Step

[0223] Dissolve 3e (3.87 g, 14.37 mmol) in THF (77.4 mL), dissolve lithium hydroxide (1.37 g, 57.49 mmol) in H2O (38.7 mL), and add the solution to the above solution. Stir the mixture at room temperature for 30 min. Add ethyl acetate (15 mL) to the reaction mixture, adjust the pH to about 2 with HCl (1N), extract the aqueous phase with ethyl acetate (38 mL x 3), dry the combined organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to get the crude product 3f (3.06 g). (aq)

[0224] MS-ESI calculated [M+H] = 260, found 260. + ​​​​

[0225] Step 4

[0226] N-hydroxysuccinimide (6.49 g, 56.37 mmol) was dissolved in N,N-dimethylformamide (36.5 mL). Trifluoroacetic anhydride (11.84 g, 56.37 mmol) was added dropwise under ice bath conditions. After stirring for 30 min, 2,4,6-trimethylpyridine (6.83 g, 56.37 mmol) was added dropwise. After the addition was complete, the mixture was stirred for another 40 min. This reaction solution was designated as A and kept for later use. Crude product 3f (3.06 g) was dissolved in N,N-dimethylformamide (36.5 mL). 2,4,6-trimethylpyridine (3.41 g, 28.15 mmol) was added dropwise under ice bath conditions. After stirring for 30 min under ice bath conditions, the above reaction solution A was added dropwise. The reaction system was allowed to warm naturally to room temperature and stirred for 24 h. Dichloromethane (180 mL) and HCl were added to the reaction solution. (aq) After stirring for 30 min at 0.7 N, 140 mL, the mixture was separated. The aqueous phase was extracted with dichloromethane (70 mL), and the organic phases were combined. The organic phase was washed with water to pH 5-7, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated by silica gel column chromatography (EA: Hexanes = 0-100%) to obtain 3 g (3.54 g), yield: 78%.

[0227] MS-ESI calculated value [M+H] + =339, the actual measured value is 339.

[0228] 1 H NMR (400MHz, CDCl3) δ6.77 (s, 2H), 4.79 (t, J = 4.8Hz, 1H), 4.49-4.40 (m, 2H) ,3.91(t,J=11.6Hz,2H),3.79-3.70(m,2H),3.45-3.34(m,1H),2.86(s,4H).

[0229] Step 5

[0230] 3 h (850 mg, 3.38 mmol) and 3 g (1.20 g, 3.55 mmol) were dissolved in N,N-dimethylformamide (8.5 mL). N,N-diisopropylethylamine (437 mg, 3.38 mmol) was added dropwise under ice bath conditions. The mixture was stirred and reacted at 0–10 °C for 2 h. The reaction solution was purified by preparative liquid chromatography to obtain 3i (740 mg), yield: 46%.

[0231] MS-ESI calculated value [M+H] + =475, the actual measured value is 475.

[0232] Step 6

[0233] Dissolve 3i (74 mg, 0.16 mmol) in N,N-dimethylformamide (1.30 mL), purge with nitrogen three times, and add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (88 mg, 0.23 mmol) under ice bath conditions. After stirring for 10 min, add 1h (132 mg, 0.16 mmol) and stir until dissolved. Then add 2,4,6-trimethylpyridine (54 mg, 0.45 mmol) and continue the reaction at 0℃–10℃ for 2 h. The reaction solution is then rinsed with HCl. (aq) (0.05N) The pH was adjusted to 6-7, and compound 3 (71 mg) was obtained by liquid phase separation and purification, yield: 34%.

[0234] MS-ESI calculated value [M+H] + =1313, the actual measured value is 1313.

[0235] Example 1.4

[0236] first step

[0237] Under a nitrogen atmosphere, 4a (5.00 g, 26.74 mmol) was dissolved in N,N-dimethylformamide (50 mL). After cooling to 0–5 °C, NaH (1.28 g, 32.09 mmol) was added, and the mixture was stirred for 10 min. Then, tert-butyl bromoacetate (6.23 g, 32.09 mmol) was added, and the mixture was stirred at 0–5 °C for 2 h. Water (200 mL) was added to the reaction solution, and the aqueous phase was extracted with ethyl acetate (200 mL). The organic phase was washed with saturated brine (200 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 4b (4.60 g), yield: 43%.

[0238] MS-ESI calculated value [M+H] + =302,304, the actual measured value is 302,304.

[0239] 1 H NMR (400MHz, CDCl3) δ8.60(d,J=2.0Hz,1H),7.84(dd,J=8.4,2.4Hz,1H),7.45(d,J=8.4Hz,1H),4.69(s,2H),4.10(s,2H),1.49(s,9H).

[0240] Step 2

[0241] Under nitrogen atmosphere, 4b (5.00 g, 16.60 mmol), 4c (3.61 g, 19.93 mmol), Pd2(dba)3(0.76 g, 0.83 mmol), BINAP (1.03 g, 1.66 mmol), cesium carbonate (13.53 g, 41.52 mmol) were dissolved in anhydrous toluene (50 mL), the mixture was heated to 80 °C and stirred for 16 h. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated by silica gel column chromatography (EA:hexanes = 0-100%) to obtain 4d (4.50 g), with a yield of 67%.

[0242] MS-ESI calculated value [M+H] + = 403, measured value 403.

[0243] Third step

[0244] 4d (2.81 g, 6.98 mmol) was dissolved in a mixed solvent of THF (28 mL) and HCl (aq) (1 N, 28 mL), and the mixture was stirred at room temperature for 3 h. Water (100 mL) was added to the reaction solution, and the aqueous phase was washed with ethyl acetate (100 mL x 2) and discarded. The aqueous phase was adjusted to pH = 8-9 with ammonia water, and then extracted with ethyl acetate (100 mL x 2). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 4e (1.43 g), with a yield of 86%.

[0245] MS-ESI calculated value [M+H] + = 239, measured value 239.

[0246] Fourth step

[0247] 4e (1.83 g, 7.68 mmol) and maleic anhydride (0.75 g, 7.68 mmol) were dissolved in acetonitrile (18.3 mL), and the mixture was stirred at room temperature for 2 h. The reaction solution was directly rotary evaporated to obtain 2.45 g of a white solid intermediate. The 2.45 g of the white solid intermediate was added to a reaction bottle, and acetic anhydride (5 mL), sodium acetate (2.09 g, 15.36 mmol) were sequentially added. The mixture was stirred at room temperature for 2 h. Water (50 mL) was added to the reaction solution, and the aqueous phase was extracted with ethyl acetate (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was separated by silica gel column chromatography (EA:hexanes = 0-100%) to obtain 4f (1.97 g), with a yield of 80%.

[0248] MS-ESI calculated value [M+H] + = 319, measured value 319.

[0249] 1H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J = 2.0 Hz, 1H), 7.83 (dd, J = 8.4, 2.4 Hz, 1H), 7.6 (d, J = 8.4 Hz, 1H), 7.24 (s, 2H), 4.67 (s, 2H), 4.15 (s, 2H), 1.44 (s, 9H).

[0250] Fifth step

[0251] Dissolve 4f (0.90 g, 2.83 mmol) in dichloromethane (9 mL), add trifluoroacetic acid (1.8 mL), stir the reaction at room temperature for 5 h. Directly spin dry the reaction, evaporate with dichloromethane (45 mL x 5), concentrate with oil pump to no obvious oil, get the crude product 4g (0.93 g).

[0252] MS-ESI calculated value [M+H] + = 263, measured value 263.

[0253] Sixth step

[0254] Into the reaction bottle, add 4g (0.06 g), 1h (0.20 g, 0.23 mmol) and N, N- dimethylformamide (4 mL) in turn, after stirring and dissolving, place in ice bath, add 2-(7- azabenzotriazol)-N, N, N', N'-tetramethyluronium hexafluorophosphate (0.13 g, 0.35 mmol) and 2, 4, 6-trimethylpyridine (0.26 g, 2.15 mmol), keep the mixed system stirring at 0-5 ℃ for 1 h. Adjust the pH of the reaction to 4-5 with HCl (aq) (0.5N) and send to the preparation of neutral aqueous system in preparative liquid phase to separate and get compound 4 (70 mg), yield: 27%.

[0255] MS-ESI calculated value [M+H] + = 1101, measured value 1101.

[0256] 1H NMR (400 MHz, DMSO-d6) δ 8.65 (t, J = 6.8 Hz, 1H), 8.60 (d, J = 8.8 Hz, 1H), 8.34 - 8.24 (m, 2H), 8.09 (d, J = 8.0 Hz, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.81 (d, J = 11.2 Hz, 1H), 7.79 (s, 1H), 7.30 - 7.14 (m, 5H), 7.04 (s, 2H), 6.68 (s, 1H), 5.91 (d, J = 16.8 Hz, 1H), 5.70 - 5.61 (m, 1H), 5.56 - 5.45 (m, 2H), 5.36 (d, J = 19.6 Hz, 1H), 5.05 (t, J = 6.0 Hz, 1H), 4.68 (d, J = 6.4 Hz, 2H), 4.59 (dd, J = 9.2, 6.0 Hz, 1H), 4.52 - 4.42 (m, 1H), 4.17 - 4.05 (m, 2H), 4.00 - 3.92 (m, 1H), 3.89 - 3.79 (m, 1H), 3.78 - 3.68 (m, 5H), 3.64 - 3.54 (m, 2H), 3.01 (dd, J = 8.8, 4.4 Hz, 1H), 2.74 (dd, J = 13.6, 9.6 Hz, 1H), 2.39 (s, 3H), 2.26 - 2.18 (m, 2H), 1.94 - 1.82 (m, 2H), 0.86 (t, J = 7.6 Hz, 3H).

[0257] Example 2. Preparation of antibody-drug conjugates

[0258] 1. Preparation of MH-ADC1

[0259] MH-ADC1

[0260] The 2A7-H1 antibody was dialyzed into 20 mM His / His-HCl pH 6.0 ± 0.2 buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, and 10 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) stock solution, 10 mM diethylene triamine pentaacetic acid (DTPA) stock solution were added in sequence, and 20 mM His / His-HCl pH 6.0 ± 0.2 buffer was added to make the final concentration of the antibody in the reaction system 20 mg / mL, the molar ratio of TCEP to antibody (1.5-3.2): 1.0, and the final concentration of DTPA 1 mM. After mixing well, the reaction was carried out in a constant temperature mixer at 20-30 °C and 400 rpm for 1.0-3.0 hours. After reduction, an appropriate amount of 5 mM linker-drug (compound 1) stock solution was added to each reaction system, and the molar ratio of linker-drug to antibody was 3.5-8.0. After mixing well, the coupling reaction was carried out in a constant temperature mixer at 20-30 °C and 400 rpm for 0.5-2.0 hours to obtain MH-ADC1, wherein Ab is the 2A7-H1 antibody. After the coupling reaction was completed, the ADC sample was dialyzed into dialysis buffer (10 mM His / His-HCl, pH 6.0 ± 0.2) and stored at ≤-60 °C or used for preparation of ADC stock solution.

[0261] The relevant process parameters for obtaining products with different DAR values and DAR value detection data are shown in Table 2. The DAR value detection method is described in Example 3.

[0262] Table 2. Process parameters for preparing MH-ADC1 samples and DAR values

[0263] Referring to MH-ADC1, compound 2, compound 3 or compound 4 was coupled respectively to prepare MH-ADC3, MH-ADC5 and MH-ADC7, wherein Ab is the 2A7-H1 antibody, and a is a number between 3 and 6.

[0264] 2. Preparation of MH-ADC2

[0265] MH-ADC2

[0266] The 2A7-H1 antibody was dialyzed into 10 mM Tris pH 8.0 ± 0.2 buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, and 10 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) stock solution, 10 mM diethylene triamine pentaacetic acid (DTPA) stock solution were added in sequence, and 10 mM Tris pH 8.0 ± 0.2 buffer was added to make the final concentration of the antibody in the reaction system 20 mg / mL, the molar ratio of TCEP to antibody (1.5-3.5): 1, and the final concentration of DTPA 1 mM. After mixing well, the reduction reaction was carried out in a constant temperature mixer at 25°C and 400 rpm for 2 hours. After the reduction was completed, an appropriate amount of 5 mM linker-drug (compound 1) stock solution was added to each reaction system, and the molar ratio of linker-drug to antibody was (3.0-8.0): 1. After mixing well, the coupling reaction was carried out in a constant temperature mixer at 22-37°C and 400 rpm for 16-24 hours to obtain MH-ADC2, wherein Ab is the 2A7-H1 antibody, and Ab is connected to the e and / or f positions of the linker through its thiol group, for example, one or more thiols on Ab are connected to one or more e positions of the linker, and one or more or another thiol is connected to one or more f positions of the linker, or the thiols on Ab are all connected to the e positions of the linker, or the thiols on Ab are all connected to the f positions of the linker. After the coupling was completed, the ADC sample was dialyzed into dialysis buffer (20 mM His / His-HCl, pH 5.2 ± 0.2) and stored at ≤-60°C or used for preparation of ADC stock solution.

[0267] It was detected that the relevant process parameters for obtaining products with different DAR values and the DAR value detection data are shown in Table 3. The DAR value detection method is described in Example 3.

[0268] Table 3. Process parameters for preparing MH-ADC2 samples and DAR values

[0269] Referring to MH-ADC2, compound 2, compound 3 or compound 4 was coupled respectively to prepare MH-ADC4, MH-ADC6, MH-ADC8, wherein Ab is the 2A7-H1 antibody, and a is a number between 3 and 6.

[0270] 3. Preparation of positive control ADC

[0271] Positive control ADC

[0272] The 2A7-H1 antibody was dialyzed into a 20 mM His / His-HCl pH 6.0 ± 0.5 buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, and 10 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) stock solution and 10 mM diethylenetriaminepentaacetic acid (DTPA) stock solution were sequentially added, and 20 mM His / His-HCl pH 6.0 ± 0.5 buffer was supplemented to make the final concentration of the antibody in the reaction system 20 mg / mL, the molar ratio of TCEP to the antibody 2.0:1, and the final concentration of DTPA 1 mM. After thorough mixing, the reaction was placed in a constant temperature mixer, and reduction was carried out at 25 °C and 400 rpm for 2 hours. After the reduction was completed, an appropriate amount of 5 mM linker-drug (Deruxtecan) stock solution was added to each reaction system, and the molar ratio of the linker-drug to the antibody was 4.5:1. After thorough mixing, the coupling reaction was carried out in a constant temperature mixer at 25 °C and 400 rpm for 1 hour to obtain a positive control ADC, wherein Ab is the 2A7-H1 antibody. After the coupling was completed, the ADC sample was dialyzed into a dialysis buffer (20 mM His / His-HCl, pH 5.2 ± 0.2, 10 mM methionine) and stored at ≤-60 °C. The DAR value (HIC-HPLC) of the positive control ADC obtained was 4.1, and the detection method is described in Example 3.

[0273] Example 3. Detection of the drug-antibody coupling ratio of antibody-drug conjugates

[0274] The average drug-antibody coupling ratio (DAR, Drug-to-antibody ratio) of the ADC was detected by high performance liquid hydrophobic chromatography (HIC-HPLC). After the ADC sample was equilibrated to room temperature and mixed, ultrapure water was used as a diluent, and the sample was diluted to a target concentration of 5 mg / mL according to the protein concentration and mixed on a vortex mixer for 20 s. The diluted sample solution was taken into an inner cannula with a pipette, and the sample number was labeled on the sample bottle. The sample detection was completed using a high performance liquid chromatograph (Agilent, 1260 Bio) and a hydrophobic chromatography column (TOSOH, TSKgel Butyl-NPR column (2.5) 4.6 mm*35 mm) after the column was equilibrated. The main method parameters are shown in Table 4, wherein the mobile phase A formula is 20 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0; and the mobile phase B formula is 20 mM sodium phosphate, 25% isopropanol (v / v), pH 7.0. The liquid chromatogram was integrated, and the percentage of ADCs with no coupling drug and 2, 4, 6, and 8 coupled drugs (DAR0%, DAR2%, DAR4%, DAR6%, and DAR8%, respectively) was calculated according to the integration results. The average drug-antibody coupling ratio was calculated using the following formula.

[0275] DAR value = (0 x DAR0% + 2 x DAR2% + 4 x DAR4% + 6 x DAR6% + 8 x DAR8%) / (DAR0% + DAR2% + DAR4% + DAR6% + DAR8%).

[0276] Table 4 Drug-antibody conjugation ratio collection method parameter table

[0277] Example 4. Detection of binding activity of antibody-drug conjugate to human tumor cells

[0278] Flow cytometry was used to detect the in vitro binding activity of antibody-drug conjugate and naked antibody to human tumor cells. The human tumor cells MX-1 (Cobioer, item number CBP60640) in the logarithmic growth phase were trypsinized, resuspended with flow buffer, counted, centrifuged, resuspended with pre-cooled flow buffer to a concentration of 2 x 10 6 After 30 min of incubation on ice in the dark, centrifugation at 1000 rpm was performed, and the supernatant was discarded. 200 μL of pre-cooled flow buffer was added to each well to resuspend the cells, which were then centrifuged and the supernatant was discarded. The secondary antibody (PE Goat anti-Human IgG Fc Secondary Antibody, Invitrogen, item number 12-4998-82) was diluted 1:200 with flow buffer, and 100 μL of the diluted secondary antibody was added to each well of the microplate. After 30 min of incubation on ice in the dark, the cells were resuspended with 200 μL of pre-cooled flow buffer, and the supernatant was discarded. After adding 100 μL of fixing solution (BD, item number 554655) to each well, the cells were fixed on ice in the dark for 10 min. After centrifugation at 1000 rpm for 5 min and discarding the supernatant, the cells were resuspended and washed twice with 200 μL of pre-cooled flow buffer. The cells were resuspended with 100 μL of pre-cooled flow buffer and the fluorescence intensity was detected using a flow cytometer (BECKMAN COULTER, CytoFLEX).

[0279] The experimental results are shown in Figure 1. The tested ADCs had strong binding activity to the tumor cell line MX-1 with high expression of B7H4, and the binding activity of MH-ADC1-2# and MH-ADC2-3# was comparable to that of the naked antibody.

[0280] Example 5. Detection of endocytosis activity of antibody-drug conjugate in human tumor cells

[0281] Logarithmic growth phase of human tumor cells MX-1 were trypsinized, resuspended with flow buffer, counted, centrifuged, resuspended with pre-cooled flow buffer to a concentration of 1 x 10 6 The ADC sample to be tested or naked antibody 2A7-H1 (both at a final concentration of 10 μg / mL) or flow buffer (blank control) was added to the cells, which were incubated on ice for 30 min in the dark. After centrifugation at 1000 rpm, the supernatant was discarded, and the cells were washed 4 times with pre-cooled cell culture medium to remove unbound antibodies or ADCs. After centrifugation and discarding the supernatant, the cells were resuspended with pre-cooled cell culture medium and added to a microplate at 100 μL / well, and incubated at 4°C and 37°C for 0 h, 1 h, 2 h, and 4 h, respectively. After centrifugation at 1000 rpm and discarding the supernatant, 200 μL of pre-cooled flow buffer was added to each well to resuspend the cells, and the cells were again centrifuged and the supernatant was discarded. After diluting the secondary antibody (PE Goat anti-Human IgG Fc Secondary Antibody, Invitrogen, Cat. No. 12-4998-82) 1:200 with flow buffer, 100 μL of the diluted secondary antibody was added to each well of the microplate, and the plate was incubated on ice for 30 min in the dark. After centrifugation and discarding the supernatant, the cells were resuspended with 200 μL of pre-cooled flow buffer, washed, and the supernatant was discarded. Then, 100 μL of fixing solution (BD, Cat. No. 554655) was added to each well, and the plate was fixed at 2-8°C for 30 min. After centrifugation at 1000 rpm for 5 min and discarding the supernatant, the cells were resuspended with 200 μL of pre-cooled flow buffer, washed, and centrifuged and the supernatant was discarded. The cells were resuspended with 100 μL of pre-cooled flow buffer, and the fluorescence intensity was detected using a flow cytometer (BECKMAN COULTER, CytoFLEX). The percentage of endocytosis of the test sample was represented by the ratio of the decrease in fluorescence intensity to the initial fluorescence intensity,

[0282] The experimental results are shown in FIG. 2. The tested ADCs had strong endocytosis activity in the tumor cell line MX-1 with high expression of B7H4, and the endocytosis activity of MH-ADC1-2# and MH-ADC2-3# was comparable to that of the naked antibody.

[0283] Example 6. Inhibition of tumor cell proliferation in vitro by antibody-drug conjugates

[0284] Human tumor cells in logarithmic growth phase were taken, resuspended with fresh complete culture medium after digestion and adjusted to appropriate concentration, added to 96-well cell culture plates, 50 μL / well. The cell culture plates were placed in a 37°C, 5% CO2 incubator for overnight culture. The next day, 50 μL of different concentrations of ADC samples to be tested or small molecule DMSO solution or buffer control were added to the corresponding wells of the cell culture plates, and then placed in the carbon dioxide incubator for 7 days. After incubation, the test plates were equilibrated to room temperature, and luminescence readings were detected using CellTiter Glo assay kit (Promega, G7558) and a multifunctional microplate reader (Spark, Tecan). The cell inhibition rate was calculated according to the following formula: survival rate (%) = (RLU ADC -RLU 空白 ) / (RLU 缓冲液 -RLU 空白 ) x 100%. The Prism Graphpad software was used to draw the pharmacodynamic inhibition rate curve and calculate the IC 50 value (see Table 5, Table 6).

[0285] Table 5. In vitro proliferation inhibition activity of antibody-drug conjugates on human tumor cells - Experiment 1

[0286] Table 6. In vitro proliferation inhibition activity of antibody-drug conjugates on human tumor cells - Experiment 2

[0287] The experimental results show that the tested ADCs have strong proliferation inhibition activity on multiple tumor cell lines with different target expression levels and B7H4 overexpression cell lines, and the proliferation inhibition activity in multiple cell lines is significantly better than that of the positive control ADC.

[0288] Example 7. Detection of bystander killing activity of antibody-drug conjugates

[0289] The stable HEK293 cell strain overexpressing human B7H4 and the control HEK293 cell strain in logarithmic growth phase were respectively taken, trypsinized, neutralized in fresh culture medium (RPMI1640+10% FBS), centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended with RPMI1640+10% FBS. After cell counting, the cell suspension was diluted according to the preset cell density, the overexpression cell strain and the control cell strain were inoculated into a 96-well cell culture plate at a ratio of 1:1, and the total volume was 50 μL. At the same time, control wells inoculated only with HEK293 cells overexpressing B7H4 and only with HEK293 control cells were set. The ADC sample was gradiently diluted, and the diluted ADC sample was added to the aforementioned wells inoculated with co-cultured cells or inoculated only with the overexpression cell strain or the control HEK293 cell strain at a preset final concentration and well plate distribution, 50 μL, mixed well, and then placed in a 5% CO2, 37°C incubator for incubation for 120 h. The cell culture plate was taken out, 100 ul / well of cell viability detection reagent was added, and a multifunctional enzyme label instrument was used for detection.

[0290] The results show that the ADC in the present disclosure has a clear bystander killing effect. Within a certain incubation concentration range, the ADC does not kill the control cells negative for target expression, but after mixing the target overexpression cells with the negative cells, the ADC also has a killing effect on the cells negative for target expression.

[0291] Example 8. In vivo pharmacodynamic detection of antibody-drug conjugate in a mouse subcutaneous transplantation tumor model of human tumor cells

[0292] The human tumor cells in logarithmic growth phase were taken, resuspended in serum-free culture medium after digestion and counting, and inoculated subcutaneously into immunodeficient mice, 100 μL per animal, to establish a mouse subcutaneous transplantation tumor model. After the tumor grew to a measurable range, the long diameter and short diameter of each tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = (a x b 2 ) / 2, where a represents the long diameter of the tumor, and b represents the short diameter of the tumor.

[0293] When the tumor volume reached an average of 100 mm 3 ~200 mm 3 , the mice were randomly grouped according to the tumor volume and the mouse weight. The tumor-bearing mice were injected with solvent control or different doses of ADC through the tail vein, single administration or once every two weeks. The long and short diameters of the tumors were measured twice a week, and the animal body weight was recorded. The tumor volumes of each group were statistically analyzed, and the tumor growth inhibition rate TGI was calculated according to the following formula: TGI = 100% x [1 - (TVt T - TV0 T ) / (TVt C - TV0 C )]. Wherein (TVt TTumor volume of the representative dosing group on the day of measurement, TV0 T Tumor volume of the representative dosing group at the time of grouping; TVt C Tumor volume of the representative solvent control group on the day of measurement, TV0 C Tumor volume of the representative solvent control group at the time of grouping.

[0294] In the human breast cancer MX-1 cell subcutaneous transplantation tumor model of nude mice, the tumor-bearing nude mice were injected with MH-ADC2-3 at 0.5 mg / kg, 1 mg / kg and 3 mg / kg (D0, the day of administration) through the tail vein, and by the end of the experiment (D18), the tumors in the 3 mg / kg dose group completely disappeared, and the tumors in the 1 mg / kg and 0.5 mg / kg dose groups were also significantly inhibited. The tumor proliferation inhibition rates TGI% of the three dose groups were 105%, 87% and 68%, respectively. The tumor growth curve is shown in Figure 3. During the experiment, the animals in each test group did not show obvious abnormalities and weight loss compared with the solvent control group (5% glucose injection), and had good tolerance. The experimental results showed that the tested ADC had excellent in vivo proliferation inhibition effect in the MX-1 nude mouse subcutaneous transplantation tumor model, and had good safety.

[0295] Example 9. Pharmacokinetic study of antibody-drug conjugate

[0296] 6-8 week old SD rats were injected with different doses of the tested ADC through the tail vein, and the volume of administration was 5 mL / kg. Blood was collected before administration (0 h) and at 5 min, 1 h, 4 h, 10 h, 24 h, 48 h, 72 h, 96 h, 168 h, 240 h, 336 h, 504 h, 672 h after the end of administration, and the serum was separated and stored at -70°C±10°C in a ultra-low temperature freezer for examination.

[0297] 2-5 year old cynomolgus monkeys were intravenously infused (fixed time 0.5 h) with different doses of the tested ADC, and blood was collected at 0 h (before administration) and at 0.5 h, 1 h, 4 h, 10 h, 24 h, 48 h, 72 h, 96 h, 168 h, 240 h, 336 h, 504 h, 672 h after the start of administration, and the serum was separated and stored at -70°C±10°C in a ultra-low temperature freezer for examination.

[0298] ADC detection: 100 μL / well of coating working solution (1 μg / mL anti-toxin antibody) was added to the enzyme-labeled plate, and after sealing the plate film, it was incubated at 2-8°C for 16-20 h. The liquid in the 96-well enzyme-labeled plate was discarded, and the plate was washed 3 times with 300 μL / well of washing solution. The 96-well plate was dried on a clean absorbent paper to remove residual liquid. 300 μL / well of blocking solution was added, and it was incubated at room temperature for 1.5-2 h. 5 μL / well of standard curve sample, blank control sample, QC sample, and sample to be tested were added to the dilution plate wells, and then 245 μL of analysis buffer was added to the corresponding dilution plate wells. The plate film was covered and placed on a shaker at room temperature at a speed of not less than 500 rpm for 10 min. The liquid in the 96-well enzyme-labeled plate was discarded, and the plate was washed 3 times with 300 μL / well of washing solution. The 96-well plate was dried on a clean absorbent paper to remove residual liquid. 100 μL / well of the above-mentioned treated sample was added according to the plate layout, and after sealing the plate film, it was incubated at room temperature at 150-300 rpm for 110-120 min. After washing the plate, 100 μL / well of detection working solution I (50 μg / mL biotin-labeled antigen) was added, and after sealing the plate film, it was incubated at room temperature at 150-300 rpm for 55-65 min. After washing the plate again and drying, 100 μL / well of detection working solution II (SA-HRP, Jackson, item number 016-030-084, 1:10,000) was added, and after sealing the plate film, it was incubated at room temperature at 150-300 rpm for 55-65 min. After washing the plate again and drying, 100 μL / well of TMB color developing working solution was added, and after sealing the plate film, it was incubated at room temperature for 7-9 min. 50 μL / well of stop solution was added and mixed gently. The 96-well enzyme-labeled plate was placed in an enzyme-labeled instrument, and the detection wavelength was 450 nm, and the reference wavelength was 620 nm. The SoftMax Pro 7.0.3Gxp software of the enzyme-labeled instrument was used for regression calculation.

[0299] Total anti-detection: 100 μL / well of coating working solution (recombinant human B7H4 protein solution, 0.5 μg / mL) was added to the enzyme-labeled plate, and after sealing the plate film, it was incubated at 2-8°C for 16-20 hours. The liquid in the 96-well enzyme-labeled plate was discarded, and the plate was washed 3 times with 300 μL / well of washing solution. The 96-well plate was dried on a clean absorbent paper to remove residual liquid. 300 μL / well of blocking solution was added, and incubated at room temperature for 1.5-2.5 hours. 5 μL / well of standard curve sample, blank control sample, QC sample and sample to be tested was added to the dilution plate well, and then 245 μL of analysis buffer was added to the corresponding dilution plate well, and the plate film was covered and placed on a shaker at room temperature at a speed of not less than 500 rpm for 10 minutes. The liquid in the 96-well enzyme-labeled plate was discarded, and the plate was washed and dried as described above. According to the plate layout, 100 μL / well of the above-mentioned treated sample was added, and the plate film was sealed and incubated at 25°C at 300 rpm for 115-125 minutes. After washing the plate again and drying, 100 μL / well of detection working solution (Mouse Anti-Human IgG1-Fc Secondary Antibody (HRP), Sino Biological, Item No. 10702-MM01T-H) was added, and the plate film was sealed and incubated at 25°C at 300 rpm for 55-65 minutes. After washing the plate again and drying, 100 μL / well of TMB color developing working solution was added, and the plate film was sealed and incubated at 25°C for 8-12 minutes. 50 μL / well of stop solution was added and mixed gently. The 96-well enzyme-labeled plate was placed in an enzyme-labeled instrument, and the detection wavelength was 450 nm and the reference wavelength was 620 nm. The SoftMax Pro 7.0.3Gxp software of the enzyme-labeled instrument was used for regression calculation.

[0300] After SD rats were given a single intravenous injection of 2, 6, 20 mg / kg MH-ADC2-3#, there was no significant difference between the ADC and total anti-curve in the serum of animals in each group, as shown in Figure 4. The exposure level of ADC and total anti in serum increased basically with the increase of dose, showing linear pharmacokinetic characteristics.

[0301] After cynomolgus monkeys were given a single intravenous injection of 1, 3, 10 mg / kg MH-ADC2-3#, there was no significant difference between the ADC and total anti-curve in the serum of animals in each group, as shown in Figure 5. The increase in the exposure level of ADC and total anti in serum was roughly equivalent to the increase in the proportion of dose, showing linear pharmacokinetic characteristics.

[0302] Example 10. Mass spectrometric drug loading analysis and ring opening confirmation of antibody-drug conjugate

[0303] The intact molecular weight of the antibody-drug conjugate was detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry, and the open ring condition of the antibody-drug conjugate was confirmed by comparing the intact molecular weight of the N-glycosylated sample with the theoretical molecular weight. 500 μg of sample was taken in an ultrafiltration centrifuge tube, centrifuged at 12000 rpm for 10 min, 5 μL of N-glycosidase and 95 μL of water were added to the ultrafiltration tube containing the sample by a pipette and mixed uniformly (enzyme: sample = 1:100), and the final concentration was about 5 mg / mL. The centrifuge tube containing the sample was placed in a 37°C constant temperature water bath for incubation for 16-20 h. 95 μL of the N-glycosylated sample was transferred to an inner tube and placed in a sample bottle, and then placed in the sample disc of the instrument. The instrument and key parameter information are shown in the following table.

[0304] Table 7. Key parameter information table

[0305] The open ring condition of the sample was confirmed by comparing the intact molecular weight of the N-glycosylated sample with the theoretical molecular weight of each peak. The detection results of MH-ADC2-3# are shown in Table 8. It is confirmed by detection that the detection results of the intact molecular weight of each component of the N-glycosylated sample are consistent with the theoretical values, and the open ring is confirmed. The intact molecular weight of each component of the N-glycosylated sample of each MH-ADC2 molecule in Example 2 and the molecules of MH-ADC4, MH-ADC6 and MH-ADC8 is also consistent with the theoretical molecular weight after the open ring, and the open ring is confirmed.

[0306] Table 8. Detection results of intact molecular weight of N-glycosylated sample (MH-ADC2-3#)

[0307] Example 11. Plasma stability study of antibody-drug conjugate

[0308] An appropriate amount of antibody-drug conjugate to be tested was added to an appropriate amount of anticoagulated plasma to make the ADC concentration in the plasma 200 μg / mL, and the sample was collected after incubation at 37°C in a biochemical incubator for different time points, and then purified by protein A chromatography or antigen-coupled magnetic beads. The purified ADC sample was ultrafiltration centrifuged and concentrated, and then subjected to deglycosylation treatment and sample detection according to the method described in Example 10. According to the molecular weight, the mass number of the conjugated small molecule drug was analyzed, the average conjugation rate (Drug-to-antibody ratio, DAR) was calculated according to the peak area percentage and the number of conjugated drugs, and the plasma stability of the ADC was studied by the change of DAR value after different incubation times. The experimental results show that the ADC (such as MH-ADC2-3#) prepared in the application has good plasma stability, and the DAR value after different incubation times has no obvious change or the change is small compared with 0 h.

[0309] A number of embodiments are described herein, but it should be understood that the description is illustrative only and is not intended to be limiting in any respect, and that various modifications can be made by persons of ordinary skill in the art without departing from the scope of the embodiments described herein. Although many possible combinations of features are shown and discussed, many other combinations of the disclosed features are possible. Unless specifically intended otherwise, any feature or element of any embodiment can be used in combination with any other feature or element of any other embodiment, or in replacement of any other feature or element of any other embodiment.

Claims

an antibody drug conjugate represented by formula (I) or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, Ab-(L-D) a Formula (I) wherein: Ab is an antibody or an antigen binding fragment thereof; L is L 1 -L 2 -L 3 -L 4 wherein L 1 is attached to Ab, L 4 is attached to D; L 1 selected from R a selected from hydrogen, Ci-C6alkyl, Ci-C6deuteroalkyl, C3-C8cycloalkyl, and C3-C8deutero- cycloalkyl; L 2 selected from -(CH2) m -X1-X2-(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)- and -(C1-C4)alkylene-C(=O)-, wherein, X1is selected from the group consisting of a bond, C3-C8cycloalkyl, 3-8 membered heterocyclyl, C6-C10aryl, and 5-9 membered heteroaryl, 10 aryl and 5-9 membered heteroaryl, X2 is selected from the group consisting of a bond, -O-, -NH-, -C(=O)-, and -C(=O)NH-, m, n, p and q are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, The -(CH2) m -X1-X2-(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)- and -(C1-C4)alkylene-C(=O)- are optionally substituted by one or more groups selected from hydrogen, halogen, hydroxyl, amino, -(C1-C4)alkylene-hydroxyl and -O-(C1-C4)alkylene-hydroxyl; L 3 selected from the group consisting of amino acid residues and peptide residues consisting of 2-10 amino acid residues; L 4 For wherein the * end is attached to D; D is R 1 and R 2 each independently is selected from hydrogen, deuterium, halogen, hydroxyl, amino, C1-C6alkyl, C1-C6deuteroalkyl, C1-C6haloalkyl, and C3-C8cycloalkyl, or, R 1 and R 2 together with the carbon atom to which they are attached form a C3-C6cycloalkyl or 3-6 membered heterocyclyl; R 3 and R 4 each independently is selected from hydrogen, deuterium, halogen, C1-C6alkyl, C1-C6deuteroalkyl, and C1-C6haloalkyl, or, R 3 and R 4 together with the carbon atom to which they are attached form a 5-6 membered heterocyclyl group optionally substituted with one or more groups selected from hydrogen, deuterium, halogen, hydroxyl, amino, C1-C6 alkyl, C1-C6 deuterated alkyl, and C1-C6 haloalkyl; a is any number between 1-10. The antibody drug conjugate of claim 1 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein, R a selected from hydrogen, Ci-C4alkyl, and Ci-C4deuteroalkyl; Preferably, R a is selected from hydrogen, methyl, ethyl, isopropyl, deuterated methyl, deuterated ethyl and deuterated isopropyl; Preferably, L 1 selected from wherein the * end is attached to L 2 is connected. The antibody drug conjugate of claim 1 or 2 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein, X1 is selected from the group consisting of a bond, 5-6 membered heterocyclyl, and 5-6 membered heteroaryl, Preferably, X1 is selected from the group consisting of a bond, 5-6 membered heterocyclyl, and 5-6 membered heteroaryl, the 5-6 membered heterocyclyl containing 1 or 2 heteroatoms selected from N and O, the 5-6 membered heteroaryl containing 1, 2 or 3 heteroatoms selected from N and O, Preferably, X1 is selected from the group consisting of a bond, pyrrolidinyl, tetrahydrofuranyl, 1,3-dioxolanyl, 1,3-dioxanyl, pyridinyl and pyrimidinyl, Preferably, X1 is selected from the group consisting of 1,3-dioxolanyl, 1,3-dioxanyl and pyridinyl; X2 is selected from the group consisting of a bond and -C(=O)NH-, Preferably, X2 is -C(=O)NH-; m is 0, 1, 2 or 3, Preferably, m is 1 or 2; n is 0, 1, 2 or 3, Preferably, n is 1; p is 0, 3, 4, 5, 6, 7, 8, 9 or 10, Preferably, p is 4 or 8; q is 0, 1, 2 or 3, Preferably, q is 1; L 2 -(CH2) m -X2-(CH2) n -(CH2OCH2) p -C(=O)-, -(CH2) m -X1-X2-(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)-, -X1-(CH2OCH2) p -C(=O)-, -(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)- and -(C1-C4)alkylene-C(=O)-, said -(CH2) m -X2-(CH2) n -(CH2OCH2) p -C(=O)-, -(CH2) m -X1-X2-(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)-, -X1-(CH2OCH2) p -C(=O)-, -(CH2) n -(CH2OCH2) p -(CH2) q -C(=O)- and -(C1-C4)alkylene-C(=O)- are optionally substituted with one or more radicals selected from the group consisting of hydrogen, hydroxy, amino, - methylene-hydroxy, -O-ethylene-hydroxy; Preferably, L 2 -CH2CH2-C(=O)NH-CH2-(CH2OCH2)8-CH2-C(=O)-, -CH2-C(=O)-, -CH2CH2-C(=O)-, -CH(CH3)-C(=O)-, -CH(CH2CH3)-C(=O)-, -CH(CH3)CH2-C(=O)- and -CH2CH(CH3)-C(=O)-, said -CH2CH2-C(=O)NH-CH2-(CH2OCH2)8-CH2-C(=O)-, -CH2-C(=O)-, -CH2CH2-C(=O)-, -CH(CH3)-C(=O)-, -CH(CH2CH3)-C(=O)-, -CH(CH3)CH2-C(=O)- and -CH2CH(CH3)-C(=O)- are optionally substituted with one or more groups selected from H, OH, NH2, CH2OH and -O-CH2CH2OH; Preferably, L 2 selected from -CH(CH2OH)-C(=0)-, -CH(CH2OCH2CH2OH)-C(=0)-, Preferably, L 2 is -CH(CH2OH)-C(=O)-; Preferably, L 2 is Preferably, L 2 is wherein the * end is attached to L 3 is connected. The antibody drug conjugate of any one of claims 1-3 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein, L 3 a peptide residue consisting of 2-4 (preferably 4) amino acid residues, wherein the amino acids are selected from the group consisting of glycine, phenylalanine, valine, alanine, lysine, citrulline, serine, glutamic acid and aspartic acid, preferably the amino acids are selected from the group consisting of glycine and phenylalanine; Preferably, L 3 is glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) (SEQ ID NO: 12); Preferably, L 3 is Preferably, L 3 is wherein the * end is attached to L 4 is connected. The antibody drug conjugate of any one of claims 1-4 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein, R 1 and R 2 each independently is selected from hydrogen, deuterium, C1-C4alkyl, C1-C4deuteroalkyl, C1-C4haloalkyl, and C3-C6cycloalkyl, Preferably, R 1 and R 2 Each is independently selected from hydrogen, deuterium, methyl, deuterated methyl, halomethyl, and cyclopropyl. Preferably, R 1 and R 2 each independently is hydrogen; or, R 1 and R 2 together with the carbon atom to which they are attached form a cyclopropyl, cyclobutyl, oxetanyl, azetanyl, Preferably, R 1 and R 2 Together with the carbon atom attached to it, they form cyclopropyl and cyclobutyl groups; R 3 and R 4 each independently is selected from hydrogen, deuterium, halogen, C1-C4alkyl, C1-C4deuteroalkyl, and C1-C4haloalkyl, R 3 and R 4 are each independently selected from the group consisting of hydrogen, deuterium, fluorine, chlorine, methyl, deuterated methyl, and halogenated methyl, Preferably, R 3 is methyl, Preferably, R 4 is fluorine; or, R 3 and R 4 with the carbon atom to which they are attached form a 5-6 membered heterocyclyl group containing 1 or 2 heteroatoms selected from O, said 5-6 membered heterocyclyl group being optionally substituted by one or more groups selected from hydrogen, deuterium, halogen and C1-C4alkyl, Preferably, R 3 and R 4 The carbon atoms connected to it together form The is optionally substituted with 1, 2 or 3 groups selected from hydrogen, deuterium and fluorine; Preferably, D is The antibody drug conjugate of any one of claims 1-5, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein, each L is each independently selected from: or each L-D is each independently selected from: The antibody drug conjugate of any one of claims 1-6 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein, Ab is an anti-B7-H4 antibody or an antigen binding fragment thereof; Preferably, the antibody or antigen binding fragment thereof, comprises: (a) the following 3 heavy chain variable region (VH) complementarity determining regions (CDRs): (a) the following 3 heavy chain variable region (VH) complementarity determining regions (CDRs): (i) a VH CDR1 having a sequence of a CDR1 contained in the VH as set forth in SEQ ID NO:7 or SEQ ID NO:8, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR1 contained in the VH; (ii) a VH CDR2 having a sequence of a CDR2 contained in the VH as set forth in SEQ ID NO:7 or SEQ ID NO:8, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR2 contained in the VH; and (iii) a VH CDR3 having a sequence of a CDR3 contained in the VH as set forth in SEQ ID NO:7 or SEQ ID NO:8, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR3 contained in the VH; and / or (b) the following 3 light chain variable region (VL) CDRs: (iv) a VL CDR1 having a sequence of a CDR1 contained in the VL as set forth in SEQ ID NO:9, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR1 contained in the VL; (v) a VL CDR2 having a sequence of a CDR2 contained in the VL as set forth in SEQ ID NO:9, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR2 contained in the VL; and (vi) a VL CDR3 having a sequence of a CDR3 contained in the VL as set forth in SEQ ID NO:9, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR3 contained in the VL; Preferably, the substitution in any of (i)-(vi) is a conservative substitution; Preferably, the CDR1, CDR2, and CDR3 contained in the heavy chain variable region (VH), and / or the CDR1, CDR2, and CDR3 contained in the light chain variable region (VL) are defined by the Kabat, Chothia, or IMGT numbering system; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) the sequence of a CDR1, CDR2, and CDR3 contained in the VH as set forth in SEQ ID NO:7 or SEQ ID NO:8; and / or (b) the sequence of a CDR1, CDR2, and CDR3 contained in the VL as set forth in SEQ ID NO:9; Preferably, the antibody or antigen-binding fragment thereof comprises: (a) the following 3 heavy chain variable region (VH) CDRs: (i) a VH CDR1 having a sequence of a CDR1 contained in the VH as set forth in SEQ ID NO:7 or SEQ ID NO:8, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR1 contained in the VH; (ii) a VH CDR2 having a sequence of a CDR2 contained in the VH as set forth in SEQ ID NO:7 or SEQ ID NO:8, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR2 contained in the VH; and (iii) a VH CDR3 having a sequence of a CDR3 contained in the VH as set forth in SEQ ID NO:7 or SEQ ID NO:8, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of the CDR3 contained in the VH; (i) a VH CDR1 consisting of the sequence set forth in SEQ ID NO:1, or a sequence having one or several (e.g. one or two) amino acid substitutions, deletions, or additions as compared to SEQ ID NO: 1, (ii) a VH CDR2 consisting of the sequence set forth in SEQ ID NO:2, or a sequence having one or several (e.g. one or two) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:2, and (iii) a VH CDR3 consisting of the sequence set forth in SEQ ID NO:3, or a sequence having one or several (e.g. one or two) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:3; and / or (b) the following 3 light chain variable region (VL) CDRs: (iv) a VL CDR1 consisting of the sequence set forth in SEQ ID NO:4, or a sequence having one or several (e.g. one or two) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:4, (v) a VL CDR2 consisting of the sequence set forth in SEQ ID NO:5, or a sequence having one or several (e.g. one or two) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:5, and (vi) a VL CDR3 consisting of the sequence set forth in SEQ ID NO:6, or a sequence having one or several (e.g. one or two) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:6; Preferably, the substitution in any of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or antigen binding fragment thereof comprises a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO:2; and, a VH CDR3 as set forth in SEQ ID NO:3; and / or, the VL of the antibody or antigen binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO:4; a VL CDR2 as set forth in SEQ ID NO:5; and, a VL CDR3 as set forth in SEQ ID NO:6; Preferably, the VH of the antibody or antigen binding fragment thereof comprises a VH CDR1 as set forth in SEQ ID NO: 1; a VH CDR2 as set forth in SEQ ID NO:2; and, a VH CDR3 as set forth in SEQ ID NO:3; and, the VL of the antibody or antigen binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO:4; a VL CDR2 as set forth in SEQ ID NO:5; and, a VL CDR3 as set forth in SEQ ID NO:6; Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region of the antibody or antigen binding fragment thereof is as set forth in SEQ ID NO: 9; Preferably, the amino acid sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain of the antibody is as set forth in SEQ ID NO: 11; Preferably, the antibody or antigen binding fragment thereof is linked to L via its thiol group. The antibody drug conjugate of any one of claims 1-7, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein, each a is independently any number between 1 and 8; Preferably, each a is independently any number between 3 and 8; Preferably, each a is independently any number between 3 and 6, such as 3-4, 4-5, 5-6, or such as about 3.1, about 3.3, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.9, about 5.1, about 5.3, about 5.

5. The antibody drug conjugate of any one of claims 1-8, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein, The antibody drug conjugate of Formula (I) is selected from: wherein, Ab is as defined in claim 1 or 7, and a is as defined in claim 1 or 8; In MH-ADC2, MH-ADC4, MH-ADC6, MH-ADC8, Ab is linked (e.g., via its thiol group) to e and / or f, such as one or more thiol groups on Ab being linked to e and another or further thiol group(s) on Ab being linked to f, or, all thiol groups on Ab being linked to e, or, all thiol groups on Ab being linked to f. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1-9, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and optionally one or more pharmaceutical excipients. A composition comprising at least one antibody drug conjugate of any one of claims 1-9, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; Preferably, the composition comprises the antibody drug conjugate MH-ADC1 as set forth in claim 9, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and the antibody drug conjugate MH-ADC2, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; Preferably, the composition comprises the antibody drug conjugate MH-ADC3 as set forth in claim 9, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and the antibody drug conjugate MH-ADC4, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; Preferably, the composition comprises the antibody drug conjugate MH-ADC5 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof as described in claim 9, and the antibody drug conjugate MH-ADC6 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; Preferably, the composition comprises the antibody drug conjugate MH-ADC7 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof as described in claim 9, and the antibody drug conjugate MH-ADC8 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. The antibody drug conjugate of any one of claims 1-9 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or the pharmaceutical composition of claim 10 or the composition of claim 11 for use in the preparation of a medicament acting on the B7-H4 target, or for use in the preparation of a medicament for treating and / or preventing a disease, Preferably, the disease is a B7-H4 related disease; Preferably, the disease is a disease related to abnormal expression of B7-H4; Preferably, the disease is a cancer or an autoimmune disease; More preferably, the disease is selected from breast cancer, ovarian cancer, endometrial cancer, and cholangiocarcinoma. Preferably, the disease is a B7-H4 related disease; Preferably, the disease is a disease related to abnormal expression of B7-H4; Preferably, the disease is a cancer or an autoimmune disease; More preferably, the disease is selected from breast cancer, ovarian cancer, endometrial cancer, and cholangiocarcinoma.

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